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Chini A, Guha P, Rishi A, Obaid M, Udden SN, Mandal SS. Discovery and functional characterization of LncRNAs associated with inflammation and macrophage activation. Methods 2024; 227:1-16. [PMID: 38703879 DOI: 10.1016/j.ymeth.2024.05.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2024] [Revised: 04/24/2024] [Accepted: 05/01/2024] [Indexed: 05/06/2024] Open
Abstract
Long noncoding RNAs (lncRNA) are emerging players in regulation of gene expression and cell signaling and their dysregulation has been implicated in a multitude of human diseases. Recent studies from our laboratory revealed that lncRNAs play critical roles in cytokine regulation, inflammation, and metabolism. We demonstrated that lncRNA HOTAIR, which is a well-known regulator of gene silencing, plays critical roles in modulation of cytokines and proinflammatory genes, and glucose metabolism in macrophages during inflammation. In addition, we recently discovered a series of novel lncRNAs that are closely associated with inflammation and macrophage activation. We termed these as long-noncoding inflammation associated RNAs (LinfRNAs). We are currently engaged in the functional characterization of these hLinfRNAs (human LinfRNAs) with a focus on their roles in inflammation, and we are investigating their potential implications in chronic inflammatory human diseases. Here, we have summarized experimental methods that have been utilized for the discovery and functional characterization of lncRNAs in inflammation and macrophage activation.
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Affiliation(s)
- Avisankar Chini
- Gene Regulation and Epigenetics Research Laboratory, Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX 76019, USA
| | - Prarthana Guha
- Gene Regulation and Epigenetics Research Laboratory, Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX 76019, USA
| | - Ashcharya Rishi
- Gene Regulation and Epigenetics Research Laboratory, Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX 76019, USA
| | - Monira Obaid
- Gene Regulation and Epigenetics Research Laboratory, Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX 76019, USA
| | - Sm Nashir Udden
- Department of Radiation Oncology, The University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA
| | - Subhrangsu S Mandal
- Gene Regulation and Epigenetics Research Laboratory, Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX 76019, USA.
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Vunnam N, Young MC, Liao EE, Lo CH, Huber E, Been M, Thomas DD, Sachs JN. Nimesulide, a COX-2 inhibitor, sensitizes pancreatic cancer cells to TRAIL-induced apoptosis by promoting DR5 clustering †. Cancer Biol Ther 2023; 24:2176692. [PMID: 36775838 PMCID: PMC9928464 DOI: 10.1080/15384047.2023.2176692] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/14/2023] Open
Abstract
Nimesulide is a nonsteroidal anti-inflammatory drug and a COX-2 inhibitor with antitumor and antiproliferative activities that induces apoptosis in oral, esophagus, breast, and pancreatic cancer cells. Despite being removed from the market due to hepatotoxicity, nimesulide is still an important research tool being used to develop new anticancer drugs. Multiple studies have been done to modify the nimesulide skeleton to develop more potent anticancer agents and related compounds are promising scaffolds for future development. As such, establishing a mechanism of action for nimesulide remains an important part of realizing its potential. Here, we show that nimesulide enhances TRAIL-induced apoptosis in resistant pancreatic cancer cells by promoting clustering of DR5 in the plasma membrane. In this way, nimesulide acts like a related compound, DuP-697, which sensitizes TRAIL-resistant colon cancer cells in a similar manner. Our approach applies a time-resolved FRET-based biosensor that monitors DR5 clustering and conformational states in the plasma membrane. We show that this tool can be used for future high-throughput screens to identify novel, nontoxic small molecule scaffolds to overcome TRAIL resistance in cancer cells.
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Affiliation(s)
- Nagamani Vunnam
- Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA
| | - Malaney C Young
- Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA
| | - Elly E Liao
- Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA
| | - Chih Hung Lo
- Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA
| | - Evan Huber
- Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA
| | - MaryJane Been
- Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA
| | - David D Thomas
- Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, USA
| | - Jonathan N Sachs
- Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA
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Goyal G, Kalonia H, Lather V. Therapeutic Potential of Catechin as an IKK-β Inhibitor for the Management of Arthritis: In vitro and In vivo Approach. JOURNAL OF PHARMACY AND BIOALLIED SCIENCES 2023; 15:172-179. [PMID: 38235046 PMCID: PMC10790745 DOI: 10.4103/jpbs.jpbs_280_23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2023] [Revised: 05/28/2023] [Accepted: 06/02/2023] [Indexed: 01/19/2024] Open
Abstract
Background Rheumatoid arthritis (RA) is associated with increased levels of cytokines, for instance, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and interleukin-1 (IL-1), which exhibit potent pro-inflammatory effects and are contributing factors to disease progression. A range of cytokines, cell adhesion molecules, and enzymes that are implicated in the debilitating effects of RA are transcribed by nuclear factor kappa. Objectives The purpose of this research was to characterize the efficacy of "catechin" as an IkappaB kinase-beta (IKK-β) inhibitor in collagen-induced arthritis (CIA) model in mice, as IKK-β is crucial in the transmission of signal-inducible NF-κβ activation. Methods Arthritis was brought on in Bagg and Albino, but it is written BALB/c (BALB/c) male mice through subcutaneous immunization with bovine type II collagen on days 0 and 21. Catechin is given orally every day after the onset of the disease. Clinical evaluation of the prevalence and severity of the condition was done throughout the trial, and biochemical testing was done at the end (day 42). Results In vitro findings of the study demonstrated catechin as a potent inhibitor of IKK-β with Half maximal Inhibitory Concentration (IC50) values of 2.90 μM and 4.358 μM in IKK-β and NF-κβ transactivation activity assay, respectively. Furthermore, catechin (dose range of 10-100 mg/kg, p.o.) was effective in reducing disease incidence and clinical signs in a dose-dependent manner, with an Effective Dose for 50% of the population (ED50) value of 79.579 mg/kg. The findings of this study demonstrate dose-dependent efficacy in terms of both disease severity (clinical scoring) and inflammatory markers (biochemical evaluation of the serum and joints). Conclusions IKK inhibitors are a prospective target for the creation of new therapeutics for arthritis and other inflammatory diseases because it has been suggested that this enzyme is crucial in the pathophysiology of RA. The finding of this study suggests that "catechin" represents a novel inhibitor of IKK-β with promising anti-inflammatory activity.
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Affiliation(s)
- Gourav Goyal
- Centre for Pharmaceutical Chemistry and Analysis, Amity Institute of Pharmacy, Amity University, Noida, Uttar Pradesh, India
| | - Harikesh Kalonia
- Wockhardt Research Centre, Wockhardt Pharmaceutical Pvt Ltd., Aurangabad, Maharashtra, India
| | - Viney Lather
- Centre for Pharmaceutical Chemistry and Analysis, Amity Institute of Pharmacy, Amity University, Noida, Uttar Pradesh, India
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Huang ZQ, Luo W, Li WX, Chen P, Wang Z, Chen RJ, Wang Y, Huang WJ, Liang G. Costunolide alleviates atherosclerosis in high-fat diet-fed ApoE -/- mice through covalently binding to IKKβ and inhibiting NF-κB-mediated inflammation. Acta Pharmacol Sin 2023; 44:58-70. [PMID: 35710877 PMCID: PMC9813247 DOI: 10.1038/s41401-022-00928-0] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/25/2022] [Accepted: 05/26/2022] [Indexed: 01/18/2023]
Abstract
Costunolide (CTD) is a sesquiterpene lactone isolated from costus root and exhibits various biological activities including anti-inflammation. Since atherosclerosis is a chronic inflammatory disease, we herein investigated the anti-atherosclerotic effects of CTD and the underlying mechanism. Atherosclerosis was induced in ApoE-/- mice by feeding them with a high-fat diet (HFD) for 8 weeks, followed by administration of CTD (10, 20 mg ·kg-1·d-1, i.g.) for 8 weeks. We showed that CTD administration dose-dependently alleviated atherosclerosis in HFD-fed ApoE-/- mice. Furthermore, we found that CTD dose-dependently reduced inflammatory responses in aortas of the mice, as CTD prevented infiltration of inflammatory cells in aortas and attenuated oxLDL uptake in macrophages, leading to reduced expression of pro-inflammatory and pro-fibrotic molecules in aortas. Similar results were observed in oxLDL-stimulated mouse primary peritoneal macrophages (MPMs) in vitro. We showed that pretreatment with CTD (2.5, 5. 10 μM) restrained oxLDL-induced inflammatory responses in MPMs by blocking pro-inflammatory NF-κB/p65 signaling pathway. We further demonstrated that CTD inactivated NF-κB via covalent binding to cysteine 179 on IKKβ, a canonical upstream regulator of NF-κB, reducing its phosphorylation and leading to conformational change in the active loop of IKKβ. Our results discover IKKβ as the target of CTD for its anti-inflammatory activity and elucidate a molecular mechanism underlying the anti-atherosclerosis effect of CTD. CTD is a potentially therapeutic candidate for retarding inflammatory atherosclerotic diseases.
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Affiliation(s)
- Zhu-Qi Huang
- Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, 325035, China
- School of Pharmaceutical Sciences, Hangzhou Medical College, Hangzhou, 311399, China
- Department of Cardiology and Medical Research Center, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325035, China
| | - Wu Luo
- Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, 325035, China
- School of Pharmaceutical Sciences, Hangzhou Medical College, Hangzhou, 311399, China
- Department of Cardiology and Medical Research Center, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325035, China
| | - Wei-Xin Li
- Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, 325035, China
| | - Pan Chen
- Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, 325035, China
| | - Zhe Wang
- Department of Pharmacy, the Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325000, China
| | - Rui-Jie Chen
- Department of Pharmacy, the Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325000, China
| | - Yi Wang
- Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, 325035, China
| | - Wei-Jian Huang
- Department of Cardiology and Medical Research Center, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325035, China.
| | - Guang Liang
- Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, 325035, China.
- School of Pharmaceutical Sciences, Hangzhou Medical College, Hangzhou, 311399, China.
- Department of Cardiology and Medical Research Center, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325035, China.
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Tseng KF, Tsai PH, Wang JS, Chen FY, Shen MY. Sesamol Attenuates Renal Inflammation and Arrests Reactive-Oxygen-Species-Mediated IL-1β Secretion via the HO-1-Induced Inhibition of the IKKα/NFκB Pathway In Vivo and In Vitro. Antioxidants (Basel) 2022; 11:antiox11122461. [PMID: 36552668 PMCID: PMC9774643 DOI: 10.3390/antiox11122461] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2022] [Revised: 11/30/2022] [Accepted: 12/10/2022] [Indexed: 12/15/2022] Open
Abstract
Chronic nephritis leads to irreversible renal fibrosis, ultimately leading to chronic kidney disease (CKD) and death. Macrophage infiltration and interleukin 1β (IL-1β) upregulation are involved in inflammation-mediated renal fibrosis and CKD. Sesamol (SM), which is extracted from sesame seeds, has antioxidant and anti-inflammatory properties. We aimed to explore whether SM mitigates macrophage-mediated renal inflammation and its underlying mechanisms. ApoE-/- mice were subjected to 5/6 nephrectomy (5/6 Nx) with or without the oral gavage of SM for eight weeks. Blood and urine samples and all the kidney remnants were collected for analysis. Additionally, THP-1 cells were used to explore the mechanism through which SM attenuates renal inflammation. Compared with the sham group, the 5/6 Nx ApoE-/- mice exhibited a significant increase in the macrophage infiltration of the kidneys (nephritis), upregulation of IL-1β, generation of reactive oxygen species, reduced creatinine clearance, and renal fibrosis. However, the administration of SM significantly alleviated these effects. SM suppressed the H2O2-induced secretion of IL-1β from the THP-1 cells via the heme oxygenase-1-induced inhibition of the IKKα-NF-κB pathway. SM attenuated renal inflammation and arrested macrophage accumulation by inhibiting IKKα, revealing a novel mechanism of the therapeutic effects of SM on renal injury and offering a potential approach to CKD treatment.
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Affiliation(s)
- Kuo-Feng Tseng
- Department of Biological Science and Technology, China Medical University, Taichung 40406, Taiwan
- Graduate Institute of Biomedical Sciences, China Medical University, Taichung 40402, Taiwan
| | - Ping-Hsuan Tsai
- Graduate Institute of Biomedical Sciences, China Medical University, Taichung 40402, Taiwan
| | - Jie-Sian Wang
- Graduate Institute of Biomedical Sciences, China Medical University, Taichung 40402, Taiwan
- Department of Internal Medicine, Division of Nephrology, China Medical University Hospital, Taichung 40402, Taiwan
| | - Fang-Yu Chen
- Graduate Institute of Biomedical Sciences, China Medical University, Taichung 40402, Taiwan
| | - Ming-Yi Shen
- Graduate Institute of Biomedical Sciences, China Medical University, Taichung 40402, Taiwan
- Department of Medical Research, China Medical University Hospital, Taichung 40402, Taiwan
- Department of Nursing, Asia University, Taichung 41354, Taiwan
- Correspondence: or ; Tel.: +886-4-2205-3366 (ext. 5809)
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Pharmacological Inhibition of IKK to Tackle Latency and Hyperinflammation in Chronic HIV-1 Infection. Int J Mol Sci 2022; 23:ijms232315000. [PMID: 36499329 PMCID: PMC9741028 DOI: 10.3390/ijms232315000] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2022] [Revised: 11/25/2022] [Accepted: 11/28/2022] [Indexed: 12/05/2022] Open
Abstract
HIV latent infection may be associated with disrupted viral RNA sensing, interferon (IFN) signaling, and/or IFN stimulating genes (ISG) activation. Here, we evaluated the use of compounds selectively targeting at the inhibitor of nuclear factor-κB (IκB) kinase (IKK) complex subunits and related kinases (TBK1) as a novel pathway to reverse HIV-1 latency in latently infected non-clonal lymphoid and myeloid cell in vitro models. IKK inhibitors (IKKis) triggered up to a 1.8-fold increase in HIV reactivation in both, myeloid and lymphoid cell models. The best-in-class IKKis, targeting TBK-1 (MRT67307) and IKKβ (TCPA-1) respectively, were also able to significantly induce viral reactivation in CD4+ T cells from people living with HIV (PLWH) ex vivo. More importantly, although none of the compounds tested showed antiviral activity, the combination of the distinct IKKis with ART did not affect the latency reactivation nor blockade of HIV infection by ART. Finally, as expected, IKKis did not upregulate cell activation markers in primary lymphocytes and innate immune signaling was blocked, resulting in downregulation of inflammatory cytokines. Overall, our results support a dual role of IKKis as immune modulators being able to tackle the HIV latent reservoir in lymphoid and myeloid cellular models and putatively control the hyperinflammatory responses in chronic HIV-1 infection.
