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1
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Winsor NJ, Bayer G, Singh O, Chan JK, Li LY, Lieng BY, Foerster E, Popovic A, Tsankov BK, Maughan H, Lemire P, Tam E, Streutker C, Chen L, Heaver SL, Ley RE, Parkinson J, Montenegro-Burke JR, Birchenough GMH, Philpott DJ, Girardin SE. Cross-kingdom-mediated detection of intestinal protozoa through NLRP6. Cell Host Microbe 2025; 33:388-407.e9. [PMID: 40043701 DOI: 10.1016/j.chom.2025.02.008] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2024] [Revised: 12/13/2024] [Accepted: 02/10/2025] [Indexed: 03/15/2025]
Abstract
Intestinal protists are detected by the host innate immune system through mechanisms that remain poorly understood. Here, we demonstrate that Tritrichomonas protozoa induce thickening of the colonic mucus in an NLRP6-, ASC-, and caspase-11-dependent manner, consistent with the activation of sentinel goblet cells. Mucus growth is recapitulated with cecal extracts from Tritrichomonas-infected mice but not purified protozoa, suggesting that NLRP6 may detect infection-induced microbial dysbiosis. In agreement, Tritrichomonas infection causes a shift in the microbiota with the expansion of Bacteroides and Prevotella, and untargeted metabolomics reveals a dramatic increase in several classes of metabolites, including sphingolipids. Finally, using a combination of gnotobiotic mice and ex vivo mucus analysis, we demonstrate that wild-type, but not sphingolipid-deficient, B. thetaiotaomicron is sufficient to induce NLRP6-dependent sentinel goblet cell function, with the greatest effect observed in female mice. Thus, we propose that NLRP6 is a sensor of intestinal protozoa infection through monitoring microbial sphingolipids.
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Affiliation(s)
- Nathaniel J Winsor
- Department of Immunology, University of Toronto, Toronto, ON, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada
| | - Giuliano Bayer
- Department of Immunology, University of Toronto, Toronto, ON, Canada
| | - Ojas Singh
- Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada
| | - Jeremy K Chan
- Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada
| | - Lu Yi Li
- Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada
| | - Brandon Y Lieng
- Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada
| | | | - Ana Popovic
- Department of Biochemistry, University of Toronto, Toronto, ON, Canada; Molecular Medicine, Hospital for Sick Children, Toronto, ON, Canada
| | - Boyan K Tsankov
- Department of Immunology, University of Toronto, Toronto, ON, Canada
| | | | - Paul Lemire
- Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada
| | - Elaine Tam
- Department of Immunology, University of Toronto, Toronto, ON, Canada
| | | | - Lina Chen
- Sunnybrook Health Sciences Centre, Toronto, ON, Canada
| | - Stacey L Heaver
- Department of Microbiome Science, Max Planck Institute for Biology, Tübingen, Germany
| | - Ruth E Ley
- Department of Microbiome Science, Max Planck Institute for Biology, Tübingen, Germany
| | - John Parkinson
- Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada; Department of Biochemistry, University of Toronto, Toronto, ON, Canada; Molecular Medicine, Hospital for Sick Children, Toronto, ON, Canada
| | - J Rafael Montenegro-Burke
- Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada; Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada
| | - George M H Birchenough
- Department of Medical Biochemistry, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden
| | - Dana J Philpott
- Department of Immunology, University of Toronto, Toronto, ON, Canada.
| | - Stephen E Girardin
- Department of Immunology, University of Toronto, Toronto, ON, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.
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2
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Lee MG, Song Y, Kang H. Exploring the complex immunomodulatory effects and gut defense via oral administration of Astragali radix water extract to normal mice. BMC Complement Med Ther 2024; 24:361. [PMID: 39375623 PMCID: PMC11460088 DOI: 10.1186/s12906-024-04667-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2024] [Accepted: 09/27/2024] [Indexed: 10/09/2024] Open
Abstract
BACKGROUND Astragali radix (AR) is one of the most widely used traditional Chinese herbal medicines. It exhibits diverse biological activities, including immunomodulatory and anti-inflammatory properties; however, some of its activities have only been demonstrated in vitro. OBJECTIVE To examine the effects of orally administered AR extract on immune cells and the intestine under physiological conditions, which bridges the gap between previously observed in vitro outcomes and in vivo results. METHODS AR extract was prepared by hot water extraction. Three separate animal experiments were conducted to isolate macrophages, splenocytes, and the small intestine epithelium. For the macrophage preparation experiment, an intraperitoneal injection of sterile thioglycolate was administered. The mice received oral AR extract at doses of 0.1, 0.5, or 2.5 g/kg for ten days. At the end of each experiment, cells or tissues were isolated. A portion of macrophages and splenocytes were analyzed for the phenotypic changes. The remaining cells were cultured and stimulated with lipopolysaccharide (LPS) or mitogen ex vivo to assess activation status, proliferation, and cytokine production. Samples of the intestine were subjected to real-time RT-PCR. RESULTS Peritoneal macrophages from AR-treated mice exhibited increased expression of scavenger receptors, including SRA and CD36. Stimulation of these macrophages ex vivo with LPS selectively modulated the inflammatory response, including reduced expression of the costimulatory molecules CD40 and CD86, which are important for T cell responses, without affecting TNF-α and IL-6 production. Splenocytes from AR-treated mice exhibited a dose-dependent increase in CD4 and CD8 T cells; however, stimulation with mitogen decreased T cell proliferation and reduced IFN-γ production, which is essential for macrophage activation. An analysis of the small intestinal epithelium revealed an attenuated antimicrobial response, including reduced IgA content in the lumen and decreased expression of mucin-2 and polymeric Ig receptor genes. CONCLUSION The response of immune cells following oral treatment with AR extract did not replicate the previously documented in vitro findings. Immune cells and intestinal epithelium from mice administered oral AR extract exhibited a selective anti-inflammatory phenotype. The overall findings indicate that the systemic effects after oral administration of AR extract include reduced sensitivity to inflammatory insults.
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Affiliation(s)
- Mi-Gi Lee
- Bio-Center, Gyeonggido Business & Science Accelerator, Suwon, 16229, Republic of Korea
| | - Youngju Song
- Department of Biomedical Science and Technology, Graduate School, Kyung Hee University, Seoul, 02447, Republic of Korea
| | - Hee Kang
- Humanitas College, Kyung Hee University, Yongin, 17104, Republic of Korea.
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3
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Matsumoto R, Ogata K, Takahashi D, Kinashi Y, Yamada T, Morita R, Tanaka K, Hattori K, Endo M, Fujimura Y, Sasaki N, Ohno H, Ishihama Y, Kimura S, Hase K. AP-1B regulates interactions of epithelial cells and intraepithelial lymphocytes in the intestine. Cell Mol Life Sci 2024; 81:425. [PMID: 39369131 PMCID: PMC11455912 DOI: 10.1007/s00018-024-05455-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2024] [Revised: 09/06/2024] [Accepted: 09/17/2024] [Indexed: 10/07/2024]
Abstract
Intraepithelial lymphocytes (IELs) reside in the epithelial layer and protect against foreign pathogens, maintaining the epithelial barrier function in the intestine. Interactions between IEL and epithelial cells are required for IELs to function effectively; however, the underlying molecular machinery remains to be elucidated. In this study, we found that intestinal epithelium-specific deficiency of the clathrin adaptor protein (AP)-1B, which regulates basolateral protein sorting, led to a massive reduction in IELs. Quantitative proteomics demonstrated that dozens of proteins, including known IEL-interacting proteins (E-cadherin, butyrophilin-like 2, and plexin B2), were decreased in the basolateral membrane of AP-1B-deficient epithelial cells. Among these proteins, CD166 interacted with CD6 on the surface of induced IEL. CD166 knockdown, using shRNA in intestinal organoid cultures, significantly inhibited IEL recruitment to the epithelial layer. These findings highlight the essential role of AP-1B-mediated basolateral sorting in IEL maintenance and survival within the epithelial layer. This study reveals a novel function of AP-1B in the intestinal immune system.
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Affiliation(s)
- Ryohtaroh Matsumoto
- Division of Biochemistry, Graduate School of Pharmaceutical Science and Faculty of Pharmacy, Keio University, 1-5-30 Shiba Koen, Minato-ku, Tokyo, 105-8512, Japan
| | - Kosuke Ogata
- Department of Molecular Systems BioAnalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan
| | - Daisuke Takahashi
- Division of Biochemistry, Graduate School of Pharmaceutical Science and Faculty of Pharmacy, Keio University, 1-5-30 Shiba Koen, Minato-ku, Tokyo, 105-8512, Japan
| | - Yusuke Kinashi
- Division of Biochemistry, Graduate School of Pharmaceutical Science and Faculty of Pharmacy, Keio University, 1-5-30 Shiba Koen, Minato-ku, Tokyo, 105-8512, Japan
| | - Takahiro Yamada
- Division of Biochemistry, Graduate School of Pharmaceutical Science and Faculty of Pharmacy, Keio University, 1-5-30 Shiba Koen, Minato-ku, Tokyo, 105-8512, Japan
| | - Ryo Morita
- Division of Biochemistry, Graduate School of Pharmaceutical Science and Faculty of Pharmacy, Keio University, 1-5-30 Shiba Koen, Minato-ku, Tokyo, 105-8512, Japan
| | - Keisuke Tanaka
- Division of Biochemistry, Graduate School of Pharmaceutical Science and Faculty of Pharmacy, Keio University, 1-5-30 Shiba Koen, Minato-ku, Tokyo, 105-8512, Japan
| | - Kouya Hattori
- Division of Biochemistry, Graduate School of Pharmaceutical Science and Faculty of Pharmacy, Keio University, 1-5-30 Shiba Koen, Minato-ku, Tokyo, 105-8512, Japan
| | - Mayumi Endo
- Division of Biochemistry, Graduate School of Pharmaceutical Science and Faculty of Pharmacy, Keio University, 1-5-30 Shiba Koen, Minato-ku, Tokyo, 105-8512, Japan
| | - Yumiko Fujimura
- Division of Biochemistry, Graduate School of Pharmaceutical Science and Faculty of Pharmacy, Keio University, 1-5-30 Shiba Koen, Minato-ku, Tokyo, 105-8512, Japan
| | - Nobuo Sasaki
- Laboratory of Mucosal Ecosystem Design, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan
| | - Hiroshi Ohno
- Laboratory for Intestinal Ecosystem, RIKEN Center for Integrative Medical Science, Yokohama, Kanagawa, Japan
| | - Yasushi Ishihama
- Department of Molecular Systems BioAnalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan
- Laboratory of Clinical and Analytical Chemistry, National Institute of Biomedical Innovation, Health and Nutrition, Ibaraki, Osaka, 567-0085, Japan
| | - Shunsuke Kimura
- Division of Biochemistry, Graduate School of Pharmaceutical Science and Faculty of Pharmacy, Keio University, 1-5-30 Shiba Koen, Minato-ku, Tokyo, 105-8512, Japan.
- PRESTO, Japan Science and Technology Agency, Saitama, 332-0012, Japan.
| | - Koji Hase
- Division of Biochemistry, Graduate School of Pharmaceutical Science and Faculty of Pharmacy, Keio University, 1-5-30 Shiba Koen, Minato-ku, Tokyo, 105-8512, Japan.
- The Institute of Fermentation Sciences (IFeS), Faculty of Food and Agricultural Sciences, Fukushima University, Kanayagawa, Fukushima, 960-1296, Japan.
- International Research and Development Center for Mucosal Vaccines, The Institute of Medical Science, The University of Tokyo (IMSUT), Tokyo, Japan.
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Lassoued N, Yero A, Jenabian MA, Soret R, Pilon N. Efficient enzyme-free method to assess the development and maturation of the innate and adaptive immune systems in the mouse colon. Sci Rep 2024; 14:11063. [PMID: 38744932 PMCID: PMC11094196 DOI: 10.1038/s41598-024-61834-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2024] [Accepted: 05/10/2024] [Indexed: 05/16/2024] Open
Abstract
Researchers who aim to globally analyze the gastrointestinal immune system via flow cytometry have many protocol options to choose from, with specifics generally tied to gut wall layers of interest. To get a clearer idea of the approach we should use on full-thickness colon samples from mice, we first undertook a systematic comparison of three tissue dissociation techniques: two based on enzymatic cocktails and the other one based on manual crushing. Using flow cytometry panels of general markers of lymphoid and myeloid cells, we found that the presence of cell-surface markers and relative cell population frequencies were more stable with the mechanical method. Both enzymatic approaches were associated with a marked decrease of several cell-surface markers. Using mechanical dissociation, we then developed two minimally overlapping panels, consisting of a total of 26 antibodies, for serial profiling of lymphoid and myeloid lineages from the mouse colon in greater detail. Here, we highlight how we accurately delineate these populations by manual gating, as well as the reproducibility of our panels on mouse spleen and whole blood. As a proof-of-principle of the usefulness of our general approach, we also report segment- and life stage-specific patterns of immune cell profiles in the colon. Overall, our data indicate that mechanical dissociation is more suitable and efficient than enzymatic methods for recovering immune cells from all colon layers at once. Additionally, our panels will provide researchers with a relatively simple tool for detailed immune cell profiling in the murine gastrointestinal tract, regardless of life stage or experimental conditions.
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Affiliation(s)
- Nejia Lassoued
- Molecular Genetics of Development Laboratory, Department of Biological Sciences, Université du Québec à Montréal, Montreal, QC, Canada
- Centre d'excellence en recherche sur les maladies orphelines - Fondation Courtois (CERMO-FC), Université du Québec à Montréal, Montreal, QC, Canada
| | - Alexis Yero
- Centre d'excellence en recherche sur les maladies orphelines - Fondation Courtois (CERMO-FC), Université du Québec à Montréal, Montreal, QC, Canada
- Human Immuno-Virology Laboratory, Department of Biological Sciences, Université du Québec à Montréal, Montreal, QC, Canada
| | - Mohammad-Ali Jenabian
- Centre d'excellence en recherche sur les maladies orphelines - Fondation Courtois (CERMO-FC), Université du Québec à Montréal, Montreal, QC, Canada
- Human Immuno-Virology Laboratory, Department of Biological Sciences, Université du Québec à Montréal, Montreal, QC, Canada
| | - Rodolphe Soret
- Molecular Genetics of Development Laboratory, Department of Biological Sciences, Université du Québec à Montréal, Montreal, QC, Canada.
- Centre d'excellence en recherche sur les maladies orphelines - Fondation Courtois (CERMO-FC), Université du Québec à Montréal, Montreal, QC, Canada.
| | - Nicolas Pilon
- Molecular Genetics of Development Laboratory, Department of Biological Sciences, Université du Québec à Montréal, Montreal, QC, Canada.
- Centre d'excellence en recherche sur les maladies orphelines - Fondation Courtois (CERMO-FC), Université du Québec à Montréal, Montreal, QC, Canada.
- Department of Pediatrics, Université de Montréal, Montreal, QC, Canada.
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Huang J, Demmler R, Mohamed Abdou M, Thoma OM, Weigmann B, Waldner MJ, Stürzl M, Naschberger E. Rapid qPCR-based quantitative immune cell phenotyping in mouse tissues. J Investig Med 2024; 72:47-56. [PMID: 37858974 DOI: 10.1177/10815589231210497] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2023]
Abstract
The immune microenvironment plays an important role in the regulation of diseases. The characterization of the cellular composition of immune cell infiltrates in diseases and respective models is a major task in pathogenesis research and diagnostics. For the assessment of immune cell populations in tissues, fluorescence-activated cell sorting (FACS) or immunohistochemistry (IHC) are the two most common techniques presently applied, but they are cost intensive, laborious, and sometimes limited by the availability of suitable antibodies. Complementary rapid qPCR-based approaches exist for the human situation but are lacking for experimental mouse models. Accordingly, we developed a robust, rapid RT-qPCR-based approach to determine and quantify the abundance of prominent immune cell populations such as T cells, helper T (Th) cells, cytotoxic T cells, Th1 cells, B cells, and macrophages in mouse tissues. The results were independently validated by the gold standards IHC and FACS in corresponding tissues and showed high concordance.
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Affiliation(s)
- Jinghao Huang
- Division of Molecular and Experimental Surgery, Translational Research Center, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany
| | - Richard Demmler
- Division of Molecular and Experimental Surgery, Translational Research Center, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany
| | - Mariam Mohamed Abdou
- Division of Molecular and Experimental Surgery, Translational Research Center, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany
| | - Oana-Maria Thoma
- Department of Medicine 1, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany
| | - Benno Weigmann
- Department of Medicine 1, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany
- Medical Immunology Campus Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), Erlangen, Germany
| | - Maximilian J Waldner
- Department of Medicine 1, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany
| | - Michael Stürzl
- Division of Molecular and Experimental Surgery, Translational Research Center, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany
- Comprehensive Cancer Center Erlangen-EMN, Universitätsklinikum Erlangen, Erlangen, Germany
| | - Elisabeth Naschberger
- Division of Molecular and Experimental Surgery, Translational Research Center, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany
- Comprehensive Cancer Center Erlangen-EMN, Universitätsklinikum Erlangen, Erlangen, Germany
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Borbet TC, Zaldaña K, Zavitsanou AM, Hines MJ, Bajwa S, Morrison T, Boehringer T, Hallisey VM, Cadwell K, Koralov SB. Temporal Tracking of Plasma Cells in vivo Using J-chain CreERT2 Reporter System. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.12.02.569736. [PMID: 38106171 PMCID: PMC10723324 DOI: 10.1101/2023.12.02.569736] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/19/2023]
Abstract
Plasma cells (PCs) are essential for humoral immunity, as they are responsible for the production of antibodies and contribute to immunological memory. Despite their importance, differentiating between long-lived and short-lived PCs in vivo remains a challenge due to a lack of specific markers to distinguish these populations. Addressing this gap, our study introduces a novel J-chain CreERT2 GFP allele (IgJCreERT2) for precise genetic studies of PCs. This model takes advantage of PC-restricted expression of the J-chain gene, enabling temporal and cell-specific tracking of PCs utilizing a tamoxifen-inducible Cre recombinase. Our in vitro and in vivo validation studies of the inducible Cre allele confirmed the fidelity and utility of this model and demonstrated the model's ability to trace the long-lived PC population in vivo following immunization. The IgJCreERT2 model allowed for detailed analysis of surface marker expression on PCs, revealing insights into PC heterogeneity and characteristics. Our findings not only validate the IgJCreERT2 mouse as a reliable tool for studying PCs but also facilitate the investigation of PC dynamics and longevity, particularly in the context of humoral immunity and vaccine responses. This model represents a significant advancement for the in-depth study of PCs in health and disease, offering a new avenue for the exploration of PC biology and immunological memory.
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Affiliation(s)
- Timothy C. Borbet
- Department of Pathology, New York University School of Medicine, New York, NY 10016, USA
| | - Kimberly Zaldaña
- Department of Pathology, New York University School of Medicine, New York, NY 10016, USA
| | - Anastasia-Maria Zavitsanou
- Department of Pathology, New York University School of Medicine, New York, NY 10016, USA
- Zuckerman Mind Brain Behavior Institute, Columbia University, New York, NY, 10027, USA
| | - Marcus J. Hines
- Department of Pathology, New York University School of Medicine, New York, NY 10016, USA
| | - Sofia Bajwa
- Department of Pathology, New York University School of Medicine, New York, NY 10016, USA
| | - Tate Morrison
- Department of Pathology, New York University School of Medicine, New York, NY 10016, USA
| | - Thomas Boehringer
- Department of Pathology, New York University School of Medicine, New York, NY 10016, USA
| | - Victoria M. Hallisey
- Department of Pathology, New York University School of Medicine, New York, NY 10016, USA
- Department of Cell Biology, New York University School of Medicine, New York, NY 10016, USA
| | - Ken Cadwell
- Division of Gastroenterology and Hepatology, Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA
| | - Sergei B. Koralov
- Department of Pathology, New York University School of Medicine, New York, NY 10016, USA
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7
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Domínguez K, Lindon AK, Gibbons J, Darch SE, Randis TM. Group B Streptococcus Drives Major Transcriptomic Changes in the Colonic Epithelium. Infect Immun 2023; 91:e0003523. [PMID: 37278645 PMCID: PMC10353456 DOI: 10.1128/iai.00035-23] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2023] [Accepted: 05/05/2023] [Indexed: 06/07/2023] Open
Abstract
Group B Streptococcus (GBS) is a leading cause of infant sepsis worldwide. Colonization of the gastrointestinal tract is a critical precursor to late-onset disease in exposed newborns. Neonatal susceptibility to GBS intestinal translocation stems from intestinal immaturity; however, the mechanisms by which GBS exploits the immature host remain unclear. β-hemolysin/cytolysin (βH/C) is a highly conserved toxin produced by GBS capable of disrupting epithelial barriers. However, its role in the pathogenesis of late-onset GBS disease is unknown. Our aim was to determine the contribution of βH/C to intestinal colonization and translocation to extraintestinal tissues. Using our established mouse model of late-onset GBS disease, we exposed animals to GBS COH-1 (WT), a βH/C-deficient mutant (KO), or vehicle control (phosphate-buffered saline [PBS]) via gavage. Blood, spleen, brain, and intestines were harvested 4 days post-exposure for determination of bacterial burden and isolation of intestinal epithelial cells. RNA sequencing was used to examine the transcriptomes of host cells followed by gene ontology enrichment and KEGG pathway analysis. A separate cohort of animals was followed longitudinally to compare colonization kinetics and mortality between WT and KO groups. We demonstrate that dissemination to extraintestinal tissues occurred only in the WT exposed animals. We observed major transcriptomic changes in the colons of colonized animals, but not in the small intestines. We noted differential expression of genes that indicated the role of βH/C in altering epithelial barrier structure and immune response signaling. Overall, our results demonstrate an important role of βH/C in the pathogenesis of late-onset GBS disease.
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Affiliation(s)
- Kristen Domínguez
- Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, Florida, USA
| | - April K. Lindon
- Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, Florida, USA
| | - Justin Gibbons
- Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, Florida, USA
| | - Sophie E. Darch
- Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, Florida, USA
| | - Tara M. Randis
- Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, Florida, USA
- Pediatrics, Morsani College of Medicine, University of South Florida, Tampa, Florida, USA
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8
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Teichman S, Wang H, Lee CR, Mohtashami M, Foerster E, Han J, Trotman-Grant AC, Winer S, Tsui H, Philpott DJ, Zúñiga-Pflücker JC. Recent Thymic Emigrants Require RBPJ-Dependent Notch Signaling to Transition into Functionally Mature Naive T Cells. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2023; 211:81-90. [PMID: 37154711 PMCID: PMC10330261 DOI: 10.4049/jimmunol.2300140] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/24/2023] [Accepted: 04/18/2023] [Indexed: 05/10/2023]
Abstract
Recent thymic emigrant (RTE) cells are nascent T cells that continue their post-thymic maturation in the periphery and dominate T cell immune responses in early life and in adults having undergone lymphodepletion regimens. However, the events that govern their maturation and their functionality as they transition to mature naive T cells have not been clearly defined. Using RBPJind mice, we were able to identify different stages of RTE maturation and interrogate their immune function using a T cell transfer model of colitis. As CD45RBlo RTE cells mature, they transition through a CD45RBint immature naive T (INT) cell population that is more immunocompetent but shows a bias toward IL-17 production at the expense of IFN-γ. Additionally, the levels of IFN-γ and IL-17 produced in INT cells are highly dependent on whether Notch signals are received during INT cell maturation or during their effector function. IL-17 production by INT cells showed a total requirement for Notch signaling. Loss of Notch signaling at any stage of INT cells resulted in an impaired colitogenic effect of INT cells. RNA sequencing of INT cells that had matured in the absence of Notch signals showed a reduced inflammatory profile compared with Notch-responsive INT cells. Overall, we have elucidated a previously unknown INT cell stage, revealed its intrinsic bias toward IL-17 production, and demonstrated a role for Notch signaling in INT cell peripheral maturation and effector function in the context of a T cell transfer model of colitis.
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Affiliation(s)
- Sintia Teichman
- Biological Sciences, Sunnybrook Research Institute, Toronto, ON, Canada
- Department of Immunology, University of Toronto, Toronto, ON, Canada
| | - Helen Wang
- Biological Sciences, Sunnybrook Research Institute, Toronto, ON, Canada
- Department of Immunology, University of Toronto, Toronto, ON, Canada
| | - Christina R. Lee
- Biological Sciences, Sunnybrook Research Institute, Toronto, ON, Canada
| | | | | | - Jianxun Han
- Biological Sciences, Sunnybrook Research Institute, Toronto, ON, Canada
| | - Ashton C. Trotman-Grant
- Biological Sciences, Sunnybrook Research Institute, Toronto, ON, Canada
- Department of Immunology, University of Toronto, Toronto, ON, Canada
| | - Shawn Winer
- Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada
| | - Hubert Tsui
- Biological Sciences, Sunnybrook Research Institute, Toronto, ON, Canada
- Department of Immunology, University of Toronto, Toronto, ON, Canada
- Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada
- Division of Hematological Pathology, Department of Laboratory Medicine and Molecular Diagnostics, Precision Diagnostics and Therapeutics Program, Sunnybrook Health Sciences Centre, Toronto, ON, Canada
| | - Dana J. Philpott
- Department of Immunology, University of Toronto, Toronto, ON, Canada
| | - Juan Carlos Zúñiga-Pflücker
- Biological Sciences, Sunnybrook Research Institute, Toronto, ON, Canada
- Department of Immunology, University of Toronto, Toronto, ON, Canada
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9
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Prior JT, Limbert VM, Horowitz RM, D'Souza SJ, Bachnak L, Godwin MS, Bauer DL, Harrell JE, Morici LA, Taylor JJ, McLachlan JB. Establishment of isotype-switched, antigen-specific B cells in multiple mucosal tissues using non-mucosal immunization. NPJ Vaccines 2023; 8:80. [PMID: 37258506 PMCID: PMC10231862 DOI: 10.1038/s41541-023-00677-z] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2022] [Accepted: 05/18/2023] [Indexed: 06/02/2023] Open
Abstract
Although most pathogens infect the human body via mucosal surfaces, very few injectable vaccines can specifically target immune cells to these tissues where their effector functions would be most desirable. We have previously shown that certain adjuvants can program vaccine-specific helper T cells to migrate to the gut, even when the vaccine is delivered non-mucosally. It is not known whether this is true for antigen-specific B cell responses. Here we show that a single intradermal vaccination with the adjuvant double mutant heat-labile toxin (dmLT) induces a robust endogenous, vaccine-specific, isotype-switched B cell response. When the vaccine was intradermally boosted, we detected non-circulating vaccine-specific B cell responses in the lamina propria of the large intestines, Peyer's patches, and lungs. When compared to the TLR9 ligand adjuvant CpG, only dmLT was able to drive the establishment of isotype-switched resident B cells in these mucosal tissues, even when the dmLT-adjuvanted vaccine was administered non-mucosally. Further, we found that the transcription factor Batf3 was important for the full germinal center reaction, isotype switching, and Peyer's patch migration of these B cells. Collectively, these data indicate that specific adjuvants can promote mucosal homing and the establishment of activated, antigen-specific B cells in mucosal tissues, even when these adjuvants are delivered by a non-mucosal route. These findings could fundamentally change the way future vaccines are formulated and delivered.
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Affiliation(s)
- John T Prior
- Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana, USA
| | - Vanessa M Limbert
- Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana, USA
| | - Rebecca M Horowitz
- Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana, USA
| | - Shaina J D'Souza
- Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana, USA
| | - Louay Bachnak
- Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana, USA
| | - Matthew S Godwin
- Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana, USA
| | - David L Bauer
- Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana, USA
| | - Jaikin E Harrell
- Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana, USA
| | - Lisa A Morici
- Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana, USA
| | - Justin J Taylor
- Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
- Department of Global Health, University of Washington, Seattle, WA, USA
- Department of Immunology, University of Washington, Seattle, WA, USA
| | - James B McLachlan
- Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana, USA.
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10
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Wong CK, Yusta B, Koehler JA, Baggio LL, McLean BA, Matthews D, Seeley RJ, Drucker DJ. Divergent roles for the gut intraepithelial lymphocyte GLP-1R in control of metabolism, microbiota, and T cell-induced inflammation. Cell Metab 2022; 34:1514-1531.e7. [PMID: 36027914 DOI: 10.1016/j.cmet.2022.08.003] [Citation(s) in RCA: 49] [Impact Index Per Article: 16.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/06/2021] [Revised: 05/31/2022] [Accepted: 08/03/2022] [Indexed: 11/03/2022]
Abstract
Gut intraepithelial lymphocytes (IELs) are thought to calibrate glucagon-like peptide 1 (GLP-1) bioavailability, thereby regulating systemic glucose and lipid metabolism. Here, we show that the gut IEL GLP-1 receptor (GLP-1R) is not required for enteroendocrine L cell GLP-1 secretion and glucose homeostasis nor for the metabolic benefits of GLP-1R agonists (GLP-1RAs). Instead, the gut IEL GLP-1R is essential for the full effects of GLP-1RAs on gut microbiota. Moreover, independent of glucose control or weight loss, the anti-inflammatory actions of GLP-1RAs require the gut IEL GLP-1R to selectively restrain local and systemic T cell-induced, but not lipopolysaccharide-induced, inflammation. Such effects are mediated by the suppression of gut IEL effector functions linked to the dampening of proximal T cell receptor signaling in a protein-kinase-A-dependent manner. These data reposition key roles of the L cell-gut IEL GLP-1R axis, revealing mechanisms linking GLP-1R activation in gut IELs to modulation of microbiota composition and control of intestinal and systemic inflammation.
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Affiliation(s)
- Chi Kin Wong
- Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, ON, Canada
| | - Bernardo Yusta
- Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, ON, Canada
| | - Jacqueline A Koehler
- Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, ON, Canada
| | - Laurie L Baggio
- Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, ON, Canada
| | - Brent A McLean
- Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, ON, Canada
| | - Dianne Matthews
- Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, ON, Canada
| | - Randy J Seeley
- Department of Surgery, University of Michigan, Ann Arbor, MI, USA
| | - Daniel J Drucker
- Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, ON, Canada; Department of Medicine, University of Toronto, Toronto, ON, Canada.
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11
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Seki N, Kimizuka T, Gondo M, Yamaguchi G, Sugiura Y, Akiyama M, Yakabe K, Uchiyama J, Higashi S, Haneda T, Suematsu M, Hase K, Kim YG. D-tryptophan suppresses enteric pathogens and pathobionts and prevents colitis by modulating microbial tryptophan metabolism. iScience 2022; 25:104838. [PMID: 35996581 PMCID: PMC9391578 DOI: 10.1016/j.isci.2022.104838] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2021] [Revised: 05/17/2022] [Accepted: 07/21/2022] [Indexed: 11/17/2022] Open
Abstract
D-Amino acids (D-AAs) have various functions in mammals and microbes. D-AAs are produced by gut microbiota and can act as potent bactericidal molecules. Thus, D-AAs regulate the ecological niche of the intestine; however, the actual impacts of D-AAs in the gut remain unknown. In this study, we show that D-Tryptophan (D-Trp) inhibits the growth of enteric pathogen and colitogenic pathobionts. The growth of Citrobacter rodentium in vitro is strongly inhibited by D-Trp treatment. Moreover, D-Trp protects mice from lethal C. rodentium infection via reduction of the pathogen. Additionally, D-Trp prevents the development of experimental colitis by the depletion of specific microbes in the intestine. D-Trp increases the intracellular level of indole acrylic acid (IA), a key molecule that determines the susceptibility of enteric microbes to D-Trp. Treatment with IA improves the survival of mice infected with C. rodentium. Hence, D-Trp could act as a gut environmental modulator that regulates intestinal homeostasis.
D-Trp inhibits the growth of Citrobacter rodentium in vitro and in vivo D-Trp suppresses experimental colitis by the depletion of specific gut microbes IA is the metabolite that determines the susceptibility of enteric microbes to D-Trp
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Affiliation(s)
- Natsumi Seki
- Research Center for Drug Discovery, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan
- Department of Biochemistry, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan
| | - Tatsuki Kimizuka
- Research Center for Drug Discovery, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan
- Department of Biochemistry, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan
| | - Monica Gondo
- Research Center for Drug Discovery, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan
- Department of Biochemistry, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan
| | - Genki Yamaguchi
- Research Center for Drug Discovery, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan
- Department of Biochemistry, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan
| | - Yuki Sugiura
- Department of Biochemistry, Keio University School of Medicine, Tokyo 160-8582, Japan
| | - Masahiro Akiyama
- Research Center for Drug Discovery, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan
| | - Kyosuke Yakabe
- Research Center for Drug Discovery, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan
- Department of Biochemistry, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan
| | - Jun Uchiyama
- Research Center for Drug Discovery, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan
- Department of Biochemistry, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan
| | - Seiichiro Higashi
- Co-Creation Center, Meiji Holdings Co., Ltd., 1-29-1 Nanakuni, Hachiouji, Tokyo 192-0919, Japan
| | - Takeshi Haneda
- Laboratory of Microbiology, School of Pharmacy, Kitasato University, Tokyo 108-8641, Japan
| | - Makoto Suematsu
- Department of Biochemistry, Keio University School of Medicine, Tokyo 160-8582, Japan
| | - Koji Hase
- Department of Biochemistry, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan
| | - Yun-Gi Kim
- Research Center for Drug Discovery, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan
- Corresponding author
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12
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Wan F, Wang H, Wang M, Lv J, Zhao M, Zhang H. Sustained release of Lactobacillus casei cell wall extract can induce a continuous and stable IgA deposition model. J Pathol 2022; 257:262-273. [PMID: 35170753 DOI: 10.1002/path.5884] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2021] [Revised: 01/30/2022] [Accepted: 02/14/2022] [Indexed: 11/08/2022]
Abstract
Mucosal immune regulation is considered a key aspect of immunopathogenesis of IgA nephropathy (IgAN). Direct experimental evidence clarifying the role of intestinal mucosa attributes in IgAN is lacking. In this study, a mouse model was established via multiple low-dose intraperitoneal injections of Lactobacillus casei cell wall extract (LCWE) emulsified with Complete Freund's Adjuvant (CFA). We found continuous and stable deposition of IgA in glomerular mesangial areas, accompanying high circulating levels of IgA and IgA-IgG complexes. Expression of key extracellular matrix components Collage IV and fibronectin also increased in the mesangial areas of LCWE-induced mice. IgA+ B220+ B-cell proportion increased in the small intestine (SI), Peyer's patches, inguinal lymph nodes, spleen, and bone marrow. The intestinal barrier was dysfunctional in the LCWE-induced mice, and consistent with this higher levels of serum zonulin (namely prehaptoglobin-2), a regulator of epithelial and endothelial barrier function were observed in patients with IgAN. Hematoxylin and eosin staining results showed immune tissues such as liver, spleen, and lymph nodes showed an inflammatory response and focal lesions. Glucocorticoid methylprednisolone treatment could alleviate serum IgA and IgA-IgG complexes level and mesangial IgA deposition. Taken together, our results indicate that we have successfully constructed a mouse model with IgA deposition in the mesangial areas of the glomeruli and provided evidence for the connection between the intestinal barrier and elevated circulating IgA and IgA-IgG in IgAN. This article is protected by copyright. All rights reserved.