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Lyu ZS, Tang SQ, Xing T, Zhou Y, Lv M, Fu HX, Wang Y, Xu LP, Zhang XH, Lee HY, Kong Y, Huang XJ. The glycolytic enzyme PFKFB3 determines bone marrow endothelial progenitor cell damage after chemotherapy and irradiation. Haematologica 2022; 107:2365-2380. [PMID: 35354250 PMCID: PMC9521251 DOI: 10.3324/haematol.2021.279756] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2021] [Accepted: 03/24/2022] [Indexed: 11/09/2022] Open
Abstract
Bone marrow (BM) endothelial progenitor cell (EPC) damage of unknown mechanism delays the repair of endothelial cells (EC) and recovery of hematopoiesis after chemo-radiotherapy. We found increased levels of the glycolytic enzyme PFKFB3 in the damaged BM EPC of patients with poor graft function, a clinical model of EPC damage-associated poor hematopoiesis after allogeneic hematopoietic stem cell transplantation. Moreover, in vitro the glycolysis inhibitor 3-(3-pyridinyl)- 1-(4-pyridinyl)-2-propen-1-one (3PO) alleviated the damaged BM EPC from patients with poor graft function. Consistently, PFKFB3 overexpression triggered BM EPC damage after 5-fluorouracil treatment and impaired hematopoiesis-supporting ability in vitro. Mechanistically, PFKFB3 facilitated pro-apoptotic transcription factor FOXO3A and expression of its downstream genes, including p21, p27, and FAS, after 5-fluorouracil treatment in vitro. Moreover, PFKFB3 induced activation of NF-κB and expression of its downstream adhesion molecule E-selectin, while it reduced hematopoietic factor SDF-1 expression, which could be rescued by FOXO3A silencing. High expression of PFKFB3 was found in damaged BM EC of murine models of chemo-radiotherapy-induced myelosuppression. Furthermore, a murine model of BM EC-specific PFKFB3 overexpression demonstrated that PFKFB3 aggravated BM EC damage, and impaired the recovery of hematopoiesis after chemotherapy in vivo, effects which could be mitigated by 3PO, indicating a critical role of PFKFB3 in regulating BM EC damage. Clinically, PFKFB3-induced FOXO3A expression and NF-κB activation were confirmed to contribute to the damaged BM EPC of patients with acute leukemia after chemotherapy. 3PO repaired the damaged BM EPC by reducing FOXO3A expression and phospho-NF-κB p65 in patients after chemotherapy. In summary, our results reveal a critical role of PFKFB3 in triggering BM EPC damage and indicate that endothelial-PFKFB3 may be a potential therapeutic target for myelosuppressive injury.
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Affiliation(s)
- Zhong-Shi Lyu
- Peking University People's Hospital, Peking University Institute of Hematology, National Clinical Research Center for Hematologic Disease, Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Collaborative Innovation Center of Hematology, Peking University, Beijing, China; Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing
| | - Shu-Qian Tang
- Peking University People's Hospital, Peking University Institute of Hematology, National Clinical Research Center for Hematologic Disease, Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Collaborative Innovation Center of Hematology, Peking University, Beijing
| | - Tong Xing
- Peking University People's Hospital, Peking University Institute of Hematology, National Clinical Research Center for Hematologic Disease, Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Collaborative Innovation Center of Hematology, Peking University, Beijing, China; Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing
| | - Yang Zhou
- Peking University People's Hospital, Peking University Institute of Hematology, National Clinical Research Center for Hematologic Disease, Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Collaborative Innovation Center of Hematology, Peking University, Beijing
| | - Meng Lv
- Peking University People's Hospital, Peking University Institute of Hematology, National Clinical Research Center for Hematologic Disease, Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Collaborative Innovation Center of Hematology, Peking University, Beijing
| | - Hai-Xia Fu
- Peking University People's Hospital, Peking University Institute of Hematology, National Clinical Research Center for Hematologic Disease, Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Collaborative Innovation Center of Hematology, Peking University, Beijing
| | - Yu Wang
- Peking University People's Hospital, Peking University Institute of Hematology, National Clinical Research Center for Hematologic Disease, Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Collaborative Innovation Center of Hematology, Peking University, Beijing
| | - Lan-Ping Xu
- Peking University People's Hospital, Peking University Institute of Hematology, National Clinical Research Center for Hematologic Disease, Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Collaborative Innovation Center of Hematology, Peking University, Beijing
| | - Xiao-Hui Zhang
- Peking University People's Hospital, Peking University Institute of Hematology, National Clinical Research Center for Hematologic Disease, Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Collaborative Innovation Center of Hematology, Peking University, Beijing
| | - Hsiang-Ying Lee
- Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China; School of Life Sciences, Peking University, Beijing
| | - Yuan Kong
- Peking University People's Hospital, Peking University Institute of Hematology, National Clinical Research Center for Hematologic Disease, Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Collaborative Innovation Center of Hematology, Peking University, Beijing.
| | - Xiao-Jun Huang
- Peking University People's Hospital, Peking University Institute of Hematology, National Clinical Research Center for Hematologic Disease, Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Collaborative Innovation Center of Hematology, Peking University, Beijing, China; Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing.
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Xu Z, Chu M. Advances in Immunosuppressive Agents Based on Signal Pathway. Front Pharmacol 2022; 13:917162. [PMID: 35694243 PMCID: PMC9178660 DOI: 10.3389/fphar.2022.917162] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2022] [Accepted: 05/02/2022] [Indexed: 12/13/2022] Open
Abstract
Immune abnormality involves in various diseases, such as infection, allergic diseases, autoimmune diseases, as well as transplantation. Several signal pathways have been demonstrated to play a central role in the immune response, including JAK/STAT, NF-κB, PI3K/AKT-mTOR, MAPK, and Keap1/Nrf2/ARE pathway, in which multiple targets have been used to develop immunosuppressive agents. In recent years, varieties of immunosuppressive agents have been approved for clinical use, such as the JAK inhibitor tofacitinib and the mTOR inhibitor everolimus, which have shown good therapeutic effects. Additionally, many immunosuppressive agents are still in clinical trials or preclinical studies. In this review, we classified the immunosuppressive agents according to the immunopharmacological mechanisms, and summarized the phase of immunosuppressive agents.
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Affiliation(s)
- Zhiqing Xu
- Department of Immunology, National Health Commission (NHC) Key Laboratory of Medical Immunology (Peking University), School of Basic Medical Sciences, Peking University, Beijing, China
- Department of Pharmacology, Jilin University, Changchun, China
| | - Ming Chu
- Department of Immunology, National Health Commission (NHC) Key Laboratory of Medical Immunology (Peking University), School of Basic Medical Sciences, Peking University, Beijing, China
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Caetano-Silva S, Simbi BH, Marr N, Hibbert A, Allen SP, Pitsillides AA. Restraint upon Embryonic Metatarsal Ex Vivo Growth by Hydrogel Reveals Interaction between Quasi-Static Load and the mTOR Pathway. Int J Mol Sci 2021; 22:ijms222413220. [PMID: 34948015 PMCID: PMC8706285 DOI: 10.3390/ijms222413220] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2021] [Revised: 12/01/2021] [Accepted: 12/02/2021] [Indexed: 11/23/2022] Open
Abstract
Mechanical cues play a vital role in limb skeletal development, yet their influence and underpinning mechanisms in the regulation of endochondral ossification (EO) processes are incompletely defined. Furthermore, interactions between endochondral growth and mechanics and the mTOR/NF-ĸB pathways are yet to be explored. An appreciation of how mechanical cues regulate EO would also clearly be beneficial in the context of fracture healing and bone diseases, where these processes are recapitulated. The study herein addresses the hypothesis that the mTOR/NF-ĸB pathways interact with mechanics to control endochondral growth. To test this, murine embryonic metatarsals were incubated ex vivo in a hydrogel, allowing for the effects of quasi-static loading on longitudinal growth to be assessed. The results showed significant restriction of metatarsal growth under quasi-static loading during a 14-day period and concentration-dependent sensitivity to hydrogel-related restriction. This study also showed that hydrogel-treated metatarsals retain their viability and do not present with increased apoptosis. Metatarsals exhibited reversal of the growth-restriction when co-incubated with mTOR compounds, whilst it was found that these compounds showed no effects under basal culture conditions. Transcriptional changes linked to endochondral growth were assessed and downregulation of Col2 and Acan was observed in hydrogel-treated metatarsi at day 7. Furthermore, cell cycle analyses confirmed the presence of chondrocytes exhibiting S-G2/M arrest. These data indicate that quasi-static load provokes chondrocyte cell cycle arrest, which is partly overcome by mTOR, with a less marked interaction for NF-ĸB regulators.
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Jaffar J, McMillan L, Wilson N, Panousis C, Hardy C, Cho HJ, Symons K, Glaspole I, Westall G, Wong M. Coagulation Factor-XII induces interleukin-6 by primary lung fibroblasts: A role in idiopathic pulmonary fibrosis? Am J Physiol Lung Cell Mol Physiol 2021; 322:L258-L272. [PMID: 34873957 DOI: 10.1152/ajplung.00165.2021] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Background The mechanisms driving idiopathic pulmonary fibrosis (IPF) remain undefined, however it is postulated that coagulation imbalances may play a role. The impact of blood-derived clotting factors, including factor XII (FXII) has not been investigated in the context of IPF. Methods Plasma levels of FXII were measured by ELISA in patients with IPF and age-matched healthy donors. Expression of FXII in human lung tissue was quantified using multiplex immunohistochemistry and western blotting. Mechanistic investigation of FXII activity was assessed in vitro on primary lung fibroblasts using qPCR and specific receptor/FXII inhibition. The functional outcome of FXII on fibroblast migration was examined by high-content image analysis. Findings Compared to 35 healthy donors, plasma levels of FXII were not higher in IPF (n=27, p>0·05). Tissue FXII was elevated in IPF (n=11) and increased numbers of FXII+ cells were found in IPF (n=8) lung tissue compared to non-diseased controls (n=6, p<0·0001). Activated FXII induced IL6 mRNA and IL-6 protein in fibroblasts that was blocked by anti-FXII antibody, CSL312. FXII-induced IL-6 production via PAR-1 and NF-kB. FXII induced migration of fibroblasts in a concentration-dependent manner. Interpretation FXII is normally confined to the circulation but leaks from damaged vessels into the lung interstitium in IPF where it 1) induces IL-6 production and 2) enhances migration of resident fibroblasts, critical events that drive chronic inflammation and therefore, contribute to fibrotic disease progression. Targeting FXII-induced fibroblastic processes in IPF may ameliorate pulmonary fibrosis. Funding National Health and Medical Research Council CRE in Lung Fibrosis and CSL Ltd.
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Affiliation(s)
- Jade Jaffar
- Department of Immunology and Pathology, Monash University, Australia.,Department of Respiratory Medicine, The Alfred Hospital, Australia
| | | | | | | | | | - Hyun Jung Cho
- Biological Optical Microscopy Platform, The University of Melbourne, Australia
| | - Karen Symons
- Department of Respiratory Medicine, The Alfred Hospital, Australia
| | - Ian Glaspole
- Department of Immunology and Pathology, Monash University, Australia.,Department of Respiratory Medicine, The Alfred Hospital, Australia
| | - Glen Westall
- Department of Immunology and Pathology, Monash University, Australia.,Department of Respiratory Medicine, The Alfred Hospital, Australia
| | - Mae Wong
- CSL Limited, Parkville, Victoria, Australia
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Fu W, Hettinghouse A, Chen Y, Hu W, Ding X, Chen M, Ding Y, Mundra J, Song W, Liu R, Yi YS, Attur M, Samuels J, Strauss E, Leucht P, Schwarzkopf R, Liu CJ. 14-3-3 epsilon is an intracellular component of TNFR2 receptor complex and its activation protects against osteoarthritis. Ann Rheum Dis 2021; 80:1615-1627. [PMID: 34226187 PMCID: PMC8595573 DOI: 10.1136/annrheumdis-2021-220000] [Citation(s) in RCA: 50] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2021] [Accepted: 06/22/2021] [Indexed: 01/28/2023]
Abstract
OBJECTIVES Osteoarthritis (OA) is the most common joint disease; however, the indeterminate nature of mechanisms by which OA develops has restrained advancement of therapeutic targets. TNF signalling has been implicated in the pathogenesis of OA. TNFR1 primarily mediates inflammation, whereas emerging evidences demonstrate that TNFR2 plays an anti-inflammatory and protective role in several diseases and conditions. This study aims to decipher TNFR2 signalling in chondrocytes and OA. METHODS Biochemical copurification and proteomics screen were performed to isolate the intracellular cofactors of TNFR2 complex. Bulk and single cell RNA-seq were employed to determine 14-3-3 epsilon (14-3-3ε) expression in human normal and OA cartilage. Transcription factor activity screen was used to isolate the transcription factors downstream of TNFR2/14-3-3ε. Various cell-based assays and genetically modified mice with naturally occurring and surgically induced OA were performed to examine the importance of this pathway in chondrocytes and OA. RESULTS Signalling molecule 14-3-3ε was identified as an intracellular component of TNFR2 complexes in chondrocytes in response to progranulin (PGRN), a growth factor known to protect against OA primarily through activating TNFR2. 14-3-3ε was downregulated in OA and its deficiency deteriorated OA. 14-3-3ε was required for PGRN regulation of chondrocyte metabolism. In addition, both global and chondrocyte-specific deletion of 14-3-3ε largely abolished PGRN's therapeutic effects against OA. Furthermore, PGRN/TNFR2/14-3-3ε signalled through activating extracellular signal-regulated kinase (ERK)-dependent Elk-1 while suppressing nuclear factor kappa B (NF-κB) in chondrocytes. CONCLUSIONS This study identifies 14-3-3ε as an inducible component of TNFR2 receptor complex in response to PGRN in chondrocytes and presents a previously unrecognised TNFR2 pathway in the pathogenesis of OA.