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Affiliation(s)
- Feng Wan
- Renal Division, Peking University First Hospital, Beijing; Peking University Institute of Nephrology; Key Laboratory of Renal Disease, Ministry of Health of China, Key Laboratory of Chronic Kidney Disease Prevention and Treatment (Peking University), Ministry of Education, Beijing, China.,Hangzhou TCM Hospital Affiliated to Zhejiang Chinese Medical University, Hangzhou, China
| | - Hui Wang
- Laboratory of Electron Microscopy, Pathological Center, Peking University First Hospital, Beijing, China
| | - Manliu Wang
- Renal Division, Peking University First Hospital, Beijing; Peking University Institute of Nephrology; Key Laboratory of Renal Disease, Ministry of Health of China, Key Laboratory of Chronic Kidney Disease Prevention and Treatment (Peking University), Ministry of Education, Beijing, China.,Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China
| | - Jicheng Lv
- Renal Division, Peking University First Hospital, Beijing; Peking University Institute of Nephrology; Key Laboratory of Renal Disease, Ministry of Health of China, Key Laboratory of Chronic Kidney Disease Prevention and Treatment (Peking University), Ministry of Education, Beijing, China.,Research Units of Diagnosis and Treatment of Immune-mediate Kidney Disease, Chinese Academy of Medical Sciences, Beijing, China
| | - MingHui Zhao
- Renal Division, Peking University First Hospital, Beijing; Peking University Institute of Nephrology; Key Laboratory of Renal Disease, Ministry of Health of China, Key Laboratory of Chronic Kidney Disease Prevention and Treatment (Peking University), Ministry of Education, Beijing, China.,Research Units of Diagnosis and Treatment of Immune-mediate Kidney Disease, Chinese Academy of Medical Sciences, Beijing, China.,Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China
| | - Hong Zhang
- Renal Division, Peking University First Hospital, Beijing; Peking University Institute of Nephrology; Key Laboratory of Renal Disease, Ministry of Health of China, Key Laboratory of Chronic Kidney Disease Prevention and Treatment (Peking University), Ministry of Education, Beijing, China.,Research Units of Diagnosis and Treatment of Immune-mediate Kidney Disease, Chinese Academy of Medical Sciences, Beijing, China
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13
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Andreuzzi E, Fejza A, Polano M, Poletto E, Camicia L, Carobolante G, Tarticchio G, Todaro F, Di Carlo E, Scarpa M, Scarpa M, Paulitti A, Capuano A, Canzonieri V, Maiero S, Fornasarig M, Cannizzaro R, Doliana R, Colombatti A, Spessotto P, Mongiat M. Colorectal cancer development is affected by the ECM molecule EMILIN-2 hinging on macrophage polarization via the TLR-4/MyD88 pathway. J Exp Clin Cancer Res 2022; 41:60. [PMID: 35148799 PMCID: PMC8840294 DOI: 10.1186/s13046-022-02271-y] [Citation(s) in RCA: 23] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2021] [Accepted: 01/22/2022] [Indexed: 12/18/2022] Open
Abstract
BACKGROUND Colorectal cancer is one of the most frequent and deadly tumors. Among the key regulators of CRC growth and progression, the microenvironment has emerged as a crucial player and as a possible route for the development of new therapeutic opportunities. More specifically, the extracellular matrix acts directly on cancer cells and indirectly affecting the behavior of stromal and inflammatory cells, as well as the bioavailability of growth factors. Among the ECM molecules, EMILIN-2 is frequently down-regulated by methylation in CRC and the purpose of this study was to verify the impact of EMILIN-2 loss in CRC development and its possible value as a prognostic biomarker. METHODS The AOM/DSS CRC protocol was applied to Emilin-2 null and wild type mice. Tumor development was monitored by endoscopy, the molecular analyses performed by IHC, IF and WB and the immune subpopulations characterized by flow cytometry. Ex vivo cultures of monocyte/macrophages from the murine models were used to verify the molecular pathways. Publicly available datasets were exploited to determine the CRC patients' expression profile; Spearman's correlation analyses and Cox regression were applied to evaluate the association with the inflammatory response; the clinical outcome was predicted by Kaplan-Meier survival curves. Pearson correlation analyses were also applied to a cohort of patients enrolled in our Institute. RESULTS In preclinical settings, loss of EMILIN-2 associated with an increased number of tumor lesions upon AOM/DSS treatment. In addition, in the early stages of the disease, the Emilin-2 knockout mice displayed a myeloid-derived suppressor cells-rich infiltrate. Instead, in the late stages, lack of EMILIN-2 associated with a decreased number of M1 macrophages, resulting in a higher percentage of the tumor-promoting M2 macrophages. Mechanistically, EMILIN-2 triggered the activation of the Toll-like Receptor 4/MyD88/NF-κB pathway, instrumental for the polarization of macrophages towards the M1 phenotype. Accordingly, dataset and immunofluorescence analyses indicated that low EMILIN-2 expression levels correlated with an increased M2/M1 ratio and with poor CRC patients' prognosis. CONCLUSIONS These novel results indicate that EMILIN-2 is a key regulator of the tumor-associated inflammatory environment and may represent a promising prognostic biomarker for CRC patients.
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Affiliation(s)
- Eva Andreuzzi
- Department of Research and Diagnosis, Division of Molecular Oncology, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, Italy.
| | - Albina Fejza
- Department of Research and Diagnosis, Division of Molecular Oncology, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, Italy
| | - Maurizio Polano
- Experimental and Clinical Pharmacology Unit, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, Italy
| | - Evelina Poletto
- Department of Research and Diagnosis, Division of Molecular Oncology, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, Italy
| | - Lucrezia Camicia
- Department of Research and Diagnosis, Division of Molecular Oncology, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, Italy
| | - Greta Carobolante
- Department of Research and Diagnosis, Division of Molecular Oncology, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, Italy
| | - Giulia Tarticchio
- Department of Research and Diagnosis, Division of Molecular Oncology, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, Italy
| | - Federico Todaro
- Department of Research and Diagnosis, Division of Molecular Oncology, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, Italy
| | - Emma Di Carlo
- Department of Medicine and Sciences of Aging, "G. d'Annunzio" University of Chieti-Pescara, Chieti, Italy.,Anatomic Pathology and Immuno-Oncology Unit, Center for Advanced Studies and Technology (CAST), "G. d'Annunzio" University of Chieti-Pescara, Chieti, Italy
| | - Melania Scarpa
- Ricerca Traslazionale Avanzata, Istituto Oncologico Veneto IOV - IRCCS, Padua, Italy
| | - Marco Scarpa
- Clinica Chirurgica I- Azienda Ospedaliera di Padova, Padua, Italy
| | - Alice Paulitti
- Department of Research and Diagnosis, Division of Molecular Oncology, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, Italy
| | - Alessandra Capuano
- Department of Research and Diagnosis, Division of Molecular Oncology, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, Italy
| | - Vincenzo Canzonieri
- Pathology, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, Italy
| | - Stefania Maiero
- Division of Oncological Gastroenterology, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, Italy
| | - Mara Fornasarig
- Division of Oncological Gastroenterology, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, Italy
| | - Renato Cannizzaro
- Division of Oncological Gastroenterology, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, Italy
| | - Roberto Doliana
- Department of Research and Diagnosis, Division of Molecular Oncology, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, Italy
| | - Alfonso Colombatti
- Department of Research and Diagnosis, Division of Molecular Oncology, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, Italy
| | - Paola Spessotto
- Department of Research and Diagnosis, Division of Molecular Oncology, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, Italy
| | - Maurizio Mongiat
- Department of Research and Diagnosis, Division of Molecular Oncology, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, Italy.
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14
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Krzywinska E, Sobecki M, Nagarajan S, Zacharjasz J, Tambuwala MM, Pelletier A, Cummins E, Gotthardt D, Fandrey J, Kerdiles YM, Peyssonnaux C, Taylor CT, Sexl V, Stockmann C. The transcription factor HIF-1α mediates plasticity of NKp46+ innate lymphoid cells in the gut. J Exp Med 2022; 219:212964. [PMID: 35024767 PMCID: PMC8763886 DOI: 10.1084/jem.20210909] [Citation(s) in RCA: 31] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2021] [Revised: 11/03/2021] [Accepted: 12/02/2021] [Indexed: 12/21/2022] Open
Abstract
Gut innate lymphoid cells (ILCs) show remarkable phenotypic diversity, yet microenvironmental factors that drive this plasticity are incompletely understood. The balance between NKp46+, IL-22-producing, group 3 ILCs (ILC3s) and interferon (IFN)-γ-producing group 1 ILCs (ILC1s) contributes to gut homeostasis. The gut mucosa is characterized by physiological hypoxia, and adaptation to low oxygen is mediated by hypoxia-inducible transcription factors (HIFs). However, the impact of HIFs on ILC phenotype and gut homeostasis is not well understood. Mice lacking the HIF-1α isoform in NKp46+ ILCs show a decrease in IFN-γ-expressing, T-bet+, NKp46+ ILC1s and a concomitant increase in IL-22-expressing, RORγt+, NKp46+ ILC3s in the gut mucosa. Single-cell RNA sequencing revealed HIF-1α as a driver of ILC phenotypes, where HIF-1α promotes the ILC1 phenotype by direct up-regulation of T-bet. Loss of HIF-1α in NKp46+ cells prevents ILC3-to-ILC1 conversion, increases the expression of IL-22-inducible genes, and confers protection against intestinal damage. Taken together, our results suggest that HIF-1α shapes the ILC phenotype in the gut.
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Affiliation(s)
| | - Michal Sobecki
- Institute of Anatomy, University of Zurich, Zurich, Switzerland
| | | | | | - Murtaza M Tambuwala
- School of Pharmacy & Pharmaceutical Sciences, Ulster University, Coleraine, UK
| | | | - Eoin Cummins
- School of Medicine, University College Dublin, Conway Institute, Dublin, Ireland
| | - Dagmar Gotthardt
- Institute of Pharmacology and Toxicology, University of Veterinary Medicine, Vienna, Austria
| | - Joachim Fandrey
- Institut für Physiologie, Universitätsklinikum Essen, Universität Duisburg-Essen, Essen, Germany
| | - Yann M Kerdiles
- Centre d'Immunologie de Marseille-Luminy, Aix Marseille Université UM2, Institut National de la Santé et de la Recherche Médicale, U1104, Centre National de la Recherche Scientifique UMR7280, Marseille, France
| | - Carole Peyssonnaux
- Université de Paris, Institut Cochin, Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, Paris, France.,Laboratory of Excellence GR-Ex, Paris, France
| | - Cormac T Taylor
- School of Medicine, University College Dublin, Conway Institute, Dublin, Ireland
| | - Veronika Sexl
- Institute of Pharmacology and Toxicology, University of Veterinary Medicine, Vienna, Austria
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15
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Stephens WZ, Kubinak JL, Ghazaryan A, Bauer KM, Bell R, Buhrke K, Chiaro TR, Weis AM, Tang WW, Monts JK, Soto R, Ekiz HA, O'Connell RM, Round JL. Epithelial-myeloid exchange of MHC class II constrains immunity and microbiota composition. Cell Rep 2021; 37:109916. [PMID: 34731608 PMCID: PMC9012449 DOI: 10.1016/j.celrep.2021.109916] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2020] [Revised: 08/13/2021] [Accepted: 10/11/2021] [Indexed: 12/12/2022] Open
Abstract
Intestinal epithelial cells (IECs) have long been understood to express high levels of major histocompatibility complex class II (MHC class II) molecules but are not considered canonical antigen-presenting cells, and the impact of IEC-MHC class II signaling on gut homeostasis remains enigmatic. As IECs serve as the primary barrier between underlying host immune cells, we reasoned that IEC-intrinsic antigen presentation may play a role in responses toward the microbiota. Mice with an IEC-intrinsic deletion of MHC class II (IECΔMHC class II) are healthy but have fewer microbial-bound IgA, regulatory T cells (Tregs), and immune repertoire selection. This was associated with increased interindividual microbiota variation and altered proportions of two taxa in the ileum where MHC class II on IECs is highest. Intestinal mononuclear phagocytes (MNPs) have similar MHC class II transcription but less surface MHC class II and are capable of acquiring MHC class II from IECs. Thus, epithelial-myeloid interactions mediate development of adaptive responses to microbial antigens within the gastrointestinal tract.
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Affiliation(s)
- W Zac Stephens
- University of Utah School of Medicine, Department of Pathology, Division of Microbiology and Immunology, Salt Lake City, UT 84112, USA
| | - Jason L Kubinak
- University of South Carolina School of Medicine, Department of Pathology, Microbiology and Immunology, Columbia, SC 29209, USA
| | - Arevik Ghazaryan
- University of Utah School of Medicine, Department of Pathology, Division of Microbiology and Immunology, Salt Lake City, UT 84112, USA
| | - Kaylyn M Bauer
- University of Utah School of Medicine, Department of Pathology, Division of Microbiology and Immunology, Salt Lake City, UT 84112, USA
| | - Rickesha Bell
- University of Utah School of Medicine, Department of Pathology, Division of Microbiology and Immunology, Salt Lake City, UT 84112, USA
| | - Kate Buhrke
- University of Utah School of Medicine, Department of Pathology, Division of Microbiology and Immunology, Salt Lake City, UT 84112, USA
| | - Tyson R Chiaro
- University of Utah School of Medicine, Department of Pathology, Division of Microbiology and Immunology, Salt Lake City, UT 84112, USA
| | - Allison M Weis
- University of Utah School of Medicine, Department of Pathology, Division of Microbiology and Immunology, Salt Lake City, UT 84112, USA
| | - William W Tang
- University of Utah School of Medicine, Department of Pathology, Division of Microbiology and Immunology, Salt Lake City, UT 84112, USA
| | - Josh K Monts
- University of Utah School of Medicine, Flow Cytometry Core, Health Sciences Center, Salt Lake City, UT 84112, USA
| | - Ray Soto
- University of Utah School of Medicine, Department of Pathology, Division of Microbiology and Immunology, Salt Lake City, UT 84112, USA
| | - H Atakan Ekiz
- University of Utah School of Medicine, Department of Pathology, Division of Microbiology and Immunology, Salt Lake City, UT 84112, USA; Izmir Institute of Technology, Molecular Biology and Genetics Department, Gulbahce, Izmir 35430, Turkey
| | - Ryan M O'Connell
- University of Utah School of Medicine, Department of Pathology, Division of Microbiology and Immunology, Salt Lake City, UT 84112, USA.
| | - June L Round
- University of Utah School of Medicine, Department of Pathology, Division of Microbiology and Immunology, Salt Lake City, UT 84112, USA.
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16
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Vomhof-DeKrey EE, Stover AD, Labuhn M, Osman MR, Basson MD. Vil-Cre specific Schlafen 3 knockout mice exhibit sex-specific differences in intestinal differentiation markers and Schlafen family members expression levels. PLoS One 2021; 16:e0259195. [PMID: 34710177 PMCID: PMC8553116 DOI: 10.1371/journal.pone.0259195] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2021] [Accepted: 10/14/2021] [Indexed: 11/25/2022] Open
Abstract
The intestinal epithelium requires self-renewal and differentiation in order to function and adapt to pathological diseases such as inflammatory bowel disease, short gut syndrome, and ulcers. The rodent Slfn3 protein and the human Slfn12 analog are known to regulate intestinal epithelial differentiation. Previous work utilizing a pan-Slfn3 knockout (KO) mouse model revealed sex-dependent gene expression disturbances in intestinal differentiation markers, metabolic pathways, Slfn family member mRNA expression, adaptive immune cell proliferation/functioning genes, and phenotypically less weight gain and sex-dependent changes in villus length and crypt depth. We have now created a Vil-Cre specific Slfn3KO (VC-Slfn3KO) mouse to further evaluate its role in intestinal differentiation. There were increases in Slfn1, Slfn2, Slfn4, and Slfn8 and decreases in Slfn5 and Slfn9 mRNA expression that were intestinal region and sex-specific. Differentiation markers, sucrase isomaltase (SI), villin 1, and dipeptidyl peptidase 4 and glucose transporters, glucose transporter 1 (Glut1), Glut2, and sodium glucose transporter 1 (SGLT1), were increased in expression in VC-Slfn3KO mice based on intestinal region and were also highly female sex-biased, except for SI in the ileum was also increased for male VC-Slfn3KO mice and SGLT1 was decreased for both sexes. Overall, the variations that we observed in these VC-Slfn3KO mice indicate a complex regulation of intestinal gene expression that is sex-dependent.
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Affiliation(s)
- Emilie E. Vomhof-DeKrey
- Department of Surgery, School of Medicine and the Health Sciences, University of North Dakota, Grand Forks, ND, United States of America
- Department of Biomedical Sciences, School of Medicine and the Health Sciences, University of North Dakota, Grand Forks, ND, United States of America
| | - Allie D. Stover
- Department of Biomedical Sciences, School of Medicine and the Health Sciences, University of North Dakota, Grand Forks, ND, United States of America
| | - Mary Labuhn
- Department of Biomedical Sciences, School of Medicine and the Health Sciences, University of North Dakota, Grand Forks, ND, United States of America
| | - Marcus R. Osman
- Department of Biomedical Sciences, School of Medicine and the Health Sciences, University of North Dakota, Grand Forks, ND, United States of America
| | - Marc D. Basson
- Department of Surgery, School of Medicine and the Health Sciences, University of North Dakota, Grand Forks, ND, United States of America
- Department of Biomedical Sciences, School of Medicine and the Health Sciences, University of North Dakota, Grand Forks, ND, United States of America
- Department of Pathology, School of Medicine and the Health Sciences, University of North Dakota, Grand Forks, ND, United States of America
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17
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Martchenko SE, Prescott D, Martchenko A, Sweeney ME, Philpott DJ, Brubaker PL. Diurnal changes in the murine small intestine are disrupted by obesogenic Western Diet feeding and microbial dysbiosis. Sci Rep 2021; 11:20571. [PMID: 34663882 PMCID: PMC8523685 DOI: 10.1038/s41598-021-98986-7] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2021] [Accepted: 09/01/2021] [Indexed: 02/07/2023] Open
Abstract
Intestinal functions demonstrate circadian rhythms thought to be entrained, in part, by an organisms’ intrinsic feeding and fasting periods as well as by the intestinal microbiome. Circadian disruption as a result of ill-timed nutrient exposure and obesogenic feeding poses an increased risk to disease. As such, the aim of this study was to assess the relationships between dietary timing, composition, and the microbiome with regard to rhythmic small intestinal structure and mucosal immunity. Rodent chow (RC)-mice exhibited time-dependent increases in small intestinal weight, villus height, and crypt depth as well as an increased proportion of CD8αα+ cells and concomitant decrease in CD8αβ+ cells at the onset of the feeding period (p < 0.05–0.001). Western diet (WD)-animals displayed disrupted time-dependent patterns in intestinal structure and lymphocyte populations (p < 0.05–0.01). Antibiotic-induced microbial depletion abrogated the time- and diet-dependent patterns in both RC- and WD-mice (p < 0.05–0.001). However, although germ-free-mice displayed altered rhythms, fecal microbial transfer from RC-mice was generally unsuccessful in restoring structural and immune changes in these animals. This study shows that adaptive changes in the small intestine at the onset of the feeding and fasting periods are disrupted by WD-feeding, and that these changes are dependent, in part, on the intestinal microbiome.
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Affiliation(s)
- Sarah E Martchenko
- Departments of Physiology, University of Toronto, Rm 3366 Medical Sciences Building, 1 King's College Circle, Toronto, ON, M5S 1A8, Canada
| | - David Prescott
- Department of Immunology, University of Toronto, Toronto, ON, Canada.,Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada
| | - Alexandre Martchenko
- Departments of Physiology, University of Toronto, Rm 3366 Medical Sciences Building, 1 King's College Circle, Toronto, ON, M5S 1A8, Canada
| | - Maegan E Sweeney
- Departments of Physiology, University of Toronto, Rm 3366 Medical Sciences Building, 1 King's College Circle, Toronto, ON, M5S 1A8, Canada
| | - Dana J Philpott
- Department of Immunology, University of Toronto, Toronto, ON, Canada
| | - Patricia L Brubaker
- Departments of Physiology, University of Toronto, Rm 3366 Medical Sciences Building, 1 King's College Circle, Toronto, ON, M5S 1A8, Canada. .,Department of Medicine, University of Toronto, Toronto, ON, Canada.
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18
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Chronic consumption of sweeteners in mice and its effect on the immune system and the small intestine microbiota. ACTA ACUST UNITED AC 2021; 41:504-530. [PMID: 34559497 PMCID: PMC8519602 DOI: 10.7705/biomedica.5806] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2020] [Indexed: 02/08/2023]
Abstract
Introduction: Sweeteners are additives used in different foods. They can be natural (sucrose and stevia) or artificial (sucralose). Currently, they are routinely consumed in multiple products and their effects on the mucosa of the small intestine and its microbiota are still controversial.
Objective: To relate the consumption of sweeteners and their effect on the immune system and the microbiota of the small intestine in CD1 mice.
Materials and methods: We used 54 three-week-old CD1 mice divided into three groups in the experiments: 1) A group of three weeks without treatment, 2) a group treated for six weeks, and 3) a group treated for 12 weeks using sucrose, sucralose, and stevia. We obtained CD19+ B lymphocytes, IgA+ antibodies, transforming growth factor-beta (TGF-b), and interleukins 12 and 17 (IL-12 and -17) from Peyer’s patches and lamina propria cells while DNA was obtained from intestinal solids to identify bacterial species.
Results: After 12 weeks, sucrose and sucralose consumption caused a reduction in bacterial communities with an increase in CD19+, a decrease in IgA+ and TGF-b, and an increase in IL-12 and -17 in the Peyer’s patches while in the lamina propria there was an increase in all parameters. In contrast, stevia led to an improvement in bacterial diversity and percentage of CD19+ lymphocytes with minimal increase in IgA+, TGF-b, and IL-12, and a decrease in IL-17.
Conclusion: Sucrose and sucralose caused negative alterations in bacterial diversity and immune parameters after 12 weeks; in contrast, stevia was beneficial for the intestinal mucosa.
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Ejima R, Akiyama M, Sato H, Tomioka S, Yakabe K, Kimizuka T, Seki N, Fujimura Y, Hirayama A, Fukuda S, Hase K, Kim YG. Seaweed Dietary Fiber Sodium Alginate Suppresses the Migration of Colonic Inflammatory Monocytes and Diet-Induced Metabolic Syndrome via the Gut Microbiota. Nutrients 2021; 13:nu13082812. [PMID: 34444972 PMCID: PMC8401899 DOI: 10.3390/nu13082812] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2021] [Revised: 08/13/2021] [Accepted: 08/13/2021] [Indexed: 12/19/2022] Open
Abstract
Metabolic syndrome (MetS) is a multifactorial chronic metabolic disorder that affects approximately one billion people worldwide. Recent studies have evaluated whether targeting the gut microbiota can prevent MetS. This study aimed to assess the ability of dietary fiber to control MetS by modulating gut microbiota composition. Sodium alginate (SA) is a seaweed-derived dietary fiber that suppresses high-fat diet (HFD)-induced MetS via an effect on the gut microbiota. We observed that SA supplementation significantly decreased body weight gain, cholesterol levels, and fat weight, while improving glucose tolerance in HFD-fed mice. SA changed the gut microbiota composition and significantly increased the abundance of Bacteroides. Antibiotic treatment completely abolished the suppressive effects of SA on MetS. Mechanistically, SA decreased the number of colonic inflammatory monocytes, which promote MetS development, in a gut microbiota-dependent manner. The abundance of Bacteroides was negatively correlated with that of inflammatory monocytes and positively correlated with the levels of several gut metabolites. The present study revealed a novel food function of SA in preventing HFD-induced MetS through its action on gut microbiota.
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Affiliation(s)
- Ryuta Ejima
- Research Center for Drug Discovery, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan; (R.E.); (M.A.); (H.S.); (S.T.); (K.Y.); (T.K.); (N.S.)
- Division of Biochemistry, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan; (Y.F.); (K.H.)
| | - Masahiro Akiyama
- Research Center for Drug Discovery, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan; (R.E.); (M.A.); (H.S.); (S.T.); (K.Y.); (T.K.); (N.S.)
| | - Hiroki Sato
- Research Center for Drug Discovery, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan; (R.E.); (M.A.); (H.S.); (S.T.); (K.Y.); (T.K.); (N.S.)
- Kaigen Pharma Co., Ltd., Osaka 541-0045, Japan
| | - Sawako Tomioka
- Research Center for Drug Discovery, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan; (R.E.); (M.A.); (H.S.); (S.T.); (K.Y.); (T.K.); (N.S.)
- Division of Biochemistry, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan; (Y.F.); (K.H.)
| | - Kyosuke Yakabe
- Research Center for Drug Discovery, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan; (R.E.); (M.A.); (H.S.); (S.T.); (K.Y.); (T.K.); (N.S.)
- Division of Biochemistry, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan; (Y.F.); (K.H.)
| | - Tatsuki Kimizuka
- Research Center for Drug Discovery, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan; (R.E.); (M.A.); (H.S.); (S.T.); (K.Y.); (T.K.); (N.S.)
- Division of Biochemistry, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan; (Y.F.); (K.H.)
| | - Natsumi Seki
- Research Center for Drug Discovery, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan; (R.E.); (M.A.); (H.S.); (S.T.); (K.Y.); (T.K.); (N.S.)
- Division of Biochemistry, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan; (Y.F.); (K.H.)
| | - Yumiko Fujimura
- Division of Biochemistry, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan; (Y.F.); (K.H.)
| | - Akiyoshi Hirayama
- Institute for Advanced Biosciences, Keio University, Yamagata 997-0052, Japan; (A.H.); (S.F.)
| | - Shinji Fukuda
- Institute for Advanced Biosciences, Keio University, Yamagata 997-0052, Japan; (A.H.); (S.F.)
- Transborder Medical Research Center, University of Tsukuba, Ibaraki 305-8575, Japan
- Intestinal Microbiota Project, Kanagawa Institute of Industrial Science and Technology, Kanagawa 210-0821, Japan
| | - Koji Hase
- Division of Biochemistry, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan; (Y.F.); (K.H.)
| | - Yun-Gi Kim
- Research Center for Drug Discovery, Faculty of Pharmacy and Graduate School of Pharmaceutical Sciences, Keio University, Tokyo 105-8512, Japan; (R.E.); (M.A.); (H.S.); (S.T.); (K.Y.); (T.K.); (N.S.)
- Correspondence:
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20
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Vomhof-DeKrey EE, Stover A, Basson MD. Microbiome diversity declines while distinct expansions of Th17, iNKT, and dendritic cell subpopulations emerge after anastomosis surgery. Gut Pathog 2021; 13:51. [PMID: 34376235 PMCID: PMC8353768 DOI: 10.1186/s13099-021-00447-z] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/19/2021] [Accepted: 07/30/2021] [Indexed: 11/26/2022] Open
Abstract
BACKGROUND Anastomotic failure causes morbidity and mortality even in technically correct anastomoses. Initial leaks must be prevented by mucosal reapproximation across the anastomosis. Healing is a concerted effort between intestinal epithelial cells (IECs), immune cells, and commensal bacteria. IEC TLR4 activation and signaling is required for mucosal healing, leading to inflammatory factor release that recruits immune cells to limit bacteria invasion. TLR4 absence leads to mucosal damage from loss in epithelial proliferation, attenuated inflammatory response, and bacteria translocation. We hypothesize after anastomosis, an imbalance in microbiota will occur due to a decrease in TLR4 expression and will lead to changes in the immune milieu. RESULTS We isolated fecal content and small intestinal leukocytes from murine, Roux-en-Y and end-to-end anastomoses, to identify microbiome changes and subsequent alterations in the regulatory and pro-inflammatory immune cells 3 days post-operative. TLR4+ IECs were impaired after anastomosis. Microbiome diversity was reduced, with Firmicutes, Bacteroidetes, and Saccharibacteria decreased and Proteobacteria increased. A distinct TCRβhi CD4+ T cells subset after anastomosis was 10-20-fold greater than in control mice. 84% were Th17 IL-17A/F+ IL-22+ and/or TNFα+. iNKT cells were increased and TCRβhi. 75% were iNKT IL-10+ and 13% iNKTh17 IL-22+. Additionally, Treg IL-10+ and IL-22+ cells were increased. A novel dendritic cell subset was identified in anastomotic regions that was CD11bhi CD103mid and was 93% IL-10+. CONCLUSIONS This anastomotic study demonstrated a decrease in IEC TLR4 expression and microbiome diversity which then coincided with increased expansion of regulatory and pro-inflammatory immune cells and cytokines. Defining the anastomotic mucosal environment could help inform innovative therapeutics to target excessive pro-inflammatory invasion and microbiome imbalance.
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Affiliation(s)
- Emilie E. Vomhof-DeKrey
- Department of Surgery, University of North Dakota School of Medicine and the Health Sciences, 1301 North Columbia Road, Stop 9037, Grand Forks, ND 58202 USA
- Department of Biomedical Sciences, University of North Dakota School of Medicine and the Health Sciences, 1301 North Columbia Road, Stop 9037, Grand Forks, ND 58202 USA
| | - Allie Stover
- Department of Biomedical Sciences, University of North Dakota School of Medicine and the Health Sciences, 1301 North Columbia Road, Stop 9037, Grand Forks, ND 58202 USA
| | - Marc D. Basson
- Department of Surgery, University of North Dakota School of Medicine and the Health Sciences, 1301 North Columbia Road, Stop 9037, Grand Forks, ND 58202 USA
- Department of Biomedical Sciences, University of North Dakota School of Medicine and the Health Sciences, 1301 North Columbia Road, Stop 9037, Grand Forks, ND 58202 USA
- Department of Pathology, University of North Dakota School of Medicine and the Health Sciences, 1301 North Columbia Road, Stop 9037, Grand Forks, ND 58202 USA
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21
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Agulla B, García-Sancho M, Sainz Á, Rodríguez-Franco F, Díaz-Regañón D, Rodríguez-Bertos A, Villaescusa A. Isolation and immunophenotyping by flow cytometry of canine peripheral blood and intraepithelial and lamina propria duodenal T lymphocytes. Vet Immunol Immunopathol 2021; 239:110305. [PMID: 34352607 DOI: 10.1016/j.vetimm.2021.110305] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2020] [Revised: 07/22/2021] [Accepted: 07/27/2021] [Indexed: 11/19/2022]
Abstract
The gut associated lymphoid tissue (GALT) effector sites play a crucial role on the pathogenesis of many immune-mediated gastrointestinal diseases. The lymphocytes at these effector sites are principally T cells which present important morphological, phenotypical and functional differences. Flow cytometry (FC) is one of the most commonly used techniques to characterize intestinal lymphocytes in human and animal models. Published studies with a focus on dogs for intraepithelial lymphocytes (IEL) immunophenotyping exist in very limited numbers. Moreover, no lamina propria lymphocytes (LPL) isolation protocols in the canine species have been described for FC evaluation. In addition to immune intestinal dysregulation, imbalances in the peripheral blood immune system have been described in both human and animal gastrointestinal disorders. The aim of this study was to provide a protocol for canine IEL and LPL isolation for FC immunophenotyping of T cells subsets. Specifically, T helper, T cytotoxic, activated Th and Tc lymphocytes, regulatory, double negative, double positive, IFN-γ and IL-4 producing T cells, and to compare their respective populations between these effector sites and with the blood stream compartment in healthy dogs. The potential relationship of these cells distributions with age, sex and breed was also evaluated. This study included sixteen healthy dogs of different sexes and breeds with a mean age of 4.55 ± 2.93 years old. The selected protocols for the three immune compartments showed proper cell yield, purity, viability, and the absence of phenotypic and functional disturbances. Histologically, an adequate separation of the duodenal epithelium from the lamina propria was also observed. All the proposed T cells subsets were identified in the three immune compartments studied, showing some statistically significant differences in their distributions at these locations that highlight the importance of their individual evaluation. This study provides an adequate method for canine small intestine IEL and LPL isolation for FC immunophenotyping and is key for future studies on the gastrointestinal immune system associated with different canine diseases.
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Affiliation(s)
- Beatriz Agulla
- Department of Animal Medicine and Surgery, College of Veterinary Medicine, Complutense University of Madrid, Avda. Puerta de Hierro s/n, 28040, Madrid, Spain.
| | - Mercedes García-Sancho
- Department of Animal Medicine and Surgery, College of Veterinary Medicine, Complutense University of Madrid, Avda. Puerta de Hierro s/n, 28040, Madrid, Spain
| | - Ángel Sainz
- Department of Animal Medicine and Surgery, College of Veterinary Medicine, Complutense University of Madrid, Avda. Puerta de Hierro s/n, 28040, Madrid, Spain
| | - Fernando Rodríguez-Franco
- Department of Animal Medicine and Surgery, College of Veterinary Medicine, Complutense University of Madrid, Avda. Puerta de Hierro s/n, 28040, Madrid, Spain
| | - David Díaz-Regañón
- Department of Animal Medicine and Surgery, College of Veterinary Medicine, Complutense University of Madrid, Avda. Puerta de Hierro s/n, 28040, Madrid, Spain
| | - Antonio Rodríguez-Bertos
- Department of Animal Medicine and Surgery, College of Veterinary Medicine, Complutense University of Madrid, Avda. Puerta de Hierro s/n, 28040, Madrid, Spain; VISAVET Health Surveillance Centre, Complutense University of Madrid, Avda. Puerta de Hierro s/n, 28040, Madrid, Spain
| | - Alejandra Villaescusa
- Department of Animal Medicine and Surgery, College of Veterinary Medicine, Complutense University of Madrid, Avda. Puerta de Hierro s/n, 28040, Madrid, Spain
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22
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Ara T, Hashimoto D, Hayase E, Noizat C, Kikuchi R, Hasegawa Y, Matsuda K, Ono S, Matsuno Y, Ebata K, Ogasawara R, Takahashi S, Ohigashi H, Yokoyama E, Matsuo K, Sugita J, Onozawa M, Okumura R, Takeda K, Teshima T. Intestinal goblet cells protect against GVHD after allogeneic stem cell transplantation via Lypd8. Sci Transl Med 2021; 12:12/550/eaaw0720. [PMID: 32611682 DOI: 10.1126/scitranslmed.aaw0720] [Citation(s) in RCA: 27] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2018] [Revised: 01/07/2020] [Accepted: 05/27/2020] [Indexed: 12/11/2022]
Abstract
Graft-versus-host disease (GVHD) and infection are major obstacles to successful allogeneic hematopoietic stem cell transplantation (HSCT). Intestinal goblet cells form the mucus layers, which spatially segregate gut microbiota from host tissues. Although it is well known that goblet cell loss is one of the histologic features of GVHD, effects of their loss in pathophysiology of GVHD remain to be elucidated. In mouse models of allogeneic HSCT, goblet cells in the colon were significantly reduced, resulting in disruption of the inner mucus layer of the colon and increased bacterial translocation into colonic mucosa. Pretransplant administration of interleukin-25 (IL-25), a growth factor for goblet cells, protected goblet cells against GVHD, prevented bacterial translocation, reduced plasma concentrations of interferon-γ (IFN-γ) and IL-6, and ameliorated GVHD. The protective role of IL-25 was dependent on Lypd8, an antimicrobial molecule produced by enterocytes in the colon that suppresses motility of flagellated bacteria. In clinical colon biopsies, low numbers of goblet cells were significantly associated with severe intestinal GVHD, increased transplant-related mortality, and poor survival after HSCT. Goblet cell loss is associated with poor transplant outcome, and administration of IL-25 represents an adjunct therapeutic strategy for GVHD by protecting goblet cells.
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Affiliation(s)
- Takahide Ara
- Department of Hematology, Hokkaido University Faculty of Medicine and Graduate School of Medicine, Sapporo 060-8638, Japan
| | - Daigo Hashimoto
- Department of Hematology, Hokkaido University Faculty of Medicine and Graduate School of Medicine, Sapporo 060-8638, Japan.
| | - Eiko Hayase
- Department of Hematology, Hokkaido University Faculty of Medicine and Graduate School of Medicine, Sapporo 060-8638, Japan
| | - Clara Noizat
- Department of Hematology, Hokkaido University Faculty of Medicine and Graduate School of Medicine, Sapporo 060-8638, Japan
| | - Ryo Kikuchi
- Department of Hematology, Hokkaido University Faculty of Medicine and Graduate School of Medicine, Sapporo 060-8638, Japan
| | - Yuta Hasegawa
- Department of Hematology, Hokkaido University Faculty of Medicine and Graduate School of Medicine, Sapporo 060-8638, Japan
| | - Kana Matsuda
- Department of Gastroenterology and Hepatology, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan
| | - Shoko Ono
- Department of Gastroenterology and Hepatology, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan
| | - Yoshihiro Matsuno
- Department of Surgical Pathology, Hokkaido University Hospital, Sapporo 060-8648, Japan
| | - Ko Ebata
- Department of Hematology, Hokkaido University Faculty of Medicine and Graduate School of Medicine, Sapporo 060-8638, Japan
| | - Reiki Ogasawara
- Department of Hematology, Hokkaido University Faculty of Medicine and Graduate School of Medicine, Sapporo 060-8638, Japan
| | - Shuichiro Takahashi
- Department of Hematology, Hokkaido University Faculty of Medicine and Graduate School of Medicine, Sapporo 060-8638, Japan
| | - Hiroyuki Ohigashi
- Department of Hematology, Hokkaido University Faculty of Medicine and Graduate School of Medicine, Sapporo 060-8638, Japan
| | - Emi Yokoyama
- Department of Hematology, Hokkaido University Faculty of Medicine and Graduate School of Medicine, Sapporo 060-8638, Japan
| | - Keitaro Matsuo
- Division of Cancer Epidemiology and Prevention, Aichi Cancer Center Research Institute, Nagoya 464-8681, Japan.,Department of Cancer Epidemiology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Junichi Sugita
- Department of Hematology, Hokkaido University Faculty of Medicine and Graduate School of Medicine, Sapporo 060-8638, Japan
| | - Masahiro Onozawa
- Department of Hematology, Hokkaido University Faculty of Medicine and Graduate School of Medicine, Sapporo 060-8638, Japan
| | - Ryu Okumura
- Department of Microbiology and Immunology, Graduate School of Medicine, WPI Immunology Frontier Research Center, Osaka University, Suita 565-0871, Japan
| | - Kiyoshi Takeda
- Department of Microbiology and Immunology, Graduate School of Medicine, WPI Immunology Frontier Research Center, Osaka University, Suita 565-0871, Japan
| | - Takanori Teshima
- Department of Hematology, Hokkaido University Faculty of Medicine and Graduate School of Medicine, Sapporo 060-8638, Japan.