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Affiliation(s)
- Wenyu Fu
- Department of Orthopaedic Surgery, NYU Grossman School of Medicine, New York, New York, USA
| | - Aubryanna Hettinghouse
- Department of Orthopaedic Surgery, NYU Grossman School of Medicine, New York, New York, USA
| | - Yujianan Chen
- Department of Orthopaedic Surgery, NYU Grossman School of Medicine, New York, New York, USA
| | - Wenhuo Hu
- Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center; Marie-Josée and Henry R. Kravis Center for Molecular Oncology, Memorial Sloan-Kettering Cancer Center, New York, New York, USA
| | - Xiang Ding
- Department of Orthopaedic Surgery, NYU Grossman School of Medicine, New York, New York, USA
| | - Meng Chen
- Department of Orthopaedic Surgery, NYU Grossman School of Medicine, New York, New York, USA
| | - Yuanjing Ding
- Department of Orthopaedic Surgery, NYU Grossman School of Medicine, New York, New York, USA
| | - Jyoti Mundra
- Department of Orthopaedic Surgery, NYU Grossman School of Medicine, New York, New York, USA
| | - Wenhao Song
- Department of Orthopaedic Surgery, NYU Grossman School of Medicine, New York, New York, USA
| | - Ronghan Liu
- Department of Orthopaedic Surgery, NYU Grossman School of Medicine, New York, New York, USA
| | - Young-Su Yi
- Department of Orthopaedic Surgery, NYU Grossman School of Medicine, New York, New York, USA
| | - Mukundan Attur
- Department of Medicine, Division of Rheumatology, NYU Grossman School of Medicine, New York, New York, USA
| | - Jonathan Samuels
- Department of Medicine, Division of Rheumatology, NYU Grossman School of Medicine, New York, New York, USA
| | - Eric Strauss
- Department of Orthopaedic Surgery, NYU Grossman School of Medicine, New York, New York, USA
| | - Philipp Leucht
- Department of Orthopaedic Surgery, NYU Grossman School of Medicine, New York, New York, USA
- Department of Cell Biology, NYU Grossman School of Medicine, New York, New York, USA
| | - Ran Schwarzkopf
- Department of Orthopaedic Surgery, NYU Grossman School of Medicine, New York, New York, USA
| | - Chuan-Ju Liu
- Department of Orthopaedic Surgery, NYU Grossman School of Medicine, New York, New York, USA
- Department of Cell Biology, NYU Grossman School of Medicine, New York, New York, USA
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12
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Gong AY, Wang Y, Li M, Zhang XT, Deng S, Chen JM, Lu E, Mathy NW, Martins GA, Strauss-Soukup JK, Chen XM. LncRNA XR_001779380 Primes Epithelial Cells for IFN-γ-Mediated Gene Transcription and Facilitates Age-Dependent Intestinal Antimicrobial Defense. mBio 2021; 12:e0212721. [PMID: 34488445 PMCID: PMC8546593 DOI: 10.1128/mbio.02127-21] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2021] [Accepted: 08/05/2021] [Indexed: 12/20/2022] Open
Abstract
Interferon (IFN) signaling is key to mucosal immunity in the gastrointestinal tract, but cellular regulatory elements that determine interferon gamma (IFN-γ)-mediated antimicrobial defense in intestinal epithelial cells are not fully understood. We report here that a long noncoding RNA (lncRNA), GenBank accession no. XR_001779380, was increased in abundance in murine intestinal epithelial cells following infection by Cryptosporidium, an important opportunistic pathogen in AIDS patients and a common cause of diarrhea in young children. Expression of XR_001779380 in infected intestinal epithelial cells was triggered by TLR4/NF-κB/Cdc42 signaling and epithelial-specific transcription factor Elf3. XR_001779380 primed epithelial cells for IFN-γ-mediated gene transcription through facilitating Stat1/Swi/Snf-associated chromatin remodeling. Interactions between XR_001779380 and Prdm1, which is expressed in neonatal but not adult intestinal epithelium, attenuated Stat1/Swi/Snf-associated chromatin remodeling induced by IFN-γ, contributing to suppression of IFN-γ-mediated epithelial defense in neonatal intestine. Our data demonstrate that XR_001779380 is an important regulator in IFN-γ-mediated gene transcription and age-associated intestinal epithelial antimicrobial defense. IMPORTANCE Epithelial cells along the mucosal surface provide the front line of defense against luminal pathogen infection in the gastrointestinal tract. These epithelial cells represent an integral component of a highly regulated communication network that can transmit essential signals to cells in the underlying intestinal mucosa that, in turn, serve as targets of mucosal immune mediators. LncRNAs are recently identified long noncoding transcripts that can regulate gene transcription through their interactions with other effect molecules. In this study, we demonstrated that lncRNA XR_001779380 was upregulated in murine intestinal epithelial cells following infection by a mucosal protozoan parasite Cryptosporidium. Expression of XR_001779380 in infected cells primed host epithelial cells for IFN-γ-mediated gene transcription, relevant to age-dependent intestinal antimicrobial defense. Our data provide new mechanistic insights into how intestinal epithelial cells orchestrate intestinal mucosal defense against microbial infection.
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Affiliation(s)
- Ai-Yu Gong
- Department of Microbial Pathogens and Immunity, Rush University Medical Center, Chicago, Illinois, USA
- Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, Nebraska, USA
| | - Yang Wang
- Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, Nebraska, USA
| | - Min Li
- Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, Nebraska, USA
| | - Xin-Tian Zhang
- Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, Nebraska, USA
| | - Silu Deng
- Department of Microbial Pathogens and Immunity, Rush University Medical Center, Chicago, Illinois, USA
- Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, Nebraska, USA
| | - Jessie M. Chen
- Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, Nebraska, USA
| | - Eugene Lu
- Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, Nebraska, USA
| | - Nicholas W. Mathy
- Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, Nebraska, USA
| | - Gislaine A. Martins
- Deptartments of Medicine and Biomedical Sciences, Research Division of Immunology Cedars-Sinai Medical Center, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA
| | | | - Xian-Ming Chen
- Department of Microbial Pathogens and Immunity, Rush University Medical Center, Chicago, Illinois, USA
- Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, Nebraska, USA
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13
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Ye M, Wang C, Zhu J, Chen M, Wang S, Li M, Lu Y, Xiao P, Zhou M, Li X, Zhou R. An NF-κB-responsive long noncoding RNA, PINT, regulates TNF-α gene transcription by scaffolding p65 and EZH2. FASEB J 2021; 35:e21667. [PMID: 34405442 DOI: 10.1096/fj.202002263r] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2020] [Revised: 03/25/2021] [Accepted: 04/30/2021] [Indexed: 12/12/2022]
Abstract
Long noncoding RNAs (lncRNAs) are central regulators of the inflammatory response and play an important role in inflammatory diseases. PINT has been reported to be involved in embryonic development and tumorigenesis. However, the potential functions of PINT in the innate immune system are largely unknown. Here, we revealed the transcriptional regulation of inflammatory genes by PINT, whose expression is primarily dependent on the NF-κB signaling pathway in human and mouse macrophage and intestinal epithelial cell lines. Functionally, PINT selectively regulates the expression of TNF-α in basal and LPS-stimulated cells. Mechanistically, PINT acts as a modular scaffold of p65 and EZH2 to coordinate their localization and specify their binding to the target genes. Further, a high expression level of PINT was detected in intestinal mucosal tissues from patients with ulcerative colitis (UC). Together, these findings demonstrate that PINT acts as an activator of inflammatory responses, highlighting the importance of this lncRNA as a potential therapeutic target in infectious diseases and inflammatory diseases.
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Affiliation(s)
- Mengling Ye
- Hubei Province Key Laboratory of Allergy and Immunology, Wuhan University School of Basic Medical Sciences, Wuhan University, Wuhan, P. R. China
- Department of Research, Tumor Hospital of Guangxi Medical University, Nanning, P. R. China
| | - Cheng Wang
- Hubei Province Key Laboratory of Allergy and Immunology, Wuhan University School of Basic Medical Sciences, Wuhan University, Wuhan, P. R. China
| | - Jie Zhu
- Hubei Province Key Laboratory of Allergy and Immunology, Wuhan University School of Basic Medical Sciences, Wuhan University, Wuhan, P. R. China
| | - Mingkai Chen
- Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan, P. R. China
| | - Shuhong Wang
- Hubei Province Key Laboratory of Allergy and Immunology, Wuhan University School of Basic Medical Sciences, Wuhan University, Wuhan, P. R. China
| | - Mingxuan Li
- Hubei Province Key Laboratory of Allergy and Immunology, Wuhan University School of Basic Medical Sciences, Wuhan University, Wuhan, P. R. China
| | - Yajing Lu
- Department of Endocrinology, Institute of geriatric medicine, Liyuan Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P. R. China
| | - Pingping Xiao
- Hubei Province Key Laboratory of Allergy and Immunology, Wuhan University School of Basic Medical Sciences, Wuhan University, Wuhan, P. R. China
| | - Mengsi Zhou
- Hubei Province Key Laboratory of Allergy and Immunology, Wuhan University School of Basic Medical Sciences, Wuhan University, Wuhan, P. R. China
| | - Xiaoqing Li
- Center for Stem Cell Research and Application, Union Hospital, Tongji Medical School, Huazhong University of Science and Technology, Wuhan, P. R. China
| | - Rui Zhou
- Hubei Province Key Laboratory of Allergy and Immunology, Wuhan University School of Basic Medical Sciences, Wuhan University, Wuhan, P. R. China
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14
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Polycomb Repressive Complex 2 Regulates Genes Necessary for Intestinal Microfold Cell (M Cell) Development. Cell Mol Gastroenterol Hepatol 2021; 12:873-889. [PMID: 34058415 PMCID: PMC8346665 DOI: 10.1016/j.jcmgh.2021.05.014] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/21/2020] [Revised: 05/21/2021] [Accepted: 05/21/2021] [Indexed: 01/22/2023]
Abstract
BACKGROUND & AIMS Microfold cells (M cells) are immunosurveillance epithelial cells located in the Peyer's patches (PPs) in the intestine and are responsible for monitoring and transcytosis of antigens, microorganisms, and pathogens. Mature M cells use the receptor glycoprotein 2 (GP2) to aid in transcytosis. Recent studies have shown transcription factors, Spi-B and SRY-Box Transcription Factor 8 (Sox8). are necessary for M-cell differentiation, but not sufficient. An exhaustive set of factors sufficient for differentiation and development of a mature GP2+ M cell remains elusive. Our aim was to understand the role of polycomb repressive complex 2 (PRC2) as an epigenetic regulator of M-cell development. Estrogen-related-receptor γ (Esrrg), identified as a PRC2-regulated gene, was studied in depth, in addition to its relationship with Spi-B and Sox8. METHODS Comparative chromatin immunoprecipitation and global run-on sequencing analysis of mouse intestinal organoids were performed in stem condition, enterocyte conditions, and receptor activator of nuclear factor κ B ligand-induced M-cell condition. Esrrg, which was identified as one of the PRC2-regulated transcription factors, was studied in wild-type mice and knocked out in intestinal organoids using guide RNA's. Sox8 null mice were used to study Esrrg and its relation to Sox8. RESULTS chromatin immunoprecipitation and global run-on sequencing analysis showed 12 novel PRC2 regulated transcription factors, PRC2-regulated Esrrg is a novel M-cell-specific transcription factor acting on a receptor activator of nuclear factor κB ligand-receptor activator of nuclear factor κB-induced nuclear factor-κB pathway, upstream of Sox8, and necessary but not sufficient for a mature M-cell marker of Gp2 expression. CONCLUSIONS PRC2 regulates a significant set of genes in M cells including Esrrg, which is critical for M-cell development and differentiation. Loss of Esrrg led to an immature M-cell phenotype lacking in Sox8 and Gp2 expression. Transcript profiling: the data have been deposited in the NCBI Gene Expression Omnibus database (GSE157629).
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15
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Intra-Articular Administration of Cramp into Mouse Knee Joint Exacerbates Experimental Osteoarthritis Progression. Int J Mol Sci 2021; 22:ijms22073429. [PMID: 33810460 PMCID: PMC8037447 DOI: 10.3390/ijms22073429] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2021] [Revised: 03/23/2021] [Accepted: 03/24/2021] [Indexed: 12/11/2022] Open
Abstract
Osteoarthritis (OA) is the most common type of arthritis and is associated with wear and tear, aging, and inflammation. Previous studies revealed that several antimicrobial peptides are up-regulated in the knee synovium of patients with OA or rheumatoid arthritis. Here, we investigated the functional effects of cathelicidin-related antimicrobial peptide (Cramp) on OA pathogenesis. We found that Cramp is highly induced by IL-1β via the NF-κB signaling pathway in mouse primary chondrocytes. Elevated Cramp was also detected in the cartilage and synovium of mice suffering from OA cartilage destruction. The treatment of chondrocytes with Cramp stimulated the expression of catabolic factors, and the knockdown of Cramp by small interfering RNA reduced chondrocyte catabolism mediated by IL-1β. Moreover, intra-articular injection of Cramp into mouse knee joints at a low dose accelerated traumatic OA progression. At high doses, Cramp affected meniscal ossification and tears, leading to cartilage degeneration. These findings demonstrate that Cramp is associated with OA pathophysiology.
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16
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He B, Zhou W, Rui Y, Liu L, Chen B, Su X. MicroRNA-574-5p Attenuates Acute Respiratory Distress Syndrome by Targeting HMGB1. Am J Respir Cell Mol Biol 2021; 64:196-207. [PMID: 33202146 PMCID: PMC7874400 DOI: 10.1165/rcmb.2020-0112oc] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Acute respiratory distress syndrome (ARDS) is a critical condition with high mortality. HMGB1 (high-mobility group protein B1) is one of the key proinflammatory factors in the ARDS “inflammatory storm.” According to previous studies, some microRNAs (miRNAs) play important roles in this process. We aimed to determine the contributing miRNAs targeting the expression and release of HMGB1. miRNA expression in the peripheral blood of patients with ARDS was measured by miRNA microarray. miRNAs targeting HMGB1 were screened and explored for further study. In LPS-induced cell and mouse ARDS models, we explored the effect of this miRNA on the expression and secretion of HMGB1 by Western blot, real-time qPCR, and ELISA. The effects of this miRNA on the NF-κB signaling pathway, proinflammatory cytokines, and NLRP3 (nod-like receptor protein 3) inflammasome were detected by Western blot and real-time qPCR. In ARDS models, microRNA-574-5p (miR-574-5p) expression could be induced by the TLR4/NF-κB pathway upon LPS stimulation. It could suppress the inflammatory response by targeting HMGB1. Enforcing the expression of miR-574-5p or HMGB1 siRNA silencing inhibits the activation of NF-κB signaling pathway and the NLRP3 inflammasome. Moreover, overexpression of HMGB1 reversed the antiinflammatory effect of miR-574-5p. In ARDS mice, overexpression of miR-574-5p suppresses alveolar leukocytes infiltration, interstitial edema, protein effusion, and inflammation. This study demonstrated that miR-574-5p provided negative feedback to LPS-induced inflammation and relieved ARDS. It may provide new therapeutic strategies for ARDS.
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Affiliation(s)
- Binchan He
- Department of Respiratory and Critical Care Medicine, Jinling Hospital, Medical School of Nanjing University, Nanjing, China.,The Second Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, China; and
| | - Wei Zhou
- Department of Respiratory and Critical Care Medicine, Jinling Hospital, Medical School of Nanjing University, Nanjing, China
| | - Yuwen Rui
- Department of Respiratory and Critical Care Medicine, Jinling Hospital, Medical School of Nanjing University, Nanjing, China
| | - Lulu Liu
- Department of Respiratory and Critical Care Medicine, Jinling Hospital, Medical School of Nanjing University, Nanjing, China
| | - Bilin Chen
- Department of Respiratory and Critical Care Medicine, Jinling Hospital, Medical School of Nanjing University, Nanjing, China
| | - Xin Su
- Department of Respiratory and Critical Care Medicine, Jinling Hospital, Medical School of Nanjing University, Nanjing, China.,Department of Respiratory and Critical Care Medicine, Jinling Hospital, The First School of Clinical Medicine, Southern Medical University, Guangzhou, Guangdong, China
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17
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Local administration of p-coumaric acid decreases lipopolysaccharide-induced acute lung injury in mice: In vitro and in silico studies. Eur J Pharmacol 2021; 897:173929. [PMID: 33561444 DOI: 10.1016/j.ejphar.2021.173929] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2020] [Revised: 01/22/2021] [Accepted: 01/29/2021] [Indexed: 01/21/2023]
Abstract
Acute lung injury (ALI) remains to cause a high rate of mortality in critically ill patients. It is known that inflammation is a key factor in the pathogenesis of lipopolysaccharide (LPS)-induced ALI, which makes it a relevant approach to the treatment of ALI. In this study, we evaluated the potential of nasally instilled p-coumaric acid to prevent LPS-induced ALI in mice, by evaluating its effects on cellular and molecular targets involved in inflammatory response via in vitro and in silico approaches. Our results demonstrated that p-coumaric acid reduced both neutrophil accumulation and pro-inflammatory cytokine abundance, and simultaneously increased IL-10 production at the site of inflammation, potentially contributing to protection against LPS-induced ALI in mice. In the in vitro experiments, we observed inhibitory effects of p-coumaric acid against IL-6 and IL-8 production in stimulated A549 cells, as well as reactive oxygen species generation by neutrophils. In addition, p-coumaric acid treatment decreased neutrophil adhesion on the TNF-α-stimulated endothelial cells. According to the in silico predictions, p-coumaric acid reached stable interactions with both the ATP-binding site of IKKβ as well as the regions within LFA-1, critical for interaction with ICAM-1, thereby suppressing the production of proinflammatory mediators and hindering the neutrophil infiltration, respectively. Collectively, these findings indicate that p-coumaric acid is a promising anti-inflammatory agent that can be used for developing a pharmaceutical drug for the treatment of ALI and other inflammatory disorders.