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23
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Gu M, Samuelson DR, Taylor CM, Molina PE, Luo M, Siggins RW, Shellito JE, Welsh DA. Alcohol-associated intestinal dysbiosis alters mucosal-associated invariant T-cell phenotype and function. Alcohol Clin Exp Res 2021; 45:934-947. [PMID: 33704802 PMCID: PMC8283808 DOI: 10.1111/acer.14589] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2020] [Revised: 02/23/2021] [Accepted: 02/24/2021] [Indexed: 02/07/2023]
Abstract
BACKGROUND Chronic alcohol consumption is associated with a compromised innate and adaptive immune responses to infectious disease. Mucosa-associated invariant T (MAIT) cells play a critical role in antibacterial host defense. However, whether alcohol-associated deficits in innate and adaptive immune responses are mediated by alterations in MAIT cells remains unclear. METHODS To investigate the impact of alcohol on MAIT cells, mice were treated with binge-on-chronic alcohol for 10 days and sacrificed at day 11. MAIT cells in the barrier organs (lung, liver, and intestine) were characterized by flow cytometry. Two additional sets of animals were used to examine the involvement of gut microbiota on alcohol-induced MAIT cell changes: (1) Cecal microbiota from alcohol-fed (AF) mice were adoptive transferred into antibiotic-pretreated mice and (2) AF mice were treated with antibiotics during the experiment. MAIT cells in the barrier organs were measured via flow cytometry. RESULTS Binge-on-chronic alcohol feeding led to a significant reduction in the abundance of MAIT cells in the barrier tissues. However, CD69 expression on tissue-associated MAIT cells was increased in AF mice compared with pair-fed (PF) mice. The expression of Th1 cytokines and the corresponding transcriptional factor was tissue specific, showing downregulation in the intestine and increases in the lung and liver in AF animals. Transplantation of fecal microbiota from AF mice resulted in a MAIT cell profile aligned to that of AF mouse donor. Antibiotic treatment abolished the MAIT cell differences between AF and PF animals. CONCLUSION MAIT cells in the intestine, liver, and lung are perturbed by alcohol use and these changes are partially attributable to alcohol-associated dysbiosis. MAIT cell dysfunction may contribute to alcohol-induced innate and adaptive immunity and consequently end-organ pathophysiology.
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Affiliation(s)
- Min Gu
- Department of Internal Medicine, Section of Pulmonary/Critical Care & Allergy/Immunology, Louisiana State University Health Science Center, New Orleans, LA, USA
| | - Derrick R. Samuelson
- Department of Internal Medicine, Section of Pulmonary/Critical Care & Allergy/Immunology, Louisiana State University Health Science Center, New Orleans, LA, USA
- Department of Internal Medicine, Division of Pulmonary, Critical Care, & Sleep, University of Nebraska Medical Center, Omaha, NE, USA
| | - Christopher M. Taylor
- Department of Microbiology, Immunology, & Parasitology, Louisiana State University Health Science Center, New Orleans, LA, USA
| | - Patricia E. Molina
- Department of Physiology, Louisiana State University Health Science Center, New Orleans, LA, USA
- Comprehensive Alcohol-HIV/AIDS Research Center, Louisiana State University Health Science Center, New Orleans, LA, USA
| | - Meng Luo
- Department of Microbiology, Immunology, & Parasitology, Louisiana State University Health Science Center, New Orleans, LA, USA
| | - Robert W. Siggins
- Department of Physiology, Louisiana State University Health Science Center, New Orleans, LA, USA
- Comprehensive Alcohol-HIV/AIDS Research Center, Louisiana State University Health Science Center, New Orleans, LA, USA
| | - Judd E. Shellito
- Department of Internal Medicine, Section of Pulmonary/Critical Care & Allergy/Immunology, Louisiana State University Health Science Center, New Orleans, LA, USA
- Department of Microbiology, Immunology, & Parasitology, Louisiana State University Health Science Center, New Orleans, LA, USA
| | - David A. Welsh
- Department of Internal Medicine, Section of Pulmonary/Critical Care & Allergy/Immunology, Louisiana State University Health Science Center, New Orleans, LA, USA
- Department of Microbiology, Immunology, & Parasitology, Louisiana State University Health Science Center, New Orleans, LA, USA
- Comprehensive Alcohol-HIV/AIDS Research Center, Louisiana State University Health Science Center, New Orleans, LA, USA
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24
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Jørgensen PB, Fenton TM, Mörbe UM, Riis LB, Jakobsen HL, Nielsen OH, Agace WW. Identification, isolation and analysis of human gut-associated lymphoid tissues. Nat Protoc 2021; 16:2051-2067. [PMID: 33619391 DOI: 10.1038/s41596-020-00482-1] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2020] [Accepted: 12/09/2020] [Indexed: 02/06/2023]
Abstract
Gut-associated lymphoid tissues (GALTs) comprise key intestinal immune inductive sites, including the Peyer's patches of the small intestine and different types of isolated lymphoid follicle (ILF) found along the length of the gut. Our understanding of human GALT is limited due to a lack of protocols for their isolation. Here we describe a technique that, uniquely among intestinal cell isolation protocols, allows identification and isolation of all human GALT, as well as GALT-free intestinal lamina propria (LP). The technique involves the mechanical separation of intestinal mucosa from the submucosa, allowing the identification and isolation of submucosal ILF (SM-ILF), LP-embedded mucosal ILF (M-ILF) and LP free of contaminating lymphoid tissue. Individual SM-ILF, M-ILF and Peyer's patch follicles can be subsequently digested for downstream cellular and molecular characterization. The technique, which takes 4-10 h, will be useful for researchers interested in intestinal immune development and function in health and disease.
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Affiliation(s)
- Peter B Jørgensen
- Department of Health Technology, Technical University of Denmark, Kongens Lyngby, Denmark
| | - Thomas M Fenton
- Department of Health Technology, Technical University of Denmark, Kongens Lyngby, Denmark
| | - Urs M Mörbe
- Department of Health Technology, Technical University of Denmark, Kongens Lyngby, Denmark
| | - Lene B Riis
- Department of Pathology, Herlev Hospital, University of Copenhagen, Herlev, Denmark
| | - Henrik L Jakobsen
- Department of Gastroenterology, Surgical Section, Herlev Hospital, University of Copenhagen, Herlev, Denmark
| | - Ole H Nielsen
- Department of Gastroenterology, Medical Section, Herlev Hospital, University of Copenhagen, Herlev, Denmark
| | - William W Agace
- Department of Health Technology, Technical University of Denmark, Kongens Lyngby, Denmark. .,Immunology Section, Lund University, Lund, Sweden.
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25
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Kahalehili HM, Newman NK, Pennington JM, Kolluri SK, Kerkvliet NI, Shulzhenko N, Morgun A, Ehrlich AK. Dietary Indole-3-Carbinol Activates AhR in the Gut, Alters Th17-Microbe Interactions, and Exacerbates Insulitis in NOD Mice. Front Immunol 2021; 11:606441. [PMID: 33552063 PMCID: PMC7858653 DOI: 10.3389/fimmu.2020.606441] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2020] [Accepted: 11/23/2020] [Indexed: 12/12/2022] Open
Abstract
The diet represents one environmental risk factor controlling the progression of type 1 diabetes (T1D) in genetically susceptible individuals. Consequently, understanding which specific nutritional components promote or prevent the development of disease could be used to make dietary recommendations in prediabetic individuals. In the current study, we hypothesized that the immunoregulatory phytochemcial, indole-3-carbinol (I3C) which is found in cruciferous vegetables, will regulate the progression of T1D in nonobese diabetic (NOD) mice. During digestion, I3C is metabolized into ligands for the aryl hydrocarbon receptor (AhR), a transcription factor that when systemically activated prevents T1D. In NOD mice, an I3C-supplemented diet led to strong AhR activation in the small intestine but minimal systemic AhR activity. In the absence of this systemic response, the dietary intervention led to exacerbated insulitis. Consistent with the compartmentalization of AhR activation, dietary I3C did not alter T helper cell differentiation in the spleen or pancreatic draining lymph nodes. Instead, dietary I3C increased the percentage of CD4+RORγt+Foxp3- (Th17 cells) in the lamina propria, intraepithelial layer, and Peyer's patches of the small intestine. The immune modulation in the gut was accompanied by alterations to the intestinal microbiome, with changes in bacterial communities observed within one week of I3C supplementation. A transkingdom network was generated to predict host-microbe interactions that were influenced by dietary I3C. Within the phylum Firmicutes, several genera (Intestinimonas, Ruminiclostridium 9, and unclassified Lachnospiraceae) were negatively regulated by I3C. Using AhR knockout mice, we validated that Intestinimonas is negatively regulated by AhR. I3C-mediated microbial dysbiosis was linked to increases in CD25high Th17 cells. Collectively, these data demonstrate that site of AhR activation and subsequent interactions with the host microbiome are important considerations in developing AhR-targeted interventions for T1D.
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MESH Headings
- Animals
- Bacteria/drug effects
- Bacteria/immunology
- Bacteria/metabolism
- Basic Helix-Loop-Helix Transcription Factors/agonists
- Basic Helix-Loop-Helix Transcription Factors/genetics
- Basic Helix-Loop-Helix Transcription Factors/metabolism
- Diabetes Mellitus, Type 1/chemically induced
- Diabetes Mellitus, Type 1/immunology
- Diabetes Mellitus, Type 1/metabolism
- Diabetes Mellitus, Type 1/microbiology
- Dietary Exposure
- Disease Models, Animal
- Disease Progression
- Dysbiosis
- Gastrointestinal Microbiome/drug effects
- Host-Pathogen Interactions
- Indoles/toxicity
- Intestine, Small/drug effects
- Intestine, Small/immunology
- Intestine, Small/metabolism
- Intestine, Small/microbiology
- Mice, Inbred NOD
- Mice, Knockout
- Receptors, Aryl Hydrocarbon/agonists
- Receptors, Aryl Hydrocarbon/genetics
- Receptors, Aryl Hydrocarbon/metabolism
- Th17 Cells/drug effects
- Th17 Cells/immunology
- Th17 Cells/metabolism
- Mice
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Affiliation(s)
- Heather M. Kahalehili
- Department of Environmental Toxicology, University of California, Davis, CA, United States
| | - Nolan K. Newman
- College of Pharmacy, Oregon State University, Corvallis, OR, United States
| | - Jamie M. Pennington
- Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR, United States
| | - Siva K. Kolluri
- Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR, United States
| | - Nancy I. Kerkvliet
- Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR, United States
| | - Natalia Shulzhenko
- Department of Biomedical Sciences, Oregon State University, Corvallis, OR, United States
| | - Andrey Morgun
- College of Pharmacy, Oregon State University, Corvallis, OR, United States
| | - Allison K. Ehrlich
- Department of Environmental Toxicology, University of California, Davis, CA, United States
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26
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Kim A, Seong KM, Choi YY, Shim S, Park S, Lee SS. Inhibition of EphA2 by Dasatinib Suppresses Radiation-Induced Intestinal Injury. Int J Mol Sci 2020; 21:ijms21239096. [PMID: 33265912 PMCID: PMC7730170 DOI: 10.3390/ijms21239096] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2020] [Revised: 11/24/2020] [Accepted: 11/25/2020] [Indexed: 12/16/2022] Open
Abstract
Radiation-induced multiorgan dysfunction is thought to result primarily from damage to the endothelial system, leading to a systemic inflammatory response that is mediated by the recruitment of leukocytes. The Eph–ephrin signaling pathway in the vascular system participates in various disease developmental processes, including cancer and inflammation. In this study, we demonstrate that radiation exposure increased intestinal inflammation via endothelial dysfunction, caused by the radiation-induced activation of EphA2, an Eph receptor tyrosine kinase, and its ligand ephrinA1. Barrier dysfunction in endothelial and epithelial cells was aggravated by vascular endothelial–cadherin disruption and leukocyte adhesion in radiation-induced inflammation both in vitro and in vivo. Among all Eph receptors and their ligands, EphA2 and ephrinA1 were required for barrier destabilization and leukocyte adhesion. Knockdown of EphA2 in endothelial cells reduced radiation-induced endothelial dysfunction. Furthermore, pharmacological inhibition of EphA2–ephrinA1 by the tyrosine kinase inhibitor dasatinib attenuated the loss of vascular integrity and leukocyte adhesion in vitro. Mice administered dasatinib exhibited resistance to radiation injury characterized by reduced barrier leakage and decreased leukocyte infiltration into the intestine. Taken together, these data suggest that dasatinib therapy represents a potential approach for the protection of radiation-mediated intestinal damage by targeting the EphA2–ephrinA1 complex.
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Affiliation(s)
- Areumnuri Kim
- Laboratory of Radiation Exposure and Therapeutics, National Radiation Emergency Medical Center, KIRAMS, Seoul 01812, Korea; (S.S.); (S.P.); (S.S.L.)
- Correspondence:
| | - Ki Moon Seong
- Laboratory of Biodosimetry, National Radiation Emergency Medical Center, KIRAMS, Seoul 01812, Korea; (K.M.S.); (Y.Y.C.)
| | - You Yeon Choi
- Laboratory of Biodosimetry, National Radiation Emergency Medical Center, KIRAMS, Seoul 01812, Korea; (K.M.S.); (Y.Y.C.)
| | - Sehwan Shim
- Laboratory of Radiation Exposure and Therapeutics, National Radiation Emergency Medical Center, KIRAMS, Seoul 01812, Korea; (S.S.); (S.P.); (S.S.L.)
| | - Sunhoo Park
- Laboratory of Radiation Exposure and Therapeutics, National Radiation Emergency Medical Center, KIRAMS, Seoul 01812, Korea; (S.S.); (S.P.); (S.S.L.)
- Laboratory of Biodosimetry, National Radiation Emergency Medical Center, KIRAMS, Seoul 01812, Korea; (K.M.S.); (Y.Y.C.)
| | - Seung Sook Lee
- Laboratory of Radiation Exposure and Therapeutics, National Radiation Emergency Medical Center, KIRAMS, Seoul 01812, Korea; (S.S.); (S.P.); (S.S.L.)
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27
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Saunders LJ, Fitzsimmons PN, Nichols JW, Gobas FAPC. In vitro-in vivo extrapolation of hepatic and gastrointestinal biotransformation rates of hydrophobic chemicals in rainbow trout. AQUATIC TOXICOLOGY (AMSTERDAM, NETHERLANDS) 2020; 228:105629. [PMID: 33002683 PMCID: PMC7962060 DOI: 10.1016/j.aquatox.2020.105629] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/11/2020] [Revised: 08/24/2020] [Accepted: 09/05/2020] [Indexed: 06/11/2023]
Abstract
Hepatic in vitro biotransformation assays, in combination with in vitro-in vivo extrapolation (IVIVE) and bioaccumulation modeling, can be used to support regulatory bioaccumulation assessments. In most applications, however, these methods ignore the possibility of extrahepatic metabolism. Here we evaluated intestinal biotransformation in rainbow trout using S9 fractions prepared from the upper intestinal (GIT) epithelium. Measured levels of activity determined using standard substrates for phase I and phase II biotransformation enzymes were within 2-fold of activities measured in hepatic S9 fractions. In vitro intrinsic clearance rates for 2-ethylhexyl-4-methoxycinnamate (EHMC; an organic sunscreen agent) and two polycyclic aromatic hydrocarbons (pyrene [PYR] and benzo(a)pyrene [BAP]) were significantly higher in liver S9 fractions than in GIT S9 fractions. For octocrylene (OCT; a second sunscreen agent), however, in vitro intrinsic clearance rates were higher in GIT S9 fractions compared to liver S9 fractions. An existing 'liver only' IVIVE model was expanded to consider biotransformation in both the liver and GIT. Relevant IVIVE scaling factors were developed by morphological, histological, and biochemical evaluation of trout intestines. For chemicals biotransformed at higher rates by hepatic S9 fractions (i.e., BAP, PYR, EHMC), the 'liver & GIT' model yielded whole-body biotransformation rate constants (kMET) that were within 1.2 to 1.4-fold of those estimated using the 'liver only' model. In contrast to these findings, the mean kMET for OCT obtained using the 'liver & GIT' model was 3.3 times higher than the mean kMET derived using the 'liver only' model and was in good agreement with empirical kMET estimates determined previously for trout (<20 % difference). The results of this study suggest that current 'liver only' IVIVE approaches may underestimate in vivo biotransformation rates for chemicals that undergo substantial biotransformation in the GIT.
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Affiliation(s)
- Leslie J Saunders
- Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada
| | | | - John W Nichols
- United States Environmental Protection Agency, Duluth, MN, USA
| | - Frank A P C Gobas
- Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada; School of Resource and Environmental Management, Simon Fraser University, Burnaby, British Columbia, Canada.