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18
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Spivack K, Muzzelo C, Hall M, Warga E, Neely C, Slepian H, Cunningham A, Tucker M, Elmer J. Enhancement of transgene expression by the β-catenin inhibitor iCRT14. Plasmid 2021; 114:102556. [PMID: 33472046 DOI: 10.1016/j.plasmid.2021.102556] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2020] [Revised: 10/08/2020] [Accepted: 10/14/2020] [Indexed: 11/30/2022]
Abstract
The innate immune response is an essential defense mechanism that allows cells to detect pathogen-associated molecular patterns (PAMPs) like endotoxin or cytosolic DNA and then induce the expression of defensive genes that restrict the replication of viruses and other pathogens. However, the therapeutic DNA used in some gene therapy treatments can also trigger the innate immune response, which activates host cell genes that may inhibit transgene expression. The goal of this study was to enhance transgene expression by inhibiting key components of the innate immune response with small molecule inhibitors (iCRT14, curcumin, Amlexanox, H-151, SC-514, & VX-702). Most of the inhibitors significantly increased transgene (luciferase) expression at least 2-fold, but the β-catenin/TCF4 inhibitor iCRT14 showed the highest enhancement (16 to 35-fold) in multiple cell lines (PC-3, MCF7, & MB49) without significantly decreasing cellular proliferation. Alternatively, cloning a β-catenin/TCF4 binding motif (TCAAAG) into the EF1α promoter also enhanced transgene expression up to 8-fold. To further investigate the role of β-catenin/TCF4 in transgene expression, mRNA-sequencing experiments were conducted to identify host cell genes that were upregulated following transfection with PEI but down-regulated after the addition of iCRT14. As expected, transfection with plasmid DNA activated the innate immune response and upregulated hundreds (687) of defensive genes, but only 7 of those genes were down-regulated in the presence of iCRT14 (e.g., PTGS2 & PLA1A). Altogether, these results show that transgene expression can be enhanced by inhibiting the innate immune response with SMIs like iCRT14, which inhibits β-catenin/TCF4 to prevent the expression of specific host cell genes.
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Affiliation(s)
- Kyle Spivack
- Villanova University, Department of Chemical & Biological Engineering, United States
| | - Christine Muzzelo
- Villanova University, Department of Chemical & Biological Engineering, United States
| | - Matthew Hall
- Villanova University, Department of Chemical & Biological Engineering, United States
| | - Eric Warga
- Villanova University, Department of Chemical & Biological Engineering, United States
| | - Christopher Neely
- Villanova University, Department of Chemical & Biological Engineering, United States
| | - Holly Slepian
- Villanova University, Department of Chemical & Biological Engineering, United States
| | - Alyssa Cunningham
- Villanova University, Department of Chemical & Biological Engineering, United States
| | - Matthew Tucker
- Villanova University, Department of Chemical & Biological Engineering, United States
| | - Jacob Elmer
- Villanova University, Department of Chemical & Biological Engineering, United States.
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19
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LncRNA HOTAIR regulates glucose transporter Glut1 expression and glucose uptake in macrophages during inflammation. Sci Rep 2021; 11:232. [PMID: 33420270 PMCID: PMC7794310 DOI: 10.1038/s41598-020-80291-4] [Citation(s) in RCA: 56] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2020] [Accepted: 12/17/2020] [Indexed: 12/11/2022] Open
Abstract
Inflammation plays central roles in the immune response. Inflammatory response normally requires higher energy and therefore is associated with glucose metabolism. Our recent study demonstrates that lncRNA HOTAIR plays key roles in NF-kB activation, cytokine expression, and inflammation. Here, we investigated if HOTAIR plays any role in the regulation of glucose metabolism in immune cells during inflammation. Our results demonstrate that LPS-induced inflammation induces the expression of glucose transporter isoform 1 (Glut1) which controls the glucose uptake in macrophages. LPS-induced Glut1 expression is regulated via NF-kB activation. Importantly, siRNA-mediated knockdown of HOTAIR suppressed the LPS-induced expression of Glut1 suggesting key roles of HOTAIR in LPS-induced Glut1 expression in macrophage. HOTAIR induces NF-kB activation, which in turn increases Glut1 expression in response to LPS. We also found that HOTAIR regulates glucose uptake in macrophages during LPS-induced inflammation and its knockdown decreases LPS-induced increased glucose uptake. HOTAIR also regulates other upstream regulators of glucose metabolism such as PTEN and HIF1α, suggesting its multimodal functions in glucose metabolism. Overall, our study demonstrated that lncRNA HOTAIR plays key roles in LPS-induced Glut1 expression and glucose uptake by activating NF-kB and hence HOTAIR regulates metabolic programming in immune cells potentially to meet the energy needs during the immune response.
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20
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Gu L, Zhu Y, Lin X, Lu B, Zhou X, Zhou F, Zhao Q, Prochownik EV, Li Y. The IKKβ-USP30-ACLY Axis Controls Lipogenesis and Tumorigenesis. Hepatology 2021; 73:160-174. [PMID: 32221968 DOI: 10.1002/hep.31249] [Citation(s) in RCA: 83] [Impact Index Per Article: 20.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/14/2019] [Revised: 01/24/2020] [Accepted: 03/14/2020] [Indexed: 12/14/2022]
Abstract
BACKGROUND AND AIMS Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death that develops as a consequence of obesity, cirrhosis, and chronic hepatitis. However, the pathways along which these changes occur remain incompletely understood. APPROACH AND RESULTS In this study, we show that the deubiquitinase USP30 is abundant in HCCs that arise in mice maintained on high-fat diets. IKKβ phosphorylated and stabilized USP30, which promoted USP30 to deubiquitinate ATP citrate lyase (ACLY) and fatty acid synthase (FASN). IKKβ also directly phosphorylated ACLY and facilitated the interaction between USP30 and ACLY and the latter's deubiquitination. In HCCs arising in DEN/CCl4 -treated mice, USP30 deletion attenuated lipogenesis, inflammation, and tumorigenesis regardless of diet. The combination of ACLY inhibitor and programmed death ligand 1 antibody largely suppressed chemical-induced hepatocarcinogenesis. The IKKβ-USP30-ACLY axis was also found to be up-regulated in human HCCs. CONCLUSIONS This study identifies an IKKβ-USP30-ACLY axis that plays an essential and wide-spread role in tumor metabolism and may be a potential therapeutic target in HCC.
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Affiliation(s)
- Li Gu
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, China.,Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University, Wuhan, China
| | - Yahui Zhu
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, China.,Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University, Wuhan, China
| | - Xi Lin
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, China.,Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University, Wuhan, China
| | - Bingjun Lu
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, China.,Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University, Wuhan, China
| | - Xinyi Zhou
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, China.,Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University, Wuhan, China
| | - Feng Zhou
- Department of Gastroenterology, Zhongnan Hospital of Wuhan University School of Medicine, Wuhan, China.,Hubei Clinical Center and Key Laboratory for Intestinal and Colorectal Diseases, Wuhan, China
| | - Qiu Zhao
- Department of Gastroenterology, Zhongnan Hospital of Wuhan University School of Medicine, Wuhan, China.,Hubei Clinical Center and Key Laboratory for Intestinal and Colorectal Diseases, Wuhan, China
| | - Edward V Prochownik
- Division of Hematology/Oncology, Children's Hospital of Pittsburgh of UPMC, The Department of Microbiology and Molecular Genetics, The Pittsburgh Liver Research Center and The Hillman Cancer Center of UPMC, The University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania
| | - Youjun Li
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan, China.,Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University, Wuhan, China
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Wang Z, Yang J, Yang Y, Pu X, Zhao J, Zhang N. Targeted and Combined TPCA-1-Gold Nanocage Therapy for In Vivo Treatment of Inflammatory Arthritis. AAPS PharmSciTech 2020; 21:298. [PMID: 33140225 DOI: 10.1208/s12249-020-01856-0] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2020] [Accepted: 10/16/2020] [Indexed: 12/22/2022] Open
Abstract
Rheumatoid arthritis (RA) is an autoimmune disease that is currently incurable. Inhibition of inflammation can prevent the deterioration of RA. 2-[(Aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1) suppresses inflammation via the inhibition of nuclear factor-κ (NF-κB) signaling pathway. Gold-based therapies have been used to treat inflammatory arthritis since the 1940s. Hyaluronic acid (HA) is a targeting ligand for CD44 receptors overexpressed on activated macrophages. Therefore, a combined therapy based on TPCA-1, gold, and HA was explored for the treatment of RA in this study. We used gold nanocages (AuNCs) to load TPCA-1 and modified the TPCA-1 (T) loaded AuNCs with HA and peptides (P) to construct an anti-inflammatory nanoparticle (HA-AuNCs/T/P). An adjuvant-induced arthritis (AIA) mice model was used to investigate the in vivo anti-inflammatory efficacy of HA-AuNCs/T/P. In vivo distribution results showed that HA-AuNCs/T/P had increased and prolonged accumulation at the inflamed paws of AIA mice. Treatment by the HA-AuNCs/T/P suppressed joint swelling and alleviated cartilage and bone damage. By loading to HA-AuNCs/T/P, the effective concentration of TPCA-1 was greatly reduced from 20 to 0.016 mg/kg mice. This study demonstrated that HA-AuNCs/T/P could effectively suppress inflammation and alleviate the symptoms of AIA mice, suggesting a great potential of HA-AuNCs/T/P for the treatment of RA.
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Dumler JS, Lichay M, Chen WH, Rennoll-Bankert KE, Park JH. Anaplasma phagocytophilum Activates NF-κB Signaling via Redundant Pathways. Front Public Health 2020; 8:558283. [PMID: 33194960 PMCID: PMC7661751 DOI: 10.3389/fpubh.2020.558283] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2020] [Accepted: 09/22/2020] [Indexed: 11/28/2022] Open
Abstract
Anaplasma phagocytophilum subverts neutrophil function permitting intracellular survival, propagation and transmission. Sustained pro-inflammatory response, recruitment of new host cells for population expansion, and delayed apoptosis are associated with prolonged nuclear presence of NF-κB. We investigated NF-κB signaling and transcriptional activity with A. phagocytophilum infection using inhibitors of NF-κB signaling pathways, and through silencing of signaling pathway genes. How inhibitors or silencing affected A. phagocytophilum growth, inflammatory response (transcription of the κB-enhanced genes CXCL8 and MMP9), and NF-κB signaling pathway gene expression were tested. Among A. phagocytophilum-infected HL-60 cells, nuclear NF-κB p50, p65, and p52 were detected by immunoblots or iTRAQ proteomics. A. phagocytophilum growth was affected most by the IKKαβ inhibitor wedelolactone (reductions of 96 to 99%) as compared with SC-514 that selectively inhibits IKKβ, illustrating a role for the non-canonical pathway. Wedelolactone inhibited transcription of both CXCL8 (p = 0.001) and MMP9 (p = 0.002) in infected cells. Compared to uninfected THP-1 cells, A. phagocytophilum infection led to >2-fold down regulation of 64 of 92 NF-κB signaling pathway genes, and >2-fold increased expression in only 4. Wedelolactone and SC-514 reversed downregulation in all 64 and 45, respectively, of the genes down-regulated by infection, but decreased expression in 1 gene with SC-514 only. Silencing of 20 NF-κB signal pathway genes increased bacterial growth in 12 (IRAK1, MAP3K1, NFKB1B, MAP3K7, TICAM2, TLR3, TRADD, TRAF3, CHUK, IRAK2, LTBR, and MALT1). Most findings support canonical pathway activation; however, the presence of NFKB2 in infected cell nuclei, selective non-canonical pathway inhibitors that dampen CXCL8 and MMP9 transcription with infection, upregulation of non-canonical pathway target genes CCL13 and CCL19, enhanced bacterial growth with TRAF3 and LTBR silencing provide evidence for non-canonical pathway signaling. Whether this impacts distinct inflammatory processes that underlie disease, and whether and how A. phagocytophilum subverts NF-κB signaling via these pathways, need to be investigated.
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Affiliation(s)
- J Stephen Dumler
- Department of Pathology, F. Edward Hébert School of Medicine, Uniformed Services University for the Health Sciences, Bethesda, MD, United States.,Division of Medical Microbiology, Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD, United States
| | - Marguerite Lichay
- Division of Medical Microbiology, Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD, United States
| | - Wan-Hsin Chen
- Division of Medical Microbiology, Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD, United States
| | - Kristen E Rennoll-Bankert
- Division of Medical Microbiology, Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD, United States
| | - Jin-Ho Park
- Division of Medical Microbiology, Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD, United States
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23
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Ma L, Zhen J, Sorisky A. Regulators of thymic stromal lymphopoietin production by human adipocytes. Cytokine 2020; 136:155284. [PMID: 32950025 DOI: 10.1016/j.cyto.2020.155284] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2020] [Revised: 09/03/2020] [Accepted: 09/04/2020] [Indexed: 01/07/2023]
Abstract
Thymic stromal lymphopoietin (TSLP) is a cytokine that is known to play a role in inflammatory conditions, especially asthma and atopic dermatitis. It is also recognized to be expressed in human adipose tissue. TSLP production from human adipocytes is stimulated by thyroid-stimulating hormone (TSH). This study aimed to identify TSH-dependent signaling routes that regulate TSLP, to determine if TSLP production is stimulated by other cytokines (IL-1β and TNF-α), and to examine if TSLP production depends on the adipose depot. Human abdominal differentiated adipocytes were stimulated with TSH, IL-1β, or TNF-α. Activation of cell signaling kinases was measured by phospho-immunoblot analysis, and TSLP in medium was assessed by ELISA. TSLP responses from abdominal subcutaneous and omental adipocytes were compared. TSH-stimulated TSLP secretion from subcutaneous adipocytes was enhanced by IBMX (raises cAMP levels) and was blocked by UO126 (inhibitor of MEK1/2-ERK1/2). TSLP secretion was stimulated by IL-1β and by TNF-α. SC-514 (inhibitor of IKKβ/NF-κB) only reduced the former. There was no effect of SB203580 (p38 MAPK inhibitor) or SP600125 (JNK inhibitor) on the stimulation by TSH, IL-1β or TNF-α. Interferon-γ inhibited TSLP responses to TSH, IL-1β, and TNF-α; IL-4 only blocked the response to TNFα. Intra-abdominal omental adipocytes also release TSLP in response to TSH, IL-1β, and TNF-α. We conclude TSLP is produced by human differentiated adipocytes derived from subcutaneous or omental depots in response to a variety of agonists. Further studies will be needed to understand what role it may play in adipose biology.