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28
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Mitochondrial bioenergetics, uncoupling protein-2 activity, and reactive oxygen species production in the small intestine of a TNBS-induced colitis rat model. Mol Cell Biochem 2020; 470:87-98. [PMID: 32394310 DOI: 10.1007/s11010-020-03749-z] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2020] [Accepted: 05/06/2020] [Indexed: 10/24/2022]
Abstract
Inflammatory bowel disease (IBD) is often associated with a decrease in energy-dependent nutrient uptake across the jejunum that serves as the main site for absorption in the small intestine. This association has prompted us to investigate the bioenergetics underlying the alterations in jejunal absorption in 2,4,6-trinitrobenzenesulfonic acid-induced colitis in rats. We have found that mitochondrial oxygen consumption did not change in state 2 and state 3 respirations but showed an increase in state 4 respiration indicating a decrease in the respiratory control ratio of jejunal mitochondria during the peak of inflammation. This decrease in the coupling state was found to be guanosine diphosphate-sensitive, hence, implicating the involvement of uncoupling protein-2 (UCP2). Furthermore, the study has reported that the production of reactive oxygen species (ROS), known to be activators of UCP2, correlated negatively with UCP2 activity. Thus, we suggest that ROS production in the jejunum might be activating UCP2 which has an antioxidant activity, and that uncoupling of the mitochondria decreases the efficiency of energy production, leading to a decrease in energy-dependent nutrient absorption. Hence, this study is the first to account for an involvement of energy production and a role for UCP2 in the absorptive abnormalities of the small intestine in animal models of colitis.
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29
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Oprescu SN, Yue F, Qiu J, Brito LF, Kuang S. Temporal Dynamics and Heterogeneity of Cell Populations during Skeletal Muscle Regeneration. iScience 2020; 23:100993. [PMID: 32248062 PMCID: PMC7125354 DOI: 10.1016/j.isci.2020.100993] [Citation(s) in RCA: 157] [Impact Index Per Article: 31.4] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2019] [Revised: 01/08/2020] [Accepted: 03/13/2020] [Indexed: 12/20/2022] Open
Abstract
Mammalian skeletal muscle possesses a unique ability to regenerate, which is primarily mediated by a population of resident muscle stem cells (MuSCs) and requires a concerted response from other supporting cell populations. Previous targeted analysis has described the involvement of various specific populations in regeneration, but an unbiased and simultaneous evaluation of all cell populations has been limited. Therefore, we used single-cell RNA-sequencing to uncover gene expression signatures of over 53,000 individual cells during skeletal muscle regeneration. Cells clustered into 25 populations and subpopulations, including a subpopulation of immune gene enriched myoblasts (immunomyoblasts) and subpopulations of fibro-adipogenic progenitors. Our analyses also uncovered striking spatiotemporal dynamics in gene expression, population composition, and cell-cell interaction during muscle regeneration. These findings provide insights into the cellular and molecular underpinning of skeletal muscle regeneration.
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Affiliation(s)
- Stephanie N Oprescu
- Department of Biological Sciences, Purdue University, 915 W State St, West Lafayette, IN 47907, USA
| | - Feng Yue
- Department of Animal Sciences, Purdue University, 270 S Russell St, West Lafayette, IN 47907, USA
| | - Jiamin Qiu
- Department of Animal Sciences, Purdue University, 270 S Russell St, West Lafayette, IN 47907, USA
| | - Luiz F Brito
- Department of Animal Sciences, Purdue University, 270 S Russell St, West Lafayette, IN 47907, USA
| | - Shihuan Kuang
- Department of Biological Sciences, Purdue University, 915 W State St, West Lafayette, IN 47907, USA; Department of Animal Sciences, Purdue University, 270 S Russell St, West Lafayette, IN 47907, USA; Center for Cancer Research, Purdue University, 201 S University St, West Lafayette, IN 47907, USA.
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30
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Webster HC, Andrusaite AT, Shergold AL, Milling SWF, Perona-Wright G. Isolation and functional characterisation of lamina propria leukocytes from helminth-infected, murine small intestine. J Immunol Methods 2020; 477:112702. [PMID: 31705860 PMCID: PMC6983935 DOI: 10.1016/j.jim.2019.112702] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2019] [Revised: 11/02/2019] [Accepted: 11/04/2019] [Indexed: 12/21/2022]
Abstract
The use of helminth infections as tools to understand the type 2 immune response is a well-established technique and important to many areas of immunological research. The phenotype and function of immune cell populations at the site of infection is a key determinant of pathogen clearance. However, infections with helminths such as the murine nematode Heligomosmoides polygryrus cause increased mucus production and thickening of the intestinal wall, which can result in extensive cell death when isolating and analysing cells from the lamina propria (LP). Populations of larger immune cells such as macrophages and dendritic cells are often trapped within mucus or dying tissues. Here we describe an optimised protocol for isolating LP leukocytes from the small intestine of H.polygyrus -infected mice, and we demonstrate phenotypic and functional identification of myeloid and CD4+ T cell subsets using cytokine staining and flow cytometry. Our protocol may provide a useful experimental method for the immunological analysis of the affected tissue site during helminth infections.
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Affiliation(s)
- Holly C Webster
- Institute of Infection, Immunity and Inflammation, University of Glasgow, 120 University Place, Glasgow G12 8TA, UK.
| | - Anna T Andrusaite
- Institute of Infection, Immunity and Inflammation, University of Glasgow, 120 University Place, Glasgow G12 8TA, UK.
| | - Amy L Shergold
- Institute of Infection, Immunity and Inflammation, University of Glasgow, 120 University Place, Glasgow G12 8TA, UK.
| | - Simon W F Milling
- Institute of Infection, Immunity and Inflammation, University of Glasgow, 120 University Place, Glasgow G12 8TA, UK.
| | - Georgia Perona-Wright
- Institute of Infection, Immunity and Inflammation, University of Glasgow, 120 University Place, Glasgow G12 8TA, UK.
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31
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Entwistle LJ, Gregory LG, Oliver RA, Branchett WJ, Puttur F, Lloyd CM. Pulmonary Group 2 Innate Lymphoid Cell Phenotype Is Context Specific: Determining the Effect of Strain, Location, and Stimuli. Front Immunol 2020; 10:3114. [PMID: 32038635 PMCID: PMC6987460 DOI: 10.3389/fimmu.2019.03114] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2019] [Accepted: 12/20/2019] [Indexed: 12/19/2022] Open
Abstract
Group 2 innate lymphoid cells (ILC2s) are enriched at mucosal sites, including the lung, and play a central role in type 2 immunity and maintaining tissue homeostasis. As a result, since their discovery in 2010, research into ILC2s has increased markedly. Numerous strategies have been used to define ILC2s by flow cytometry, often utilizing different combinations of surface markers despite their expression being variable and context-dependent. In this study, we sought to generate a comprehensive characterization of pulmonary ILC2s, identifying stable and context specific markers from different pulmonary compartments following different airway exposures in different strains of mice. Our analysis revealed that pulmonary ILC2 surface marker phenotype is heterogeneous and is influenced by mouse strain, pulmonary location, in vivo treatment/exposure and ex vivo stimulation. Therefore, we propose that a lineage negative cell expressing CD45 and Gata3 defines an ILC2, and subsequent surface marker expression should be used to describe their phenotype in context-specific scenarios.
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Affiliation(s)
- Lewis J Entwistle
- Inflammation, Repair and Development, National Heart and Lung Institute, Imperial College London, London, United Kingdom
| | - Lisa G Gregory
- Inflammation, Repair and Development, National Heart and Lung Institute, Imperial College London, London, United Kingdom
| | - Robert A Oliver
- Inflammation, Repair and Development, National Heart and Lung Institute, Imperial College London, London, United Kingdom
| | - William J Branchett
- Inflammation, Repair and Development, National Heart and Lung Institute, Imperial College London, London, United Kingdom
| | - Franz Puttur
- Inflammation, Repair and Development, National Heart and Lung Institute, Imperial College London, London, United Kingdom
| | - Clare M Lloyd
- Inflammation, Repair and Development, National Heart and Lung Institute, Imperial College London, London, United Kingdom
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32
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Sofi MH, Johnson BM, Gudi RR, Jolly A, Gaudreau MC, Vasu C. Polysaccharide A-Dependent Opposing Effects of Mucosal and Systemic Exposures to Human Gut Commensal Bacteroides fragilis in Type 1 Diabetes. Diabetes 2019; 68:1975-1989. [PMID: 31311801 PMCID: PMC6754247 DOI: 10.2337/db19-0211] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/28/2019] [Accepted: 07/11/2019] [Indexed: 12/12/2022]
Abstract
Bacteroides fragilis (BF) is an integral component of the human colonic commensal microbiota. BF is also the most commonly isolated organism from clinical cases of intra-abdominal abscesses, suggesting its potential to induce proinflammatory responses upon accessing the systemic compartment. Hence, we examined the impact of mucosal and systemic exposures to BF on type 1 diabetes (T1D) incidence in NOD mice. The impact of intestinal exposure to BF under a chemically induced enhanced gut permeability condition, which permits microbial translocation, in T1D was also examined. While oral administration of heat-killed (HK) BF to prediabetic mice caused enhanced immune regulation and suppression of autoimmunity, resulting in delayed hyperglycemia, mice that received HK BF by intravenous injection showed rapid disease progression. Importantly, polysaccharide A-deficient BF failed to produce these opposing effects upon oral and systemic deliveries. Furthermore, BF-induced modulation of disease progression was observed in wild-type, but not TLR2-deficient, NOD mice. Interestingly, oral administration of BF under enhanced gut permeability conditions resulted in accelerated disease progression and rapid onset of hyperglycemia in NOD mice. Overall, these observations suggest that BF-like gut commensals can cause proinflammatory responses upon gaining access to the systemic compartment and contribute to T1D in at-risk subjects.
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Affiliation(s)
- M Hanief Sofi
- Microbiology and Immunology, College of Medicine, Medical University of South Carolina, Charleston, SC
| | - Benjamin M Johnson
- Microbiology and Immunology, College of Medicine, Medical University of South Carolina, Charleston, SC
| | - Radhika R Gudi
- Microbiology and Immunology, College of Medicine, Medical University of South Carolina, Charleston, SC
| | - Amy Jolly
- Microbiology and Immunology, College of Medicine, Medical University of South Carolina, Charleston, SC
| | - Marie-Claude Gaudreau
- Microbiology and Immunology, College of Medicine, Medical University of South Carolina, Charleston, SC
| | - Chenthamarakshan Vasu
- Microbiology and Immunology, College of Medicine, Medical University of South Carolina, Charleston, SC
- Surgery, College of Medicine, Medical University of South Carolina, Charleston, SC
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33
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McManus D, Novaira HJ, Hamers AAJ, Pillai AB. Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E. J Vis Exp 2019. [PMID: 31609324 DOI: 10.3791/59821] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.
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Affiliation(s)
- Duneia McManus
- Department of Pediatrics, Division of Hematology / Oncology and Bone Marrow Transplantation, University of Miami Miller School of Medicine; Batchelor Children's Research Institute, University of Miami Miller School of Medicine; Department of Microbiology & Immunology, University of Miami Miller School of Medicine; Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine
| | - Horacio J Novaira
- Department of Pediatrics, Division of Hematology / Oncology and Bone Marrow Transplantation, University of Miami Miller School of Medicine; Batchelor Children's Research Institute, University of Miami Miller School of Medicine; Department of Microbiology & Immunology, University of Miami Miller School of Medicine; Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine
| | - Anouk A J Hamers
- Department of Pediatrics, Division of Hematology / Oncology and Bone Marrow Transplantation, University of Miami Miller School of Medicine; Batchelor Children's Research Institute, University of Miami Miller School of Medicine; Department of Microbiology & Immunology, University of Miami Miller School of Medicine; Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine
| | - Asha B Pillai
- Department of Pediatrics, Division of Hematology / Oncology and Bone Marrow Transplantation, University of Miami Miller School of Medicine; Batchelor Children's Research Institute, University of Miami Miller School of Medicine; Department of Microbiology & Immunology, University of Miami Miller School of Medicine; Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine; Holtz Children's Hospital, University of Miami Miller School of Medicine;
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34
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Multiplex staining depicts the immune infiltrate in colitis-induced colon cancer model. Sci Rep 2019; 9:12645. [PMID: 31477791 PMCID: PMC6718623 DOI: 10.1038/s41598-019-49164-3] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2019] [Accepted: 08/20/2019] [Indexed: 12/24/2022] Open
Abstract
Assessment of the host immune response pattern is of increasing importance as highly prognostic and diagnostic, in immune-related diseases and in some types of cancer. Chronic inflammation is a major hallmark in colon cancer formation, but, despite the extent of local inflammatory infiltrate has been demonstrated to be extremely informative, its evaluation is not routinely assessed due to the complexity and limitations of classical immunohistochemistry (IHC). In the last years, technological advance helped in bypassing technical limits, setting up multiplex IHC (mIHC) based on tyramide signal amplification (TSA) method and designing software suited to aid pathologists in cell scoring analysis. Several studies verified the efficacy of this method, but they were restricted to the analysis of human samples. In the era of translational medicine the use of animal models to depict human pathologies, in a more complete and complex approach, is really crucial. Nevertheless, the optimization and validation of this method to species other than human is still poor. We took advantage of Multispectral Imaging System to identify the immunoprofile of Dextran Sulphate Sodium (DSS)-treated mouse colon. We optimized a protocol to sequentially stain formalin fixed paraffin embedded murine colon samples for CD3, CD8a, CD4, and CD4R5B0 antigens. With this approach we obtained a detailed lymphocyte profile, while preserving the morphological tissue context, generally lost with techniques like gene expression profiling or flow cytometry. This study, comparing the results obtained by mIHC with immunophenotyping performed with cytofluorimetric and standard IHC methods validates the potentiality and the applicability of this innovative approach.
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35
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Jochems SP, de Ruiter K, Solórzano C, Voskamp A, Mitsi E, Nikolaou E, Carniel BF, Pojar S, German EL, Reiné J, Soares-Schanoski A, Hill H, Robinson R, Hyder-Wright AD, Weight CM, Durrenberger PF, Heyderman RS, Gordon SB, Smits HH, Urban BC, Rylance J, Collins AM, Wilkie MD, Lazarova L, Leong SC, Yazdanbakhsh M, Ferreira DM. Innate and adaptive nasal mucosal immune responses following experimental human pneumococcal colonization. J Clin Invest 2019; 129:4523-4538. [PMID: 31361601 PMCID: PMC6763269 DOI: 10.1172/jci128865] [Citation(s) in RCA: 27] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Streptococcus pneumoniae (Spn) is a common cause of respiratory infection, but also frequently colonizes the nasopharynx in the absence of disease. We used mass cytometry to study immune cells from nasal biopsy samples collected following experimental human pneumococcal challenge in order to identify immunological mechanisms of control of Spn colonization. Using 37 markers, we characterized 293 nasal immune cell clusters, of which 7 were associated with Spn colonization. B cell and CD161+CD8+ T cell clusters were significantly lower in colonized than in noncolonized subjects. By following a second cohort before and after pneumococcal challenge we observed that B cells were depleted from the nasal mucosa upon Spn colonization. This associated with an expansion of Spn polysaccharide–specific and total plasmablasts in blood. Moreover, increased responses of blood mucosa-associated invariant T (MAIT) cells against in vitro stimulation with pneumococcus prior to challenge associated with protection against establishment of Spn colonization and with increased mucosal MAIT cell populations. These results implicate MAIT cells in the protection against pneumococcal colonization and demonstrate that colonization affects mucosal and circulating B cell populations.
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Affiliation(s)
- Simon P Jochems
- Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.,Department of Parasitology, Leiden University Medical Center, Leiden, Netherlands
| | - Karin de Ruiter
- Department of Parasitology, Leiden University Medical Center, Leiden, Netherlands
| | - Carla Solórzano
- Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, United Kingdom
| | - Astrid Voskamp
- Department of Parasitology, Leiden University Medical Center, Leiden, Netherlands
| | - Elena Mitsi
- Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, United Kingdom
| | - Elissavet Nikolaou
- Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, United Kingdom
| | - Beatriz F Carniel
- Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, United Kingdom
| | - Sherin Pojar
- Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, United Kingdom
| | - Esther L German
- Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, United Kingdom
| | - Jesús Reiné
- Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, United Kingdom
| | | | - Helen Hill
- Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.,Royal Liverpool and Broadgreen University Hospital, Liverpool, United Kingdom
| | - Rachel Robinson
- Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.,Royal Liverpool and Broadgreen University Hospital, Liverpool, United Kingdom
| | - Angela D Hyder-Wright
- Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.,Royal Liverpool and Broadgreen University Hospital, Liverpool, United Kingdom
| | | | - Pascal F Durrenberger
- Centre for Inflammation and Tissue Repair, University College London, London, United Kingdom
| | | | - Stephen B Gordon
- Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.,Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Blantyre, Malawi
| | - Hermelijn H Smits
- Department of Parasitology, Leiden University Medical Center, Leiden, Netherlands
| | - Britta C Urban
- Department of Parasitology, Liverpool School of Tropical Medicine, Liverpool, United Kingdom
| | - Jamie Rylance
- Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, United Kingdom
| | - Andrea M Collins
- Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.,Royal Liverpool and Broadgreen University Hospital, Liverpool, United Kingdom.,Aintree University Hospital NHS Foundation Trust, Liverpool, United Kingdom
| | - Mark D Wilkie
- Royal Liverpool and Broadgreen University Hospital, Liverpool, United Kingdom
| | - Lepa Lazarova
- Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.,Royal Liverpool and Broadgreen University Hospital, Liverpool, United Kingdom
| | - Samuel C Leong
- Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.,Department of Otorhinolaryngology - Head and Neck Surgery, Aintree University Hospital NHS Foundation Trust, Liverpool, United Kingdom
| | - Maria Yazdanbakhsh
- Department of Parasitology, Leiden University Medical Center, Leiden, Netherlands
| | - Daniela M Ferreira
- Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool, United Kingdom
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36
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Wollam J, Riopel M, Xu YJ, Johnson AMF, Ofrecio JM, Ying W, El Ouarrat D, Chan LS, Han AW, Mahmood NA, Ryan CN, Lee YS, Watrous JD, Chordia MD, Pan D, Jain M, Olefsky JM. Microbiota-Produced N-Formyl Peptide fMLF Promotes Obesity-Induced Glucose Intolerance. Diabetes 2019; 68:1415-1426. [PMID: 31010956 PMCID: PMC6609982 DOI: 10.2337/db18-1307] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/18/2018] [Accepted: 04/08/2019] [Indexed: 12/14/2022]
Abstract
The composition of the gastrointestinal microbiota and associated metabolites changes dramatically with diet and the development of obesity. Although many correlations have been described, specific mechanistic links between these changes and glucose homeostasis remain to be defined. Here we show that blood and intestinal levels of the microbiota-produced N-formyl peptide, formyl-methionyl-leucyl-phenylalanine, are elevated in high-fat diet-induced obese mice. Genetic or pharmacological inhibition of the N-formyl peptide receptor Fpr1 leads to increased insulin levels and improved glucose tolerance, dependent upon glucagon-like peptide 1. Obese Fpr1 knockout mice also display an altered microbiome, exemplifying the dynamic relationship between host metabolism and microbiota. Overall, we describe a new mechanism by which the gut microbiota can modulate glucose metabolism, providing a potential approach for the treatment of metabolic disease.