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Affiliation(s)
- Loretta Ma
- Chronic Disease Program, Ottawa Hospital Research Institute, 501 Smyth Rd, Ottawa, Ontario K1H 8L6, Canada
| | - Jamie Zhen
- Chronic Disease Program, Ottawa Hospital Research Institute, 501 Smyth Rd, Ottawa, Ontario K1H 8L6, Canada
| | - Alexander Sorisky
- Chronic Disease Program, Ottawa Hospital Research Institute, 501 Smyth Rd, Ottawa, Ontario K1H 8L6, Canada; Department of Medicine and Biochemistry, Microbiology & Immunology, University of Ottawa, 501 Smyth Rd, Ottawa, Ontario K1H 8L6, Canada.
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24
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Sehnert B, Burkhardt H, Dübel S, Voll RE. Cell-Type Targeted NF-kappaB Inhibition for the Treatment of Inflammatory Diseases. Cells 2020; 9:E1627. [PMID: 32640727 PMCID: PMC7407293 DOI: 10.3390/cells9071627] [Citation(s) in RCA: 37] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2020] [Revised: 06/30/2020] [Accepted: 07/02/2020] [Indexed: 12/29/2022] Open
Abstract
Deregulated NF-k activation is not only involved in cancer but also contributes to the pathogenesis of chronic inflammatory diseases like rheumatoid arthritis (RA) and multiple sclerosis (MS). Ideally, therapeutic NF-KappaB inhibition should only take place in those cell types that are involved in disease pathogenesis to maintain physiological cell functions in all other cells. In contrast, unselective NF-kappaB inhibition in all cells results in multiple adverse effects, a major hindrance in drug development. Hitherto, various substances exist to inhibit different steps of NF-kappaB signaling. However, powerful tools for cell-type specific NF-kappaB inhibition are not yet established. Here, we review the role of NF-kappaB in inflammatory diseases, current strategies for drug delivery and NF-kappaB inhibition and point out the "sneaking ligand" approach. Sneaking ligand fusion proteins (SLFPs) are recombinant proteins with modular architecture consisting of three domains. The prototype SLC1 binds specifically to the activated endothelium and blocks canonical NF-kappaB activation. In vivo, SLC1 attenuated clinical and histological signs of experimental arthritides. The SLFP architecture allows an easy exchange of binding and effector domains and represents an attractive approach to study disease-relevant biological targets in a broad range of diseases. In vivo, SLFP treatment might increase therapeutic efficacy while minimizing adverse effects.
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Affiliation(s)
- Bettina Sehnert
- Department of Rheumatology and Clinical Immunology, Medical Center—University of Freiburg, Faculty of Medicine, University of Freiburg, 79098 Freiburg, Germany
| | - Harald Burkhardt
- Division of Rheumatology, University Hospital Frankfurt, Goethe University, and Branch for Translational Medicine and Pharmacology TMP, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, 60590 Frankfurt am Main, Germany;
| | - Stefan Dübel
- Institute of Biochemistry and Biotechnology, Technical University Braunschweig, 38106 Braunschweig, Germany;
| | - Reinhard E. Voll
- Department of Rheumatology and Clinical Immunology, Medical Center—University of Freiburg, Faculty of Medicine, University of Freiburg, 79098 Freiburg, Germany
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25
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Yang Y, Delalio LJ, Best AK, Macal E, Milstein J, Donnelly I, Miller AM, McBride M, Shu X, Koval M, Isakson BE, Johnstone SR. Endothelial Pannexin 1 Channels Control Inflammation by Regulating Intracellular Calcium. THE JOURNAL OF IMMUNOLOGY 2020; 204:2995-3007. [PMID: 32312847 PMCID: PMC7336877 DOI: 10.4049/jimmunol.1901089] [Citation(s) in RCA: 56] [Impact Index Per Article: 11.2] [Reference Citation Analysis] [Abstract] [Subscribe] [Scholar Register] [Received: 09/10/2019] [Accepted: 03/27/2020] [Indexed: 12/26/2022]
Abstract
The proinflammatory cytokine IL-1β is a significant risk factor in cardiovascular disease that can be targeted to reduce major cardiovascular events. IL-1β expression and release are tightly controlled by changes in intracellular Ca2+ ([Ca2+]i), which has been associated with ATP release and purinergic signaling. Despite this, the mechanisms that regulate these changes have not been identified. The pannexin 1 (Panx1) channels have canonically been implicated in ATP release, especially during inflammation. We examined Panx1 in human umbilical vein endothelial cells following treatment with the proinflammatory cytokine TNF-α. Analysis by whole transcriptome sequencing and immunoblot identified a dramatic increase in Panx1 mRNA and protein expression that is regulated in an NF-κB-dependent manner. Furthermore, genetic inhibition of Panx1 reduced the expression and release of IL-1β. We initially hypothesized that increased Panx1-mediated ATP release acted in a paracrine fashion to control cytokine expression. However, our data demonstrate that IL-1β expression was not altered after direct ATP stimulation in human umbilical vein endothelial cells. Because Panx1 forms a large pore channel, we hypothesized it may permit Ca2+ diffusion into the cell to regulate IL-1β. High-throughput flow cytometric analysis demonstrated that TNF-α treatments lead to elevated [Ca2+]i, corresponding with Panx1 membrane localization. Genetic or pharmacological inhibition of Panx1 reduced TNF-α-associated increases in [Ca2+]i, blocked phosphorylation of the NF-κB-p65 protein, and reduced IL-1β transcription. Taken together, the data in our study provide the first evidence, to our knowledge, that [Ca2+]i regulation via the Panx1 channel induces a feed-forward effect on NF-κB to regulate IL-1β synthesis and release in endothelium during inflammation.
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Affiliation(s)
- Yang Yang
- Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VA 22908.,Department of Pharmacology, Dalian Medical University, Dalian 116044, China
| | - Leon J Delalio
- Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VA 22908
| | - Angela K Best
- Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VA 22908
| | - Edgar Macal
- Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VA 22908
| | - Jenna Milstein
- Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VA 22908
| | - Iona Donnelly
- British Heart Foundation Cardiovascular Research Centre, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, United Kingdom
| | - Ashley M Miller
- British Heart Foundation Cardiovascular Research Centre, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, United Kingdom
| | - Martin McBride
- British Heart Foundation Cardiovascular Research Centre, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, United Kingdom
| | - Xiaohong Shu
- Department of Pharmacology, Dalian Medical University, Dalian 116044, China
| | - Michael Koval
- Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322.,Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322; and
| | - Brant E Isakson
- Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VA 22908; .,Department of Molecular Physiology and Biophysics, University of Virginia School of Medicine, Charlottesville, VA 22908
| | - Scott R Johnstone
- Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VA 22908;
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26
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Zhou Z, Qi J, Zhao J, Lim CW, Kim J, Kim B. Dual TBK1/IKKɛ inhibitor amlexanox attenuates the severity of hepatotoxin-induced liver fibrosis and biliary fibrosis in mice. J Cell Mol Med 2020; 24:1383-1398. [PMID: 31821710 PMCID: PMC6991653 DOI: 10.1111/jcmm.14817] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2019] [Revised: 09/23/2019] [Accepted: 10/26/2019] [Indexed: 12/31/2022] Open
Abstract
Although numerous studies have suggested that canonical IκB kinases (IKK) play a key role in the progression of liver fibrosis, the role of non-canonical IKKε and TANK-binding kinase 1 (TBK1) on the development and progression of liver fibrosis remains unclear. To demonstrate such issue, repeated injection of CCl4 was used to induce hepatotoxin-mediated chronic liver injury and biliary fibrosis was induced by 0.1% diethoxycarbonyl-1, 4-dihydrocollidine diet feeding for 4 weeks. Mice were orally administered with amlexanox (25, 50, and 100 mg/kg) during experimental period. Significantly increased levels of TBK1 and IKKε were observed in fibrotic livers or hepatic stellate cells (HSCs) isolated from fibrotic livers. Interestingly, amlexanox treatment significantly inhibited the phosphorylation of TBK1 and IKKε accompanied by reduced liver injury as confirmed by histopathologic analysis, decreased serum biochemical levels and fibro-inflammatory responses. Additionally, treatment of amlexanox promoted the fibrosis resolution. In accordance with these findings, amlexanox treatment suppressed HSC activation and its related fibrogenic responses by partially inhibiting signal transducer and activator of transcription 3. Furthermore, amlexanox decreased the activation and inflammatory responses in Kupffer cells. Collectively, we found that inhibition of the TBK1 and IKKε by amlexanox is a promising therapeutic strategy to cure liver fibrosis.
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Affiliation(s)
- Zixiong Zhou
- Biosafety Research Institute and Laboratory of Pathology (BK21 Plus Program)College of Veterinary MedicineJeonbuk National UniversityIksanKorea
| | - Jing Qi
- Biosafety Research Institute and Laboratory of Pathology (BK21 Plus Program)College of Veterinary MedicineJeonbuk National UniversityIksanKorea
| | - Jing Zhao
- Biosafety Research Institute and Laboratory of Pathology (BK21 Plus Program)College of Veterinary MedicineJeonbuk National UniversityIksanKorea
| | - Chae Woong Lim
- Biosafety Research Institute and Laboratory of Pathology (BK21 Plus Program)College of Veterinary MedicineJeonbuk National UniversityIksanKorea
| | - Jong‐Won Kim
- Biosafety Research Institute and Laboratory of Pathology (BK21 Plus Program)College of Veterinary MedicineJeonbuk National UniversityIksanKorea
| | - Bumseok Kim
- Biosafety Research Institute and Laboratory of Pathology (BK21 Plus Program)College of Veterinary MedicineJeonbuk National UniversityIksanKorea
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27
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Ryan KM, Boyle NT, Harkin A, Connor TJ. Dexamethasone attenuates inflammatory-mediated suppression of β 2-adrenoceptor expression in rat primary mixed glia. J Neuroimmunol 2019; 338:577082. [PMID: 31707103 DOI: 10.1016/j.jneuroim.2019.577082] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2019] [Revised: 10/04/2019] [Accepted: 10/04/2019] [Indexed: 11/29/2022]
Abstract
β2-adrenoceptors are G-protein coupled receptors expressed on both astrocytes and microglia that play a key role in mediating the anti-inflammatory actions of noradrenaline in the CNS. Here the effect of an inflammatory stimulus (LPS + IFN-γ) was examined on glial β2-adrenoceptor expression and function. Exposure of glia to LPS + IFN-γ decreased β2-adrenoceptor mRNA and agonist-stimulated production of the intracellular second messenger cAMP. Pre-treatment with the synthetic glucocorticoid and potent anti-inflammatory agent dexamethasone prevented the LPS + IFN-γ-induced suppression of β2-adrenoceptor mRNA expression. These results raise the possibility that inflammation-mediated β2-adrenoceptor downregulation in glia may dampen the innate anti-inflammatory properties of noradrenaline in the CNS.
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Affiliation(s)
- Karen M Ryan
- Neuroimmunology Research Group, Department of Physiology, Trinity College Institute of Neuroscience & School of Medicine, Trinity College, Dublin 2, Ireland
| | - Noreen T Boyle
- Neuroimmunology Research Group, Department of Physiology, Trinity College Institute of Neuroscience & School of Medicine, Trinity College, Dublin 2, Ireland
| | - Andrew Harkin
- Neuropsychopharmacology Research Group, Trinity College Institute of Neuroscience, School of Pharmacy and Pharmaceutical Sciences, Trinity College, Dublin 2, Ireland.
| | - Thomas J Connor
- Neuroimmunology Research Group, Department of Physiology, Trinity College Institute of Neuroscience & School of Medicine, Trinity College, Dublin 2, Ireland
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28
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Gnani D, Crippa S, della Volpe L, Rossella V, Conti A, Lettera E, Rivis S, Ometti M, Fraschini G, Bernardo ME, Di Micco R. An early-senescence state in aged mesenchymal stromal cells contributes to hematopoietic stem and progenitor cell clonogenic impairment through the activation of a pro-inflammatory program. Aging Cell 2019; 18:e12933. [PMID: 30828977 PMCID: PMC6516180 DOI: 10.1111/acel.12933] [Citation(s) in RCA: 133] [Impact Index Per Article: 22.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2018] [Revised: 01/22/2019] [Accepted: 02/02/2019] [Indexed: 12/14/2022] Open
Abstract
Hematopoietic stem and progenitor cells (HSPC) reside in the bone marrow (BM) niche and serve as a reservoir for mature blood cells throughout life. Aging in the BM is characterized by low‐grade chronic inflammation that could contribute to the reduced functionality of aged HSPC. Mesenchymal stromal cells (MSC) in the BM support HSPC self‐renewal. However, changes in MSC function with age and the crosstalk between MSC and HSPC remain understudied. Here, we conducted an extensive characterization of senescence features in BM‐derived MSC from young and aged healthy donors. Aged MSC displayed an enlarged senescent‐like morphology, a delayed clonogenic potential and reduced proliferation ability when compared to younger counterparts. Of note, the observed proliferation delay was associated with increased levels of SA‐β‐galactosidase (SA‐β‐Gal) and lipofuscin in aged MSC at early passages and a modest but consistent accumulation of physical DNA damage and DNA damage response (DDR) activation. Consistent with the establishment of a senescence‐like state in aged MSC, we detected an increase in pro‐inflammatory senescence‐associated secretory phenotype (SASP) factors, both at the transcript and protein levels. Conversely, the immunomodulatory properties of aged MSC were significantly reduced. Importantly, exposure of young HSPC to factors secreted by aged MSC induced pro‐inflammatory genes in HSPC and impaired HSPC clonogenic potential in a SASP‐dependent manner. Altogether, our results reveal that BM‐derived MSC from aged healthy donors display features of senescence and that, during aging, MSC‐associated secretomes contribute to activate an inflammatory transcriptional program in HSPC that may ultimately impair their functionality.
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Affiliation(s)
- Daniela Gnani
- San Raffaele Telethon Institute for Gene Therapy Milan Italy
| | - Stefania Crippa
- San Raffaele Telethon Institute for Gene Therapy Milan Italy
| | - Lucrezia della Volpe
- San Raffaele Telethon Institute for Gene Therapy Milan Italy
- Vita‐Salute San Raffaele University Milan Italy
| | | | - Anastasia Conti
- San Raffaele Telethon Institute for Gene Therapy Milan Italy
| | - Emanuele Lettera
- San Raffaele Telethon Institute for Gene Therapy Milan Italy
- Vita‐Salute San Raffaele University Milan Italy
| | - Silvia Rivis
- San Raffaele Telethon Institute for Gene Therapy Milan Italy
| | - Marco Ometti
- Department of Orthopedics and Traumatology San Raffaele Hospital Scientific Institute Milan Italy
| | - Gianfranco Fraschini
- Department of Orthopedics and Traumatology San Raffaele Hospital Scientific Institute Milan Italy
| | - Maria Ester Bernardo
- San Raffaele Telethon Institute for Gene Therapy Milan Italy
- Pediatric Immunohematology and Bone Marrow Transplantation Unit San Raffaele Scientific Institute Milan Italy
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29
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Choi WS, Yang JI, Kim W, Kim HE, Kim SK, Won Y, Son YO, Chun CH, Chun JS. Critical role for arginase II in osteoarthritis pathogenesis. Ann Rheum Dis 2019; 78:421-428. [PMID: 30610061 PMCID: PMC6390026 DOI: 10.1136/annrheumdis-2018-214282] [Citation(s) in RCA: 32] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2018] [Revised: 12/04/2018] [Accepted: 12/05/2018] [Indexed: 12/19/2022]
Abstract
OBJECTIVE Osteoarthritis (OA) appears to be associated with various metabolic disorders, but the potential contribution of amino acid metabolism to OA pathogenesis has not been clearly elucidated. Here, we explored whether alterations in the amino acid metabolism of chondrocytes could regulate OA pathogenesis. METHODS Expression profiles of amino acid metabolism-regulating genes in primary-culture passage 0 mouse chondrocytes were examined by microarray analysis, and selected genes were further characterised in mouse OA chondrocytes and OA cartilage of human and mouse models. Experimental OA in mice was induced by destabilisation of the medial meniscus (DMM) or intra-articular (IA) injection of adenoviruses expressing catabolic regulators. The functional consequences of arginase II (Arg-II) were examined in Arg2-/- mice and those subjected to IA injection of an adenovirus encoding Arg-II (Ad-Arg-II). RESULTS The gene encoding Arg-II, an arginine-metabolising enzyme, was specifically upregulated in chondrocytes under various pathological conditions and in OA cartilage from human patients with OA and various mouse models. Adenovirus-mediated overexpression of Arg-II in mouse joint tissues caused OA pathogenesis, whereas genetic ablation of Arg2 in mice (Arg2-/-) abolished all manifestations of DMM-induced OA. Mechanistically, Arg-II appears to cause OA cartilage destruction at least partly by upregulating the expression of matrix-degrading enzymes (matrix metalloproteinase 3 [MMP3] and MMP13) in chondrocytes via the nuclear factor (NF)-κB pathway. CONCLUSIONS Our results indicate that Arg-II is a crucial regulator of OA pathogenesis in mice. Although chondrocytes of human and mouse do not identically, but similarly, respond to Arg-II, our results suggest that Arg-II could be a therapeutic target of OA pathogenesis.
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Affiliation(s)
- Wan-Su Choi
- National Creative Research Initiatives Center for Osteoarthritis Pathogenesis and School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, Republic of Korea
| | - Jeong-In Yang
- National Creative Research Initiatives Center for Osteoarthritis Pathogenesis and School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, Republic of Korea
| | - Wihak Kim
- National Creative Research Initiatives Center for Osteoarthritis Pathogenesis and School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, Republic of Korea
| | - Hyo-Eun Kim
- National Creative Research Initiatives Center for Osteoarthritis Pathogenesis and School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, Republic of Korea
| | - Seul-Ki Kim
- National Creative Research Initiatives Center for Osteoarthritis Pathogenesis and School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, Republic of Korea
| | - Yoonkyung Won
- National Creative Research Initiatives Center for Osteoarthritis Pathogenesis and School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, Republic of Korea
| | - Young-Ok Son
- National Creative Research Initiatives Center for Osteoarthritis Pathogenesis and School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, Republic of Korea
| | - Churl-Hong Chun
- Department of Orthopedic Surgery, Wonkwang University School of Medicine, Iksan, Republic of Korea
| | - Jang-Soo Chun
- National Creative Research Initiatives Center for Osteoarthritis Pathogenesis and School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju, Republic of Korea
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30
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Reactive oxygen species modulate macrophage immunosuppressive phenotype through the up-regulation of PD-L1. Proc Natl Acad Sci U S A 2019; 116:4326-4335. [PMID: 30770442 DOI: 10.1073/pnas.1819473116] [Citation(s) in RCA: 143] [Impact Index Per Article: 23.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
The combination of immune checkpoint blockade with chemotherapy is currently under investigation as a promising strategy for the treatment of triple negative breast cancer (TNBC). Tumor-associated macrophages (TAMs) are the most prominent component of the breast cancer microenvironment because they influence tumor progression and the response to therapies. Here we show that macrophages acquire an immunosuppressive phenotype and increase the expression of programmed death ligand-1 (PD-L1) when treated with reactive oxygen species (ROS) inducers such as the glutathione synthesis inhibitor, buthionine sulphoximine (BSO), and paclitaxel. Mechanistically, these agents cause accumulation of ROS that in turn activate NF-κB signaling to promote PD-L1 transcription and the release of immunosuppressive chemokines. Systemic in vivo administration of paclitaxel promotes PD-L1 accumulation on the surface of TAMS in a mouse model of TNBC, consistent with in vitro results. Combinatorial treatment with paclitaxel and an anti-mouse PD-L1 blocking antibody significantly improved the therapeutic efficacy of paclitaxel by reducing tumor burden and increasing the number of tumor-associated cytotoxic T cells. Our results provide a strong rationale for the use of anti-PD-L1 blockade in the treatment of TNBC patients. Furthermore, interrogation of chemotherapy-induced PD-L1 expression in TAMs is warranted to define appropriate patient selection in the use of PD-L1 blockade.
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31
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Rahimova N, Babazada H, Higuchi Y, Yamashita F, Hashida M. Development of mKO2 fusion proteins for real-time imaging and mechanistic investigation of the degradation kinetics of human IκBα in living cells. BIOCHIMICA ET BIOPHYSICA ACTA. MOLECULAR CELL RESEARCH 2019; 1866:190-198. [PMID: 30391277 DOI: 10.1016/j.bbamcr.2018.10.018] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/22/2018] [Revised: 10/03/2018] [Accepted: 10/28/2018] [Indexed: 12/11/2022]
Abstract
In resting cells, the nuclear factor kappa B (NF-κB) family of transcription factors is stabilized by complexation with the cytoplasmic inhibitor of kappa B alpha (IκBα). Extracellular stimuli, such as tumor necrosis factor alpha (TNFα) or bacterial lipopolysaccharide activate NF-κB through IκBα phosphorylation and ubiquitin-proteasomal degradation. Herein, we developed a novel biosensor, by fusing the monomeric fluorescent protein Kusabira-Orange 2 to IκBα (mKO2-IκBα), to study the dynamics and structure-activity relationship of IκBα degradation. Site-specific deletion studies on the IκBα sequence revealed that the C-terminal PEST domain is required in signal-induced proteasomal degradation of IκBα and functions independently from ankyrin repeats. Using deletion mutants, we show that IκBα ankyrin repeats do not affect IκBα degradability but affect its degradation rate. We demonstrate, by both real-time confocal microscopy and western blot analysis, that the half-life of mKO2-IκBα in response to TNFα is approximately 35 min, which is similar to the half-life of endogenous IκBα. Using this biosensor we also show that selective proteasome inhibitors, such as lactacystin and MG132, inhibit degradation and affect the kinetics of IκBα in a dose-dependent manner. The techniques described here can have a range of possible applications, such as facilitating studies associated with IκBα dynamics and biochemical characteristics, as well as the screening of potential proteasome inhibitors.
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Affiliation(s)
- Nilufar Rahimova
- Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, Yoshidashimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
| | - Hasan Babazada
- Department of Anesthesiology and Critical Care, Perelman School of Medicine, University of Pennsylvania, 312 John Morgan Building, 3620 Hamilton Walk, Philadelphia, PA 19104, USA
| | - Yuriko Higuchi
- Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, Yoshidashimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
| | - Fumiyoshi Yamashita
- Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, Yoshidashimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan
| | - Mitsuru Hashida
- Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, Yoshidashimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan; Institute for Integrated Cell-Material Sciences, Kyoto University, Yoshidaushinomiya-cho, Sakyo-ku, Kyoto 606-8501, Japan.
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Bhatti FUR, Hasty KA, Cho H. Anti-inflammatory role of TPCA-1 encapsulated nanosomes in porcine chondrocytes against TNF-α stimulation. Inflammopharmacology 2019; 27:1011-1019. [PMID: 30600473 DOI: 10.1007/s10787-018-0542-5] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2018] [Accepted: 10/20/2018] [Indexed: 11/28/2022]
Abstract
In this study, we evaluated the hypothesis that immunonanosomes carrying the drug [5-(p-Fluorophenyl)-2-ureido]thiophene-3-carboxamide (TPCA-1) will help in reducing nuclear factor-kappaB (NF-κB)-associated inflammation in porcine chondrocytes against tumor necrosis factor-alpha (TNF-α)-induced stress. The nanosomes were tagged with monoclonal anti-type II collagen (MabCII) antibody to specifically target the exposed type II collagen in cartilage matrix. TPCA-1 at a concentration of 10 µM significantly reduced expression of the matrix-degrading enzyme, Matrix metalloproteinase-13 (MMP-13) and blocked the p65 nuclear translocation. In comparison to the TPCA-1 solution alone, the TPCA-1 nanosomes were found to be more effective in reducing the cellular toxicity, oxidative stress and inflammation in chondrocytes treated with TNF-α. In addition, TPCA-1 nanosomes were more effective in reducing the gene expression of hypoxia-inducible factor-2alpha (HIF-2α) that in turn is associated with the regulation of MMP-13 gene. TPCA-1 nanosomes significantly reduced expression of both these genes. The data also showed that TPCA-1 did not attenuate the down-regulated gene expression levels of anabolic genes aggrecan (ACAN) and collagen type II alpha (COL2A1). In conclusion, this study showed that TPCA-1 nanosomes carrying a dose of 10 µM TPCA-1 can effectively increase the survival of cultured porcine chondrocytes against TNF-α-induced stress. The findings of this study could be used to develop nanosome-based drug delivery systems (DDSs) for animal model of OA. Moreover, the approach presented here can be further utilized in other studies for targeted delivery of the drug of interest at a cellular level.
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Affiliation(s)
- Fazal Ur Rehman Bhatti
- Department of Orthopaedic Surgery and Biomedical Engineering, University of Tennessee Health Science Center, Research 151, VAMC, 1030 Jefferson Ave, Memphis, TN, 38104, USA.,VA Medical Center, Memphis, TN, USA
| | - Karen A Hasty
- Department of Orthopaedic Surgery and Biomedical Engineering, University of Tennessee Health Science Center, Research 151, VAMC, 1030 Jefferson Ave, Memphis, TN, 38104, USA. .,Department of Orthopaedic Surgery and Biomedical Engineering, University of Tennessee Health Science Center-Campbell Clinic, Research 151, VAMC, 1030 Jefferson Ave, Memphis, TN, 38104, USA. .,VA Medical Center, Memphis, TN, USA.
| | - Hongsik Cho
- Department of Orthopaedic Surgery and Biomedical Engineering, University of Tennessee Health Science Center, Research 151, VAMC, 1030 Jefferson Ave, Memphis, TN, 38104, USA. .,Department of Orthopaedic Surgery and Biomedical Engineering, University of Tennessee Health Science Center-Campbell Clinic, Research 151, VAMC, 1030 Jefferson Ave, Memphis, TN, 38104, USA. .,VA Medical Center, Memphis, TN, USA.
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Gurney LRI, Taggart J, Tong WC, Jones AT, Robson SC, Taggart MJ. Inhibition of Inflammatory Changes in Human Myometrial Cells by Cell Penetrating Peptide and Small Molecule Inhibitors of NFκB. Front Immunol 2018; 9:2966. [PMID: 30619324 PMCID: PMC6307458 DOI: 10.3389/fimmu.2018.02966] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2018] [Accepted: 12/03/2018] [Indexed: 01/17/2023] Open
Abstract
Complications arising from Preterm Birth are the leading causes of neonatal death globally. Current therapeutic strategies to prevent Preterm Birth are yet to demonstrate success in terms of reducing this neonatal disease burden. Upregulation of intracellular inflammatory pathways in uterine cells, including those involving nuclear factor kappa-B (NFκB), have been causally linked to both human term and preterm labor, but the barrier presented by the cell membrane presents an obstacle to interventions aimed at dampening these inflammatory responses. Cell penetrating peptides (CPPs) are novel vectors that can traverse cell membranes without the need for recognition by cell surface receptors and offer the ability to deliver therapeutic cargo internal to cell membranes. Using a human uterine cell culture inflammatory model, this study aimed to test the effectiveness of CPP-cargo delivery to inhibit inflammatory responses, comparing this effect with a small molecule inhibitor (Sc514) that has a similar intracellular target of action within the NFκB pathway (the IKK complex). The CPP Penetratin, conjugated to rhodamine, was able to enter uterine cells within a 60 min timeframe as assessed by live confocal microscopy, this phenomena was not observed with the use of a rhodamine-conjugated inert control peptide (GC(GS)4). Penetratin CPP conjugated to an IKK-inhibitory peptide (Pen-NBD) demonstrated ability to inhibit both the IL1β-induced expression of the inflammatory protein COX2 and dampen the expression of a bespoke array of inflammatory genes. Truncation of the CPP vector rendered the CPP-cargo conjugate much less effective, demonstrating the importance of careful vector selection. The small molecule inhibitor Sc514 also demonstrated ability to inhibit COX2 protein responses and a broad down-regulatory effect on uterine cell inflammatory gene expression. These results support the further exploration of either CPP-based or small molecular treatment strategies to dampen gestational cell inflammatory responses in the context of preterm birth. The work underlines both the importance of careful selection of CPP vector-cargo combinations and basic testing over a broad time and concentration range to ensure effective responses. Further work should demonstrate the effectiveness of CPP-linked cargos to dampen alternative pathways of inflammation linked to Preterm Birth such as MAP Kinase or AP1.