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Affiliation(s)
- Joshua Wollam
- Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Diego, La Jolla, CA
| | - Matthew Riopel
- Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Diego, La Jolla, CA
| | - Yong-Jiang Xu
- Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Diego, La Jolla, CA
- Department of Pharmacology, University of California, San Diego, La Jolla, CA
| | - Andrew M F Johnson
- Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Diego, La Jolla, CA
| | - Jachelle M Ofrecio
- Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Diego, La Jolla, CA
| | - Wei Ying
- Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Diego, La Jolla, CA
| | - Dalila El Ouarrat
- Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Diego, La Jolla, CA
| | | | | | | | | | - Yun Sok Lee
- Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Diego, La Jolla, CA
| | - Jeramie D Watrous
- Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Diego, La Jolla, CA
- Department of Pharmacology, University of California, San Diego, La Jolla, CA
| | - Mahendra D Chordia
- Department of Radiology and Medical Imaging, University of Virginia, Charlottesville, VA
| | - Dongfeng Pan
- Department of Radiology and Medical Imaging, University of Virginia, Charlottesville, VA
| | - Mohit Jain
- Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Diego, La Jolla, CA
- Department of Pharmacology, University of California, San Diego, La Jolla, CA
| | - Jerrold M Olefsky
- Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Diego, La Jolla, CA
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Trachsel J, Briggs C, Gabler NK, Allen HK, Loving CL. Dietary Resistant Potato Starch Alters Intestinal Microbial Communities and Their Metabolites, and Markers of Immune Regulation and Barrier Function in Swine. Front Immunol 2019; 10:1381. [PMID: 31275319 PMCID: PMC6593117 DOI: 10.3389/fimmu.2019.01381] [Citation(s) in RCA: 40] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2018] [Accepted: 05/31/2019] [Indexed: 01/10/2023] Open
Abstract
Interactions between diet, the microbiota, and the host set the ecological conditions in the gut and have broad implications for health. Prebiotics are dietary compounds that may shift conditions toward health by promoting the growth of beneficial microbes that produce metabolites capable of modulating host cells. This study's objective was to assess how a dietary prebiotic could impact host tissues via modulation of the intestinal microbiota. Pigs fed a diet amended with 5% resistant potato starch (RPS) exhibited alterations associated with gut health relative to swine fed an unamended control diet (CON). RPS intake increased abundances of anaerobic Clostridia in feces and several tissues, as well as intestinal concentrations of butyrate. Functional gene amplicons suggested bacteria similar to Anaerostipes hadrus were stimulated by RPS intake. The CON treatment exhibited increased abundances of several genera of Proteobacteria (which utilize respiratory metabolisms) in several intestinal locations. RPS intake increased the abundance of regulatory T cells in the cecum, but not periphery, and cecal immune status alterations were indicative of enhanced mucosal defenses. A network analysis of host and microbial changes in the cecum revealed that regulatory T cells positively correlated with butyrate concentration, luminal IgA concentration, expression of IL-6 and DEF1B, and several mucosa-associated bacterial taxa. Thus, the administration of RPS modulated the microbiota and host immune status, altering markers of cecal barrier function and immunological tolerance, and suggesting a reduced niche for bacterial respiration.
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Affiliation(s)
- Julian Trachsel
- Food Safety and Enteric Pathogens Research Unit, National Animal Disease Center, Agricultural Research Service, Ames, IA, United States.,Interdepartmental Microbiology Graduate Program, Iowa State University, Ames, IA, United States
| | - Cassidy Briggs
- Food Safety and Enteric Pathogens Research Unit, National Animal Disease Center, Agricultural Research Service, Ames, IA, United States.,Summer Scholar Research Program, College of Veterinary Medicine, Iowa State University, Ames, IA, United States
| | - Nicholas K Gabler
- Department of Animal Science, Iowa State University, Ames, IA, United States
| | - Heather K Allen
- Food Safety and Enteric Pathogens Research Unit, National Animal Disease Center, Agricultural Research Service, Ames, IA, United States
| | - Crystal L Loving
- Food Safety and Enteric Pathogens Research Unit, National Animal Disease Center, Agricultural Research Service, Ames, IA, United States
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38
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Arthur CM, Nalbant D, Feldman HA, Saeedi BJ, Matthews J, Robinson BS, Kamili NA, Bennett A, Cress GA, Sola-Visner M, Jones RM, Zimmerman MB, Neish AS, Patel RM, Nopoulos P, Georgieff MK, Roback JD, Widness JA, Josephson CD, Stowell SR. Anemia induces gut inflammation and injury in an animal model of preterm infants. Transfusion 2019; 59:1233-1245. [PMID: 30897226 DOI: 10.1111/trf.15254] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2018] [Revised: 11/12/2018] [Accepted: 11/28/2018] [Indexed: 12/15/2022]
Abstract
BACKGROUND While very low birth weight (VLBW) infants often require multiple red blood cell transfusions, efforts to minimize transfusion-associated risks have resulted in more restrictive neonatal transfusion practices. However, whether restrictive transfusion strategies limit transfusions without increasing morbidity and mortality in this population remains unclear. Recent epidemiologic studies suggest that severe anemia may be an important risk factor for the development of necrotizing enterocolitis (NEC). However, the mechanism whereby anemia may lead to NEC remains unknown. STUDY DESIGN AND METHODS The potential impact of anemia on neonatal inflammation and intestinal barrier disruption, two well-characterized predisposing features of NEC, was defined by correlation of hemoglobin values to cytokine levels in premature infants and by direct evaluation of intestinal hypoxia, inflammation and gut barrier disruption using a pre-clinical neonatal murine model of phlebotomy-induced anemia (PIA). RESULTS Increasing severity of anemia in the preterm infant correlated with the level of IFN-gamma, a key pro-inflammatory cytokine that may predispose an infant to NEC. Gradual induction of PIA in a pre-clinical model resulted in significant hypoxia throughout the intestinal mucosa, including areas where intestinal macrophages reside. PIA-induced hypoxia significantly increased macrophage pro-inflammatory cytokine levels, while reducing tight junction protein ZO-1 expression and increasing intestinal barrier permeability. Macrophage depletion reversed the impact of anemia on intestinal ZO-1 expression and barrier function. CONCLUSIONS Taken together, these results suggest that anemia can increase intestinal inflammation and barrier disruption likely through altered macrophage function, leading to the type of predisposing intestinal injury that may increase the risk for NEC.
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Affiliation(s)
- Connie M Arthur
- Departments of Pathology and Laboratory Medicine, Center for Transfusion and Cellular Therapies, Atlanta, Georgia
| | - Demet Nalbant
- Department of Pediatrics, University of Iowa, Iowa City, Iowa
| | - Henry A Feldman
- Division of Newborn Medicine, Boston Children's Hospital, Boston, Massachusetts
| | - Bejan J Saeedi
- Departments of Pathology and Laboratory Medicine, Center for Transfusion and Cellular Therapies, Atlanta, Georgia
| | - Jason Matthews
- Departments of Pathology and Laboratory Medicine, Center for Transfusion and Cellular Therapies, Atlanta, Georgia
| | - Brian S Robinson
- Departments of Pathology and Laboratory Medicine, Center for Transfusion and Cellular Therapies, Atlanta, Georgia
| | - Nourine A Kamili
- Departments of Pathology and Laboratory Medicine, Center for Transfusion and Cellular Therapies, Atlanta, Georgia
| | - Ashley Bennett
- Departments of Pathology and Laboratory Medicine, Center for Transfusion and Cellular Therapies, Atlanta, Georgia
| | | | - Martha Sola-Visner
- Division of Newborn Medicine, Boston Children's Hospital, Boston, Massachusetts
| | - Rheinallt M Jones
- Departments of Pathology and Laboratory Medicine, Center for Transfusion and Cellular Therapies, Atlanta, Georgia
| | | | - Andrew S Neish
- Departments of Pathology and Laboratory Medicine, Center for Transfusion and Cellular Therapies, Atlanta, Georgia
| | - Ravi M Patel
- Pediatrics, Emory University School of Medicine, Atlanta, Georgia
| | - Peggy Nopoulos
- Department of Psychiatry, College of Medicine, University of Iowa, Iowa City, Iowa
| | - Michael K Georgieff
- Department of Pediatrics, School of Medicine, University of Minnesota, Minneapolis, Minnesota
| | - John D Roback
- Departments of Pathology and Laboratory Medicine, Center for Transfusion and Cellular Therapies, Atlanta, Georgia
| | - John A Widness
- Department of Pediatrics, University of Iowa, Iowa City, Iowa
| | - Cassandra D Josephson
- Departments of Pathology and Laboratory Medicine, Center for Transfusion and Cellular Therapies, Atlanta, Georgia.,Pediatrics, Emory University School of Medicine, Atlanta, Georgia
| | - Sean R Stowell
- Departments of Pathology and Laboratory Medicine, Center for Transfusion and Cellular Therapies, Atlanta, Georgia
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39
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Martínez-López M, Iborra S, Conde-Garrosa R, Mastrangelo A, Danne C, Mann ER, Reid DM, Gaboriau-Routhiau V, Chaparro M, Lorenzo MP, Minnerup L, Saz-Leal P, Slack E, Kemp B, Gisbert JP, Dzionek A, Robinson MJ, Rupérez FJ, Cerf-Bensussan N, Brown GD, Bernardo D, LeibundGut-Landmann S, Sancho D. Microbiota Sensing by Mincle-Syk Axis in Dendritic Cells Regulates Interleukin-17 and -22 Production and Promotes Intestinal Barrier Integrity. Immunity 2019; 50:446-461.e9. [PMID: 30709742 PMCID: PMC6382412 DOI: 10.1016/j.immuni.2018.12.020] [Citation(s) in RCA: 162] [Impact Index Per Article: 27.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2017] [Revised: 07/30/2018] [Accepted: 12/17/2018] [Indexed: 12/15/2022]
Abstract
Production of interleukin-17 (IL-17) and IL-22 by T helper 17 (Th17) cells and group 3 innate lymphoid cells (ILC3s) in response to the gut microbiota ensures maintenance of intestinal barrier function. Here, we examined the mechanisms whereby the immune system detects microbiota in the steady state. A Syk-kinase-coupled signaling pathway in dendritic cells (DCs) was critical for commensal-dependent production of IL-17 and IL-22 by CD4+ T cells. The Syk-coupled C-type lectin receptor Mincle detected mucosal-resident commensals in the Peyer's patches (PPs), triggered IL-6 and IL-23p19 expression, and thereby regulated function of intestinal Th17- and IL-17-secreting ILCs. Mice deficient in Mincle or with selective depletion of Syk in CD11c+ cells had impaired production of intestinal RegIIIγ and IgA and increased systemic translocation of gut microbiota. Consequently, Mincle deficiency led to liver inflammation and deregulated lipid metabolism. Thus, sensing of commensals by Mincle and Syk signaling in CD11c+ cells reinforces intestinal immune barrier and promotes host-microbiota mutualism, preventing systemic inflammation.
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Affiliation(s)
- María Martínez-López
- Immunobiology Laboratory, Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Melchor Fernández Almagro 3, Madrid 28029, Spain
| | - Salvador Iborra
- Immunobiology Laboratory, Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Melchor Fernández Almagro 3, Madrid 28029, Spain; Department of Immunology, School of Medicine, Universidad Complutense de Madrid, 12 de Octubre Health Research Institute (imas12), Madrid, Spain.
| | - Ruth Conde-Garrosa
- Immunobiology Laboratory, Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Melchor Fernández Almagro 3, Madrid 28029, Spain
| | - Annalaura Mastrangelo
- Immunobiology Laboratory, Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Melchor Fernández Almagro 3, Madrid 28029, Spain
| | - Camille Danne
- Kennedy Institute of Rheumatology, University of Oxford, Oxford, UK
| | - Elizabeth R Mann
- Lydia Becker Institute of Immunology and Inflammation, University of Manchester, Manchester, UK; Manchester Collaborative Centre for Inflammation Research, Faculty of Biology, Medicine, and Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK
| | - Delyth M Reid
- Medical Research Council Centre for Medical Mycology at the University of Aberdeen, Aberdeen Fungal Group, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK
| | - Valérie Gaboriau-Routhiau
- INRA Micalis Institut, UMR1319, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France; INSERM UMR1163, Institut Imagine, Laboratory of Intestinal Immunity, 75015 Paris, France; Université Paris Descartes-Sorbonne Paris Cité, 75006 Paris, France
| | - Maria Chaparro
- Gastroenterology Unit, Hospital Universitario de La Princesa, Instituto de Investigación Sanitaria Princesa (IIS-IP), Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Diego de León 62, Madrid 28006, Spain
| | - María P Lorenzo
- Centro de Metabolómica y Bioanálisis (CEMBIO), Facultad de Farmacia, Universidad San Pablo-CEU, Urbanización Montepríncipe, km 0, M501, Alcorcón 28925, Spain
| | | | - Paula Saz-Leal
- Immunobiology Laboratory, Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Melchor Fernández Almagro 3, Madrid 28029, Spain
| | - Emma Slack
- Institute of Food, Nutrition, and Health, ETH Zurich, Vladimir-Prelog-Weg 4, Zürich 8093, Switzerland
| | | | - Javier P Gisbert
- Gastroenterology Unit, Hospital Universitario de La Princesa, Instituto de Investigación Sanitaria Princesa (IIS-IP), Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Diego de León 62, Madrid 28006, Spain
| | | | | | - Francisco J Rupérez
- Centro de Metabolómica y Bioanálisis (CEMBIO), Facultad de Farmacia, Universidad San Pablo-CEU, Urbanización Montepríncipe, km 0, M501, Alcorcón 28925, Spain
| | - Nadine Cerf-Bensussan
- INSERM UMR1163, Institut Imagine, Laboratory of Intestinal Immunity, 75015 Paris, France; Université Paris Descartes-Sorbonne Paris Cité, 75006 Paris, France
| | - Gordon D Brown
- Medical Research Council Centre for Medical Mycology at the University of Aberdeen, Aberdeen Fungal Group, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK
| | - David Bernardo
- Gastroenterology Unit, Hospital Universitario de La Princesa, Instituto de Investigación Sanitaria Princesa (IIS-IP), Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Diego de León 62, Madrid 28006, Spain
| | - Salomé LeibundGut-Landmann
- Section of Immunology, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 266a Zurich 8057, Switzerland
| | - David Sancho
- Immunobiology Laboratory, Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Melchor Fernández Almagro 3, Madrid 28029, Spain.
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40
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Baal N, Cunningham S, Obermann HL, Thomas J, Lippitsch A, Dietert K, Gruber AD, Kaufmann A, Michel G, Nist A, Stiewe T, Rupp O, Goesmann A, Zukunft S, Fleming I, Bein G, Lohmeyer J, Bauer S, Hackstein H. ADAR1 Is Required for Dendritic Cell Subset Homeostasis and Alveolar Macrophage Function. THE JOURNAL OF IMMUNOLOGY 2019; 202:1099-1111. [DOI: 10.4049/jimmunol.1800269] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/02/2018] [Accepted: 12/07/2018] [Indexed: 11/19/2022]
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41
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In vivo imaging reveals unique neutrophil transendothelial migration patterns in inflamed intestines. Mucosal Immunol 2018; 11:1571-1581. [PMID: 30104624 PMCID: PMC6279495 DOI: 10.1038/s41385-018-0069-5] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2018] [Revised: 06/28/2018] [Accepted: 06/28/2018] [Indexed: 02/04/2023]
Abstract
Neutrophil (PMN) infiltration of the intestinal mucosa is a hallmark of gastrointestinal inflammation, with significant implications for host defense, injury and repair. However, phenotypic and mechanistic aspects of PMN recruitment in inflamed intestines have not been explored in vivo. Using novel epithelial/PMN fluorescence reporter mice, advanced intravital imaging and 3D reconstruction analysis, we mapped the microvasculature architecture across the intestinal layers and determined that in response to Salmonella/endotoxin-induced inflammation, PMN transendothelial migration (TEM) was restricted to submucosal vessels. PMN TEM was not observed in villus or crypt vessels, proximal to the epithelium that underlies the intestinal lumen, and was partially dependent on (C-X-C motif) ligands 1 (CXCL1) and 2 (CXCL2) expression, which was found to be elevated in the submucosa layer. Restricted PMN extravasation at the submucosa and subsequent PMN interstitial migration may serve as a novel regulatory step of PMN effector function and recruitment to the luminal space in inflamed intestines.
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42
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Oral consumption of cinnamon enhances the expression of immunity and lipid absorption genes in the small intestinal epithelium and alters the gut microbiota in normal mice. J Funct Foods 2018. [DOI: 10.1016/j.jff.2018.08.013] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
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43
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Attaya A, Wang T, Zou J, Herath T, Adams A, Secombes CJ, Yoon S. Gene expression analysis of isolated salmonid GALT leucocytes in response to PAMPs and recombinant cytokines. FISH & SHELLFISH IMMUNOLOGY 2018; 80:426-436. [PMID: 29906623 DOI: 10.1016/j.fsi.2018.06.022] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/29/2018] [Revised: 05/30/2018] [Accepted: 06/11/2018] [Indexed: 06/08/2023]
Abstract
Increased knowledge of the immune response of the intestine, a physiologically critical organ involved in absorption, secretion and homeostasis in a non-sterile environment, is needed to better understand the mechanisms involved in the induction of long-lasting immunity and, subsequently, the development of efficacious gastrointestinal immunization approaches. To this end, analysis of isolated gut cells will give an insight into the cell types present and their immune capability. Hence, in this study we first optimised a method for salmonid gut leucocyte isolation and characterised the cells on the basis of their expression of a range of selected cell markers associated with T & B cells and dendritic cells. The GALT leucocytes were then stimulated with a variety of PAMPs, recombinant cytokines and PHA, as a means to help characterise the diversity of the immune repertoire present in such cells. The stimulants tested were designed to examine the nature of the antibacterial, antiviral and T cell type responses in the cells (at the transcript level) using a panel of genes relevant to innate and adaptive immunity. The results showed distinct responses to the stimulants, with a clear delineation seen between the stimulant used (eg viral or bacterial PAMP) and the pathway elicited. The changes in the expression patterns of the immune genes in these cells indicates that the salmonid intestine contains a good repertoire of competent immune cells able to respond to different pathogen types. Such information may aid the development of efficient priming by oral vaccination in salmonids.