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Affiliation(s)
- Leo R. I. Gurney
- Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom
| | - Julie Taggart
- Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom
| | - Wing-Chiu Tong
- Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom
| | - Arwyn T. Jones
- School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, United Kingdom
| | - Stephen C. Robson
- Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom
| | - Michael J. Taggart
- Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom
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Bloom MJ, Saksena SD, Swain GP, Behar MS, Yankeelov TE, Sorace AG. The effects of IKK-beta inhibition on early NF-kappa-B activation and transcription of downstream genes. Cell Signal 2018; 55:17-25. [PMID: 30543861 DOI: 10.1016/j.cellsig.2018.12.004] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2018] [Revised: 12/05/2018] [Accepted: 12/10/2018] [Indexed: 02/07/2023]
Abstract
Small molecule approaches targeting the nuclear factor kappa B (NF-kB) pathway, a regulator of inflammation, have thus far proven unsuccessful in the clinic in part due to the complex pleiotropic nature of this network. Downstream effects depend on multiple factors including stimulus-specific temporal patterns of NF-kB activity. Despite considerable advances, genome-level impact of changes in temporal NF-kB activity caused by inhibitors and their stimulus dependency remains unexplored. This study evaluates the effects of pathway inhibitors on early NF-κB activity and downstream gene transcription. 3T3 fibroblasts were treated with SC-514, an inhibitor targeted to the NF-kB pathway, prior to stimulation with interleukin 1 beta (IL-1β) or tumor necrosis factor alpha (TNF-α). Stimulus induced NF-κB activation was quantified using immunofluorescence imaging over 90-minutes and gene expression tracked over 6-hours using mRNA TagSeq. When stimulated with IL-1β or TNF-α, significant differences (P < 0.05, two-way ANOVA), were observed in the temporal profiles of NF-κB activation between treated and untreated cells. Increasing numbers of differentially expressed genes (P < 0.01) were observed at higher inhibitor concentrations. Individual gene expression profiles varied in an inhibitor concentration and stimulus-dependent manner. The results in this study demonstrate small molecule inhibitors acting on pleiotropic pathway components can alter signal dynamics in a stimulus-dependent manner and affect gene response in complex ways.
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Affiliation(s)
- Meghan J Bloom
- Biomedical Engineering, The University of Texas, Austin, TX, USA.
| | - Sachit D Saksena
- Computational and Systems Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.
| | - George P Swain
- Biomedical Engineering, The University of Texas, Austin, TX, USA.
| | - Marcelo S Behar
- Biomedical Engineering, The University of Texas, Austin, TX, USA
| | - Thomas E Yankeelov
- Biomedical Engineering, The University of Texas, Austin, TX, USA; Diagnostic Medicine, The University of Texas, Austin, TX, USA; Livestrong Cancer Institutes, The University of Texas, Austin, TX, USA; Oncology, The University of Texas, Austin, TX, USA; Institute for Computational and Engineering Sciences, The University of Texas, Austin, TX, USA.
| | - Anna G Sorace
- Biomedical Engineering, The University of Texas, Austin, TX, USA; Diagnostic Medicine, The University of Texas, Austin, TX, USA; Livestrong Cancer Institutes, The University of Texas, Austin, TX, USA; Oncology, The University of Texas, Austin, TX, USA.
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Li M, Gong AY, Zhang XT, Wang Y, Mathy NW, Martins GA, Strauss-Soukup JK, Chen XM. Induction of a Long Noncoding RNA Transcript, NR_045064, Promotes Defense Gene Transcription and Facilitates Intestinal Epithelial Cell Responses against Cryptosporidium Infection. THE JOURNAL OF IMMUNOLOGY 2018; 201:3630-3640. [PMID: 30446564 DOI: 10.4049/jimmunol.1800566] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/20/2018] [Accepted: 10/20/2018] [Indexed: 12/26/2022]
Abstract
Cryptosporidium is an important opportunistic intestinal pathogen for immunocompromised individuals and a common cause of diarrhea in young children in developing countries. Gastrointestinal epithelial cells play a central role in activating and orchestrating host immune responses against Cryptosporidium infection, but underlying molecular mechanisms are not fully understood. We report in this paper that C. parvum infection causes significant alterations in long noncoding RNA (lncRNA) expression profiles in murine intestinal epithelial cells. Transcription of a panel of lncRNA genes, including NR_045064, in infected cells is controlled by the NF-κB signaling. Functionally, inhibition of NR_045064 induction increases parasite burden in intestinal epithelial cells. Induction of NR_045064 enhances the transcription of selected defense genes in host cells following C. parvum infection. Epigenetic histone modifications are involved in NR_045064-mediated transcription of associated defense genes in infected host cells. Moreover, the p300/MLL-associated chromatin remodeling is involved in NR_045064-mediated transcription of associated defense genes in intestinal epithelial cells following C. parvum infection. Expression of NR_045064 and associated genes is also identified in intestinal epithelium in C57BL/6J mice following phosphorothioate oligodeoxynucleotide or LPS stimulation. Our data demonstrate that lncRNAs, such as NR_045064, play a role in regulating epithelial defense against microbial infection.
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Affiliation(s)
- Min Li
- Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, NE 68178
| | - Ai-Yu Gong
- Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, NE 68178
| | - Xin-Tian Zhang
- Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, NE 68178
| | - Yang Wang
- Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, NE 68178
| | - Nicholas W Mathy
- Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, NE 68178
| | - Gislaine A Martins
- Research Division of Immunology, Department of Medicine, Cedars-Sinai Medical Center, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90048.,Research Division of Immunology, Department of Biomedical Sciences, Cedars-Sinai Medical Center, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90048; and
| | | | - Xian-Ming Chen
- Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, NE 68178;
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LncRNA HOTAIR regulates lipopolysaccharide-induced cytokine expression and inflammatory response in macrophages. Sci Rep 2018; 8:15670. [PMID: 30353135 PMCID: PMC6199307 DOI: 10.1038/s41598-018-33722-2] [Citation(s) in RCA: 75] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2018] [Accepted: 10/02/2018] [Indexed: 12/16/2022] Open
Abstract
Long noncoding RNAs (lncRNAs) are emerging as major regulators of a variety of cell signaling processes. Many lncRNAs are expressed in immune cells and appear to play critical roles in the regulation of immune response. Here, we have investigated the potential role of a well-known lncRNA, HOTAIR, in inflammatory and immune response. Our studies demonstrate that HOTAIR expression is induced in immune cells (macrophages) upon treatment with lipopolysaccharide (LPS). Knockdown of HOTAIR reduces NF-κB-mediated inflammatory gene and cytokine expression in macrophages. Inhibition of NF-κB resulted in down-regulation of LPS-induced expression of HOTAIR as well as IL-6 and iNOS expression. We further demonstrated that HOTAIR regulates activation of NF-κB and its target genes (IL-6 and iNOS) expression via facilitating the degradation of IκBα. HOTAIR knockdown reduces the expression of NF-κB target gene expression via inhibiting the recruitment of NF-κB and associated cofactors at the target gene promoters. Taken together, our findings suggest that HOTAIR is a critical player in NF-κB activation in macrophages suggesting its potential functions in inflammatory and immune response.
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Prescott JA, Cook SJ. Targeting IKKβ in Cancer: Challenges and Opportunities for the Therapeutic Utilisation of IKKβ Inhibitors. Cells 2018; 7:cells7090115. [PMID: 30142927 PMCID: PMC6162708 DOI: 10.3390/cells7090115] [Citation(s) in RCA: 88] [Impact Index Per Article: 12.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2018] [Revised: 08/15/2018] [Accepted: 08/19/2018] [Indexed: 02/08/2023] Open
Abstract
Deregulated NF-κB signalling is implicated in the pathogenesis of numerous human inflammatory disorders and malignancies. Consequently, the NF-κB pathway has attracted attention as an attractive therapeutic target for drug discovery. As the primary, druggable mediator of canonical NF-κB signalling the IKKβ protein kinase has been the historical focus of drug development pipelines. Thousands of compounds with activity against IKKβ have been characterised, with many demonstrating promising efficacy in pre-clinical models of cancer and inflammatory disease. However, severe on-target toxicities and other safety concerns associated with systemic IKKβ inhibition have thus far prevented the clinical approval of any IKKβ inhibitors. This review will discuss the potential reasons for the lack of clinical success of IKKβ inhibitors to date, the challenges associated with their therapeutic use, realistic opportunities for their future utilisation, and the alternative strategies to inhibit NF-κB signalling that may overcome some of the limitations associated with IKKβ inhibition.
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Affiliation(s)
- Jack A Prescott
- Signalling Laboratory, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK.
| | - Simon J Cook
- Signalling Laboratory, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK.
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Basu S, Jalodia K, Ranjan S, Yeh JRJ, Peterson RT, Sachidanandan C. Small Molecule Inhibitors of NFkB Reverse Iron Overload and Hepcidin Deregulation in a Zebrafish Model for Hereditary Hemochromatosis Type 3. ACS Chem Biol 2018; 13:2143-2152. [PMID: 29897731 DOI: 10.1021/acschembio.8b00317] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Hereditary hemochromatosis (HH) is one of the most common genetic disorders in Caucasian populations, with no viable therapeutic options except phlebotomy. We describe a zebrafish model of human HH (HH) created by targeted mutagenesis of the gene encoding transferrin receptor 2 ( tfr2). TFR2 mutations in humans lead to HH Type 3, a rare but severe form of the disease. The tfr2 mutant model in zebrafish recapitulates the defining features of HH3: iron overload and suppression of hepcidin, the iron regulatory hormone. Using in vivo chemical screens in zebrafish embryos, we identify a new small molecule inducer of hepcidin: SC-514, a specific chemical inhibitor of NFkB signaling. Using independent small molecule inhibitors of the NFkB pathway, we demonstrate that inhibition of NFkB signaling causes induction of hepcidin transcription and reduction of iron overload in the HH3 model. This first successful chemical intervention for hereditary hemochromatosis may also have relevance in treatment of other very prevalent iron regulatory iron overload disorders such as thalassemia.
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Affiliation(s)
- Sandeep Basu
- CSIR-Institute of Genomics and Integrative Biology, New Delhi, 110025, India
- Academy of Scientific and Innovative Research (AcSIR), New Delhi, 110025, India
| | - Kanika Jalodia
- CSIR-Institute of Genomics and Integrative Biology, New Delhi, 110025, India
| | - Shashi Ranjan
- CSIR-Institute of Genomics and Integrative Biology, New Delhi, 110025, India
| | - Jing-Ruey J. Yeh
- Cardiovascular Research Center, Massachusetts General Hospital & Harvard Medical School, Boston, United States
| | - Randall T. Peterson
- Cardiovascular Research Center, Massachusetts General Hospital & Harvard Medical School, Boston, United States
- Department of Pharmacology and Toxicology, College of Pharmacy, University of Utah, Salt Lake City, United States
| | - Chetana Sachidanandan
- CSIR-Institute of Genomics and Integrative Biology, New Delhi, 110025, India
- Academy of Scientific and Innovative Research (AcSIR), New Delhi, 110025, India
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Proteasome inhibition induces IKK-dependent interleukin-8 expression in triple negative breast cancer cells: Opportunity for combination therapy. PLoS One 2018; 13:e0201858. [PMID: 30089134 PMCID: PMC6082561 DOI: 10.1371/journal.pone.0201858] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2018] [Accepted: 07/23/2018] [Indexed: 12/14/2022] Open
Abstract
Triple negative breast cancer (TNBC) cells express increased levels of the pro-inflammatory and pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8), which promotes their proliferation and migration. Because TNBC patients are unresponsive to current targeted therapies, new therapeutic strategies are urgently needed. While proteasome inhibition by bortezomib (BZ) or carfilzomib (CZ) has been effective in treating hematological malignancies, it has been less effective in solid tumors, including TNBC, but the mechanisms are incompletely understood. Here we report that proteasome inhibition significantly increases expression of IL-8, and its receptors CXCR1 and CXCR2, in TNBC cells. Suppression or neutralization of the BZ-induced IL-8 potentiates the BZ cytotoxic and anti-proliferative effect in TNBC cells. The IL-8 expression induced by proteasome inhibition in TNBC cells is mediated by IκB kinase (IKK), increased nuclear accumulation of p65 NFκB, and by IKK-dependent p65 recruitment to IL-8 promoter. Importantly, inhibition of IKK activity significantly decreases proliferation, migration, and invasion of BZ-treated TNBC cells. These data provide the first evidence demonstrating that proteasome inhibition increases the IL-8 signaling in TNBC cells, and suggesting that IKK inhibitors may increase effectiveness of proteasome inhibitors in treating TNBC.
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Choi MC, MaruYama T, Chun CH, Park Y. Alleviation of Murine Osteoarthritis by Cartilage-Specific Deletion of IκBζ. Arthritis Rheumatol 2018; 70:1440-1449. [PMID: 29604191 DOI: 10.1002/art.40514] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2017] [Accepted: 03/22/2018] [Indexed: 01/06/2023]
Abstract
OBJECTIVE IκBζ, an atypical IκB family member, regulates gene expression in the nucleus as a transcriptional cofactor. Although IκBζ has been extensively studied in the immune system, its specific roles in osteoarthritis (OA) are currently unknown. The objective of this study was to investigate the potential role of IκBζ in chondrocyte catabolism and OA pathogenesis. We also determined the molecular mechanism underlying its relationship to the transcription factor NF-κB. METHODS We determined expression levels of IκBζ in mouse chondrocytes treated with interleukin-1β (IL-1β), in human OA cartilage, and in mouse experimental OA cartilage. Adenovirus-mediated overexpression and small interfering RNA knockdown of IκBζ were performed to determine the impact of IκBζ on catabolic gene expression in vitro. Cartilage-specific IκBζ-transgenic and -knockout mice were generated and used for in vivo studies. Experimental and spontaneous OA were induced by surgical destabilization of the medial meniscus and by aging, respectively. Coimmunoprecipitation assay was used to examine the association between IκBζ and NF-κB subunits. RESULTS IκBζ was highly up-regulated in chondrocytes in response to IL-1β and in OA cartilage of human and mouse knee joints. Overexpression of IκBζ in chondrocytes promoted spontaneous OA development by activating chondrocyte catabolism. Genetic ablation of IκBζ in chondrocytes abolished catabolic gene induction by IL-1β and protected against the development of experimental OA. IκBζ formed complexes with NF-κB members to regulate catabolic factor expression. CONCLUSION These findings demonstrate a critical role for IκBζ in OA pathogenesis. Inhibition of IκBζ function might be an effective therapeutic approach for OA treatment.
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Affiliation(s)
- Moon-Chang Choi
- Gwangju Institute of Science and Technology and Chosun University, Gwangju, Republic of Korea
| | | | - Churl-Hong Chun
- Wonkwang University School of Medicine, Iksan, Republic of Korea
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Benzo(a)pyrene in Cigarette Smoke Enhances HIV-1 Replication through NF-κB Activation via CYP-Mediated Oxidative Stress Pathway. Sci Rep 2018; 8:10394. [PMID: 29991690 PMCID: PMC6039513 DOI: 10.1038/s41598-018-28500-z] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2018] [Accepted: 06/14/2018] [Indexed: 02/06/2023] Open
Abstract
Smoking aggravates HIV-1 pathogenesis and leads to decreased responses to antiretroviral therapy. In this study, we aim to find a molecular mechanism that would explain smoking-induced HIV-1 replication. Benzo(a)pyrene (BaP), a major carcinogen in cigarette, requires metabolic activation through cytochrome P450s (CYPs) to exert its toxic effects. We hypothesized that CYP-mediated BaP metabolism generates reactive oxygen species (ROS), and the resultant oxidative stress aggravates HIV-1 replication. As expected, we observed ~3 to 4-fold increase in HIV-1 replication in U1 cells and human primary macrophages after chronic BaP exposure. We also observed ~30-fold increase in the expression of CYP1A1 at mRNA level, ~2.5-fold increase in its enzymatic activity as well as elevated ROS and cytotoxicity in U1 cells. The knock-down of the CYP1A1 gene using siRNA and treatment with selective CYP inhibitors and antioxidants significantly reduced HIV-1 replication. Further, we observed a nuclear translocation of NF-κB subunits (p50 and p65) after chronic BaP exposure, which was reduced by treatment with siRNA and antioxidants/CYP inhibitors. Suppression of NF-κB pathway using specific NF-κB inhibitors also significantly reduced HIV-1 replication. Altogether, our results suggest that BaP enhances HIV-1 replication in macrophages by a CYP-mediated oxidative stress pathway followed by the NF-κB pathway.