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Affiliation(s)
- A Attaya
- Scottish Fish Immunology Research Centre, Institute of Biological and Environmental Sciences, University of Aberdeen, Aberdeen, AB24 2TZ, UK
| | - T Wang
- Scottish Fish Immunology Research Centre, Institute of Biological and Environmental Sciences, University of Aberdeen, Aberdeen, AB24 2TZ, UK
| | - J Zou
- Scottish Fish Immunology Research Centre, Institute of Biological and Environmental Sciences, University of Aberdeen, Aberdeen, AB24 2TZ, UK
| | - T Herath
- Institute of Aquaculture, University of Stirling, Stirling, FK9 4LA, UK
| | - A Adams
- Institute of Aquaculture, University of Stirling, Stirling, FK9 4LA, UK
| | - C J Secombes
- Scottish Fish Immunology Research Centre, Institute of Biological and Environmental Sciences, University of Aberdeen, Aberdeen, AB24 2TZ, UK.
| | - S Yoon
- Scottish Fish Immunology Research Centre, Institute of Biological and Environmental Sciences, University of Aberdeen, Aberdeen, AB24 2TZ, UK.
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44
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Zai K, Hirota M, Yamada T, Ishihara N, Mori T, Kishimura A, Suzuki K, Hase K, Katayama Y. Therapeutic effect of vitamin D 3-containing nanostructured lipid carriers on inflammatory bowel disease. J Control Release 2018; 286:94-102. [PMID: 30017723 DOI: 10.1016/j.jconrel.2018.07.019] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2018] [Revised: 06/19/2018] [Accepted: 07/10/2018] [Indexed: 12/11/2022]
Abstract
The active form of vitamin D3, 1,25(OH)2D3 has been found to exert multiple effects on the suppression of progression of inflammatory bowel disease (IBD). Vitamin D3 has been gathering attention as a therapy for IBD. However, the clinical trials conducted to date revealed that a relatively high dosage of vitamin D3 was required to see a significant therapeutic effect. Thus, effective formulation and delivery of vitamin D3 to colonic inflammatory lesions will be required. Herein we describe the preparation of a nanostructured lipid carrier (NLC) for the encapsulation of 1,25(OH)2D3 for colonic delivery via oral administration. The optimized fabrication procedure enabled the incorporation of 1,25(OH)2D3 in the NLC by minimizing the destruction of chemically unstable 1,25(OH)2D3. The obtained NLCs orally delivered 1,25(OH)2D3 to the colon in mice and maintained a high concentration of 1,25(OH)2D3 in the colonic tissue for at least 12 h. The NLC showed multiple effects on the suppression of symptoms of colitis induced by dextran sodium sulfate, namely maintaining crypt structure, reducing the tissue concentration of inflammatory cytokines, suppressing the infiltration of polymorphonuclear leukocytes, and augmenting anti-inflammatory CX3CR1high macrophages. Our NLCs containing 1,25(OH)2D3 may be an alternative treatment for IBD therapy.
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Affiliation(s)
- Khadijah Zai
- Department of Applied Chemistry, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan
| | - Masato Hirota
- Division of Biochemistry, Faculty of Pharmacy, Keio University, Tokyo 105-8512, Japan
| | - Takahiro Yamada
- Division of Biochemistry, Faculty of Pharmacy, Keio University, Tokyo 105-8512, Japan
| | - Narumi Ishihara
- Division of Biochemistry, Faculty of Pharmacy, Keio University, Tokyo 105-8512, Japan
| | - Takeshi Mori
- Department of Applied Chemistry, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan; Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan; Center for Future Chemistry, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan
| | - Akihiro Kishimura
- Department of Applied Chemistry, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan; Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan; Center for Future Chemistry, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan; International Research Center for Molecular Systems, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan
| | - Koichiro Suzuki
- Division of Biochemistry, Faculty of Pharmacy, Keio University, Tokyo 105-8512, Japan
| | - Koji Hase
- Division of Biochemistry, Faculty of Pharmacy, Keio University, Tokyo 105-8512, Japan; Division of Mucosal Barrierology, International Research and Development Center for Mucosal Vaccines, The Institute of Medical Science the University of Tokyo, Tokyo 108-8639, Japan.
| | - Yoshiki Katayama
- Department of Applied Chemistry, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan; Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan; Center for Future Chemistry, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan; International Research Center for Molecular Systems, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan; Centre for Advanced Medicine Innovation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan; Department of Biomedical Engineering, Chung Yuan Christian University, 200 Chung Pei Rd., Chung Li 32023, Taiwan, PR China.
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Immune quiescence in the oral mucosa is maintained by a uniquely large population of highly activated Foxp3 + regulatory T cells. Mucosal Immunol 2018; 11:1092-1102. [PMID: 29743613 PMCID: PMC6035783 DOI: 10.1038/s41385-018-0027-2] [Citation(s) in RCA: 37] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2017] [Revised: 03/05/2018] [Accepted: 03/29/2018] [Indexed: 02/07/2023]
Abstract
The oral mucosa is a critical barrier tissue that protects the oral cavity against invading pathogens and foreign antigens. Interestingly, inflammation in the oral cavity is rarely observed, indicating that overt immune activation in this site is actively suppressed. Whether Foxp3+ Treg cells are involved in controlling immunity of the oral mucosa, however, is not fully understood. Here, we show that the oral mucosa is highly enriched in Foxp3+ Treg cells, and that oral mucosa Treg cells are phenotypically distinct from those of LN or spleen, as they expressed copious amounts of the tissue-retention molecule CD103 and unusually high-levels of CTLA4. Acute depletion of Foxp3+ Treg cells had catastrophic effects, resulting in marked infiltration of activated effector T cells that were associated with autoimmunity and tissue destruction of the oral mucosa. Moreover, adoptive transfer of naive CD4 T cells revealed that the oral mucosa is highly ineffective in inducing Foxp3+ Treg cells in situ, so that it depends on recruitment and migration of exogenous Treg cells to populate this mucosal site. Collectively, these results demonstrate a previously unappreciated role and a distinct developmental pathway for Foxp3+ Treg cells in the oral mucosa, which are essential to control local tissue immunity.
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46
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Nusse YM, Savage AK, Marangoni P, Rosendahl-Huber AKM, Landman TA, de Sauvage FJ, Locksley RM, Klein OD. Parasitic helminths induce fetal-like reversion in the intestinal stem cell niche. Nature 2018. [PMID: 29950724 DOI: 10.1038/s41586‐018‐0257‐1] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Epithelial surfaces form critical barriers to the outside world and are continuously renewed by adult stem cells1. Whereas dynamics of epithelial stem cells during homeostasis are increasingly well understood, how stem cells are redirected from a tissue-maintenance program to initiate repair after injury remains unclear. Here we examined infection by Heligmosomoides polygyrus, a co-evolved pathosymbiont of mice, to assess the epithelial response to disruption of the mucosal barrier. H. polygyrus disrupts tissue integrity by penetrating the duodenal mucosa, where it develops while surrounded by a multicellular granulomatous infiltrate2. Crypts overlying larvae-associated granulomas did not express intestinal stem cell markers, including Lgr53, in spite of continued epithelial proliferation. Granuloma-associated Lgr5- crypt epithelium activated an interferon-gamma (IFN-γ)-dependent transcriptional program, highlighted by Sca-1 expression, and IFN-γ-producing immune cells were found in granulomas. A similar epithelial response accompanied systemic activation of immune cells, intestinal irradiation, or ablation of Lgr5+ intestinal stem cells. When cultured in vitro, granuloma-associated crypt cells formed spheroids similar to those formed by fetal epithelium, and a sub-population of H. polygyrus-induced cells activated a fetal-like transcriptional program, demonstrating that adult intestinal tissues can repurpose aspects of fetal development. Therefore, re-initiation of the developmental program represents a fundamental mechanism by which the intestinal crypt can remodel itself to sustain function after injury.
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Affiliation(s)
- Ysbrand M Nusse
- Biomedical Sciences Graduate Program, University of California, San Francisco, CA, USA.,Program in Craniofacial Biology and Department of Orofacial Sciences, University of California, San Francisco, CA, USA
| | - Adam K Savage
- Howard Hughes Medical Institute and Departments of Medicine and Microbiology & Immunology, University of California, San Francisco, CA, USA
| | - Pauline Marangoni
- Program in Craniofacial Biology and Department of Orofacial Sciences, University of California, San Francisco, CA, USA
| | - Axel K M Rosendahl-Huber
- Program in Craniofacial Biology and Department of Orofacial Sciences, University of California, San Francisco, CA, USA
| | - Tyler A Landman
- Program in Craniofacial Biology and Department of Orofacial Sciences, University of California, San Francisco, CA, USA
| | | | - Richard M Locksley
- Howard Hughes Medical Institute and Departments of Medicine and Microbiology & Immunology, University of California, San Francisco, CA, USA.
| | - Ophir D Klein
- Program in Craniofacial Biology and Department of Orofacial Sciences, University of California, San Francisco, CA, USA. .,Department of Pediatrics and Institute for Human Genetics, University of California, San Francisco, CA, USA.
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47
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Parasitic helminths induce fetal-like reversion in the intestinal stem cell niche. Nature 2018; 559:109-113. [PMID: 29950724 PMCID: PMC6042247 DOI: 10.1038/s41586-018-0257-1] [Citation(s) in RCA: 226] [Impact Index Per Article: 32.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2018] [Accepted: 05/09/2018] [Indexed: 12/18/2022]
Abstract
Epithelial surfaces form critical barriers to the outside world and are continuously renewed by adult stem cells1. Whereas epithelial stem cell dynamics during homeostasis are increasingly well understood, how stem cells are redirected from a tissue-maintenance program to initiate repair after injury remains unclear. Here, we examined infection by Heligmosomoides polygyrus (Hp), a co-evolved pathosymbiont of mice, to assess the epithelial response to disruption of the mucosal barrier. Hp disrupts tissue integrity by penetrating the duodenal mucosa, where it develops while surrounded by a multicellular granulomatous infiltrate2. Unexpectedly, intestinal stem cell (ISC) markers, including Lgr53, were lost in crypts overlying larvae-associated granulomas, despite continued epithelial proliferation. Granuloma-associated Lgr5− crypt epithelia activated an interferon-gamma (IFNγ)-dependent transcriptional program, highlighted by Sca-1 expression, and IFNγ-producing immune cells were found in granulomas. A similar epithelial response accompanied systemic activation of immune cells, intestinal irradiation, or ablation of Lgr5+ ISCs. Granuloma-associated crypt cells generated fetal-like spheroids in culture, and a sub-population of Hp-induced cells activated a fetal-like transcriptional program, demonstrating that adult intestinal tissues can repurpose aspects of fetal development. Thus, re-initiation of the developmental program represents a fundamental mechanism by which the intestinal crypt can remodel to sustain function after injury.
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48
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Newton JM, Hanoteau A, Sikora AG. Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors. J Vis Exp 2018. [PMID: 29939180 DOI: 10.3791/57685] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023] Open
Abstract
The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for immunotherapies. Recent clinical advances in immunotherapy highlight the need for reproducible methods to accurately and thoroughly characterize the tumor and its associated immune infiltrate. Tumor enzymatic digestion and flow cytometric analysis allow broad characterization of numerous immune cell subsets and phenotypes; however, depth of analysis is often limited by fluorophore restrictions on panel design and the need to acquire large tumor samples to observe rare immune populations of interest. Thus, we have developed an effective and high throughput method for separating and enriching the tumor immune infiltrate from the non-immune tumor components. The described tumor digestion and centrifugal density-based separation technique allows separate characterization of tumor and tumor immune infiltrate fractions and preserves cellular viability, and thus, provides a broad characterization of the tumor immunologic state. This method was used to characterize the extensive spatial immune heterogeneity in solid tumors, which further demonstrates the need for consistent whole tumor immunologic profiling techniques. Overall, this method provides an effective and adaptable technique for the immunologic characterization of subcutaneous solid murine tumors; as such, this tool can be used to better characterize the tumoral immunologic features and in the preclinical evaluation of novel immunotherapeutic strategies.
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Affiliation(s)
- Jared M Newton
- Department of Otolaryngology-Head and Neck Surgery, Baylor College of Medicine; Interdepartmental Graduate Program in Translational Biology and Molecular Medicine, Baylor College of Medicine
| | - Aurelie Hanoteau
- Department of Otolaryngology-Head and Neck Surgery, Baylor College of Medicine
| | - Andrew G Sikora
- Department of Otolaryngology-Head and Neck Surgery, Baylor College of Medicine;
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49
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iNOS- and NOX1-dependent ROS production maintains bacterial homeostasis in the ileum of mice. Mucosal Immunol 2018; 11:774-784. [PMID: 29210363 DOI: 10.1038/mi.2017.106] [Citation(s) in RCA: 61] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2017] [Accepted: 10/17/2017] [Indexed: 02/04/2023]
Abstract
The intestinal epithelial cells constitute the first line of defense against gut microbes, which includes secretion of various antimicrobial substances. Reactive oxygen species (ROS) are well characterized as part of the innate phagocytic immunity; however, a role in controlling microorganisms in the gut lumen is less clear. Here, we show a role for nitric oxide synthase (iNOS)- and NOX1-produced ROS in maintaining homeostasis of the gut microbiota. In vivo imaging revealed distinctly high levels of ROS in the ileum of normal healthy mice, regulated in accordance with the amount of gut bacteria. The ROS level was dependent on the nitric oxide and superoxide producers iNOS and NOX1, respectively, suggesting peroxynitrite as the effector molecule. In the ileum of iNOS- and NOX1-deficient mice, the bacterial load is increased and the composition is more cecum like. Our data suggest a unique role of ileum in maintaining homeostasis of gut microbes through production of ROS with potential importance for preventing reflux from the large intestine, bacterial overgrowth, and translocation.
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50
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Lorentsen KJ, Cho JJ, Luo X, Zuniga AN, Urban JF, Zhou L, Gharaibeh R, Jobin C, Kladde MP, Avram D. Bcl11b is essential for licensing Th2 differentiation during helminth infection and allergic asthma. Nat Commun 2018; 9:1679. [PMID: 29700302 PMCID: PMC5920086 DOI: 10.1038/s41467-018-04111-0] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2017] [Accepted: 04/03/2018] [Indexed: 12/24/2022] Open
Abstract
During helminth infection and allergic asthma, naive CD4+ T-cells differentiate into cytokine-producing Type-2 helper (Th2) cells that resolve the infection or induce asthma-associated pathology. Mechanisms regulating the Th2 differentiation in vivo remain poorly understood. Here we report that mice lacking Bcl11b in mature T-cells have a diminished capacity to mount Th2 responses during helminth infection and allergic asthma, showing reduced Th2 cytokines and Gata3, and elevated Runx3. We provide evidence that Bcl11b is required to maintain chromatin accessibility at Th2-cytokine promoters and locus-control regions, and binds the Il4 HS IV silencer, reducing its accessibility. Bcl11b also binds Gata3-intronic and downstream-noncoding sites, sustaining the Gata3 expression. In addition, Bcl11b binds and deactivates upstream enhancers at Runx3 locus, restricting the Runx3 expression and its availability to act at the Il4 HS IV silencer. Thus, our results establish novel roles for Bcl11b in the regulatory loop that licenses Th2 program in vivo.
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Affiliation(s)
- Kyle J Lorentsen
- Department of Medicine, Division of Pulmonary Medicine, College of Medicine, University of Florida, 1600 SW Archer Rd, Gainesville, FL, 32610, USA
| | - Jonathan J Cho
- Department of Medicine, Division of Pulmonary Medicine, College of Medicine, University of Florida, 1600 SW Archer Rd, Gainesville, FL, 32610, USA.,Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL, 32610, USA
| | - Xiaoping Luo
- Department of Medicine, Division of Pulmonary Medicine, College of Medicine, University of Florida, 1600 SW Archer Rd, Gainesville, FL, 32610, USA.,Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL, 32610, USA
| | - Ashley N Zuniga
- Department of Medicine, Division of Pulmonary Medicine, College of Medicine, University of Florida, 1600 SW Archer Rd, Gainesville, FL, 32610, USA.,Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL, 32610, USA
| | - Joseph F Urban
- Beltsville Human Nutrition Research Center, Agricultural Research Service, Diet, Genomic and Immunology Laboratory, US Department of Agriculture, Beltsville, MD, 20705, USA
| | - Liang Zhou
- Department of Infectious Diseases and Immunology, College of Veterinary Medicine, University of Florida, 2015 SW 16th Ave, Gainesville, FL, 32608, USA.,UF Health Cancer Center, University of Florida, Gainesville, FL, 32610, USA
| | - Raad Gharaibeh
- UF Health Cancer Center, University of Florida, Gainesville, FL, 32610, USA.,Department of Medicine, Division of Gastroenterology, College of Medicine, University of Florida, 2033 Mowry Rd., CGRC 461, Gainesville, FL, 32610, USA
| | - Christian Jobin
- UF Health Cancer Center, University of Florida, Gainesville, FL, 32610, USA.,Department of Medicine, Division of Gastroenterology, College of Medicine, University of Florida, 2033 Mowry Rd., CGRC 461, Gainesville, FL, 32610, USA
| | - Michael P Kladde
- UF Health Cancer Center, University of Florida, Gainesville, FL, 32610, USA.,Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, 2033 Mowry Rd., CGRC 359, Gainesville, FL, 32610, USA
| | - Dorina Avram
- Department of Medicine, Division of Pulmonary Medicine, College of Medicine, University of Florida, 1600 SW Archer Rd, Gainesville, FL, 32610, USA. .,Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL, 32610, USA. .,UF Health Cancer Center, University of Florida, Gainesville, FL, 32610, USA.
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