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KH-type splicing regulatory protein is regulated by nuclear factor-κB signaling to mediate innate immunity in Caco-2 cells infected by Salmonella enteritidis. Folia Microbiol (Praha) 2018; 63:669-676. [DOI: 10.1007/s12223-018-0606-3] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2017] [Accepted: 04/25/2018] [Indexed: 02/04/2023]
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Targeting IκappaB kinases for cancer therapy. Semin Cancer Biol 2018; 56:12-24. [PMID: 29486318 DOI: 10.1016/j.semcancer.2018.02.007] [Citation(s) in RCA: 37] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2017] [Revised: 02/18/2018] [Accepted: 02/21/2018] [Indexed: 01/05/2023]
Abstract
The inhibitory kappa B kinases (IKKs) and IKK related kinases are crucial regulators of the pro-inflammatory transcription factor, nuclear factor kappa B (NF-κB). The dysregulation in the activities of these kinases has been reported in several cancer types. These kinases are known to regulate survival, proliferation, invasion, angiogenesis, and metastasis of cancer cells. Thus, IKK and IKK related kinases have emerged as an attractive target for the development of cancer therapeutics. Several IKK inhibitors have been developed, few of which have advanced to the clinic. These inhibitors target IKK either directly or indirectly by modulating the activities of other signaling molecules. Some inhibitors suppress IKK activity by disrupting the protein-protein interaction in the IKK complex. The inhibition of IKK has also been shown to enhance the efficacy of conventional chemotherapeutic agents. Because IKK and NF-κB are the key components of innate immunity, suppressing IKK is associated with the risk of immune suppression. Furthermore, IKK inhibitors may hit other signaling molecules and thus may produce off-target effects. Recent studies suggest that multiple cytoplasmic and nuclear proteins distinct from NF-κB and inhibitory κB are also substrates of IKK. In this review, we discuss the utility of IKK inhibitors for cancer therapy. The limitations associated with the intervention of IKK are also discussed.
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Kanaya T, Sakakibara S, Jinnohara T, Hachisuka M, Tachibana N, Hidano S, Kobayashi T, Kimura S, Iwanaga T, Nakagawa T, Katsuno T, Kato N, Akiyama T, Sato T, Williams IR, Ohno H. Development of intestinal M cells and follicle-associated epithelium is regulated by TRAF6-mediated NF-κB signaling. J Exp Med 2018; 215:501-519. [PMID: 29339448 PMCID: PMC5789402 DOI: 10.1084/jem.20160659] [Citation(s) in RCA: 61] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2016] [Revised: 11/05/2017] [Accepted: 12/13/2017] [Indexed: 12/31/2022] Open
Abstract
TRAF6 is essential for RANK-mediated NF-κB activation and is involved in the development of several types of cells. Kanaya et al. demonstrate that RANK–TRAF6-mediated NF-κB is essential for the development of M cells and FAE. M cells are located in the follicle-associated epithelium (FAE) that covers Peyer’s patches (PPs) and are responsible for the uptake of intestinal antigens. The differentiation of M cells is initiated by receptor activator of NF-κB. However, the intracellular pathways involved in M cell differentiation are still elusive. In this study, we demonstrate that the NF-κB pathway activated by RANK is essential for M cell differentiation using in vitro organoid culture. Overexpression of NF-κB transcription factors enhances the expression of M cell–associated molecules but is not sufficient to complete M cell differentiation. Furthermore, we evaluated the requirement for tumor necrosis factor receptor–associated factor 6 (TRAF6). Conditional deletion of TRAF6 in the intestinal epithelium causes a complete loss of M cells in PPs, resulting in impaired antigen uptake into PPs. In addition, the expression of FAE-associated genes is almost silenced in TRAF6-deficient mice. This study thus demonstrates the crucial role of TRAF6-mediated NF-κB signaling in the development of M cells and FAE.
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Affiliation(s)
- Takashi Kanaya
- Laboratory for Intestinal Ecosystem, RIKEN Center for Integrative Medical Sciences, Kanagawa, Japan.,Division of Immunobiology, Department of Medical Life Science, Graduate School of Medical Life Science, Yokohama City University, Kanagawa, Japan
| | - Sayuri Sakakibara
- Laboratory for Intestinal Ecosystem, RIKEN Center for Integrative Medical Sciences, Kanagawa, Japan
| | - Toshi Jinnohara
- Laboratory for Intestinal Ecosystem, RIKEN Center for Integrative Medical Sciences, Kanagawa, Japan.,Division of Immunobiology, Department of Medical Life Science, Graduate School of Medical Life Science, Yokohama City University, Kanagawa, Japan
| | - Masami Hachisuka
- Laboratory for Intestinal Ecosystem, RIKEN Center for Integrative Medical Sciences, Kanagawa, Japan.,Division of Immunobiology, Department of Medical Life Science, Graduate School of Medical Life Science, Yokohama City University, Kanagawa, Japan
| | - Naoko Tachibana
- Laboratory for Intestinal Ecosystem, RIKEN Center for Integrative Medical Sciences, Kanagawa, Japan
| | - Shinya Hidano
- Department of Infectious Diseases Control, Faculty of Medicine, Oita University, Oita, Japan
| | - Takashi Kobayashi
- Department of Infectious Diseases Control, Faculty of Medicine, Oita University, Oita, Japan
| | - Shunsuke Kimura
- Laboratory of Histology and Cytology, Graduate School of Medicine, Hokkaido University, Sapporo, Japan
| | - Toshihiko Iwanaga
- Laboratory of Histology and Cytology, Graduate School of Medicine, Hokkaido University, Sapporo, Japan
| | - Tomoo Nakagawa
- Department of Gastroenterology, Graduate School of Medicine, Chiba University, Chiba, Japan
| | - Tatsuro Katsuno
- Department of Gastroenterology, Graduate School of Medicine, Chiba University, Chiba, Japan
| | - Naoya Kato
- Department of Gastroenterology, Graduate School of Medicine, Chiba University, Chiba, Japan
| | - Taishin Akiyama
- Laboratory for Immune Homeostasis, RIKEN Center for Integrative Medical Sciences, Kanagawa, Japan
| | - Toshiro Sato
- Department of Gastroenterology, Keio University School of Medicine, Tokyo, Japan
| | - Ifor R Williams
- Department of Pathology, Emory University School of Medicine, Atlanta, GA
| | - Hiroshi Ohno
- Laboratory for Intestinal Ecosystem, RIKEN Center for Integrative Medical Sciences, Kanagawa, Japan .,Division of Immunobiology, Department of Medical Life Science, Graduate School of Medical Life Science, Yokohama City University, Kanagawa, Japan
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45
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Hill DR, Huang S, Nagy MS, Yadagiri VK, Fields C, Mukherjee D, Bons B, Dedhia PH, Chin AM, Tsai YH, Thodla S, Schmidt TM, Walk S, Young VB, Spence JR. Bacterial colonization stimulates a complex physiological response in the immature human intestinal epithelium. eLife 2017; 6:29132. [PMID: 29110754 PMCID: PMC5711377 DOI: 10.7554/elife.29132] [Citation(s) in RCA: 126] [Impact Index Per Article: 15.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2017] [Accepted: 10/29/2017] [Indexed: 12/19/2022] Open
Abstract
The human gastrointestinal tract is immature at birth, yet must adapt to dramatic changes such as oral nutrition and microbial colonization. The confluence of these factors can lead to severe inflammatory disease in premature infants; however, investigating complex environment-host interactions is difficult due to limited access to immature human tissue. Here, we demonstrate that the epithelium of human pluripotent stem-cell-derived human intestinal organoids is globally similar to the immature human epithelium and we utilize HIOs to investigate complex host-microbe interactions in this naive epithelium. Our findings demonstrate that the immature epithelium is intrinsically capable of establishing a stable host-microbe symbiosis. Microbial colonization leads to complex contact and hypoxia driven responses resulting in increased antimicrobial peptide production, maturation of the mucus layer, and improved barrier function. These studies lay the groundwork for an improved mechanistic understanding of how colonization influences development of the immature human intestine.
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Affiliation(s)
- David R Hill
- Division of Gastroenterology, Department of Internal Medicine, University of Michigan, Ann Arbor, United States
| | - Sha Huang
- Division of Gastroenterology, Department of Internal Medicine, University of Michigan, Ann Arbor, United States
| | - Melinda S Nagy
- Division of Gastroenterology, Department of Internal Medicine, University of Michigan, Ann Arbor, United States
| | - Veda K Yadagiri
- Division of Gastroenterology, Department of Internal Medicine, University of Michigan, Ann Arbor, United States
| | - Courtney Fields
- Division of Infectious Disease, Department of Internal Medicine, University of Michigan, Ann Arbor, United States
| | - Dishari Mukherjee
- Department of Microbiology and Immunology, University of Michigan, Ann Arbor, United States
| | - Brooke Bons
- Division of Infectious Disease, Department of Internal Medicine, University of Michigan, Ann Arbor, United States
| | - Priya H Dedhia
- Department of Surgery, University of Michigan, Ann Arbor, United States
| | - Alana M Chin
- Division of Gastroenterology, Department of Internal Medicine, University of Michigan, Ann Arbor, United States
| | - Yu-Hwai Tsai
- Division of Gastroenterology, Department of Internal Medicine, University of Michigan, Ann Arbor, United States
| | - Shrikar Thodla
- Division of Gastroenterology, Department of Internal Medicine, University of Michigan, Ann Arbor, United States
| | - Thomas M Schmidt
- Department of Microbiology and Immunology, University of Michigan, Ann Arbor, United States
| | - Seth Walk
- Department of Microbiology and Immunology, Montana State University, Bozeman, United States
| | - Vincent B Young
- Division of Infectious Disease, Department of Internal Medicine, University of Michigan, Ann Arbor, United States
| | - Jason R Spence
- Division of Gastroenterology, Department of Internal Medicine, University of Michigan, Ann Arbor, United States.,Department of Cell andDevelopmental Biology, University of Michigan, Ann Arbor, United States
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46
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Protease-Mediated Suppression of DRG Neuron Excitability by Commensal Bacteria. J Neurosci 2017; 37:11758-11768. [PMID: 29089436 DOI: 10.1523/jneurosci.1672-17.2017] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2017] [Revised: 10/23/2017] [Indexed: 12/27/2022] Open
Abstract
Peripheral pain signaling reflects a balance of pronociceptive and antinociceptive influences; the contribution by the gastrointestinal microbiota to this balance has received little attention. Disorders, such as inflammatory bowel disease and irritable bowel syndrome, are associated with exaggerated visceral nociceptive actions that may involve altered microbial signaling, particularly given the evidence for bacterial dysbiosis. Thus, we tested whether a community of commensal gastrointestinal bacteria derived from a healthy human donor (microbial ecosystem therapeutics; MET-1) can affect the excitability of male mouse DRG neurons. MET-1 reduced the excitability of DRG neurons by significantly increasing rheobase, decreasing responses to capsaicin (2 μm) and reducing action potential discharge from colonic afferent nerves. The increase in rheobase was accompanied by an increase in the amplitude of voltage-gated K+ currents. A mixture of bacterial protease inhibitors abrogated the effect of MET-1 effects on DRG neuron rheobase. A serine protease inhibitor but not inhibitors of cysteine proteases, acid proteases, metalloproteases, or aminopeptidases abolished the effects of MET-1. The serine protease cathepsin G recapitulated the effects of MET-1 on DRG neurons. Inhibition of protease-activated receptor-4 (PAR-4), but not PAR-2, blocked the effects of MET-1. Furthermore, Faecalibacterium prausnitzii recapitulated the effects of MET-1 on excitability of DRG neurons. We conclude that serine proteases derived from commensal bacteria can directly impact the excitability of DRG neurons, through PAR-4 activation. The ability of microbiota-neuronal interactions to modulate afferent signaling suggests that therapies that induce or correct microbial dysbiosis may impact visceral pain.SIGNIFICANCE STATEMENT Commercially available probiotics have the potential to modify visceral pain. Here we show that secretory products from gastrointestinal microbiota derived from a human donor signal to DRG neurons. Their secretory products contain serine proteases that suppress excitability via activation of protease-activated receptor-4. Moreover, from this community of commensal microbes, Faecalibacterium prausnitzii strain 16-6-I 40 fastidious anaerobe agar had the greatest effect. Our study suggests that therapies that induce or correct microbial dysbiosis may affect the excitability of primary afferent neurons, many of which are nociceptive. Furthermore, identification of the bacterial strains capable of suppressing sensory neuron excitability, and their mechanisms of action, may allow therapeutic relief for patients with gastrointestinal diseases associated with pain.
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Lu Y, Liu X, Xie M, Liu M, Ye M, Li M, Chen XM, Li X, Zhou R. The NF-κB-Responsive Long Noncoding RNA FIRRE Regulates Posttranscriptional Regulation of Inflammatory Gene Expression through Interacting with hnRNPU. THE JOURNAL OF IMMUNOLOGY 2017; 199:3571-3582. [PMID: 28993514 DOI: 10.4049/jimmunol.1700091] [Citation(s) in RCA: 108] [Impact Index Per Article: 13.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/18/2017] [Accepted: 09/11/2017] [Indexed: 11/19/2022]
Abstract
Long noncoding RNAs, a newly identified class of noncoding RNAs, are important regulators of gene expression in innate immunity. We report in this study that the transcription of FIRRE, a conserved long noncoding RNA between humans and mice, is controlled by NF-κB signaling in macrophages and intestinal epithelial cells. Functionally, FIRRE appears to positively regulate the expression of several inflammatory genes in macrophages or intestinal epithelial cells in response to LPS stimulation via posttranscriptional mechanisms. Specifically, FIRRE physically interacts with heterogeneous nuclear ribonucleoproteins U, regulating the stability of mRNAs of selected inflammatory genes through targeting the AU-rich elements of their mRNAs in cells following LPS stimulation. Therefore, our data indicate a new regulatory role for NF-κB-responsive FIRRE in the posttranscriptional regulation of inflammatory genes in the innate immune system.
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Affiliation(s)
- Yajing Lu
- Hubei Province Key Laboratory of Allergy and Immunology, School of Basic Medical Sciences, Wuhan University, Wuhan, Hubei 430071, China.,Department of Endocrinology, Institute of Geriatric Medicine, Liyuan Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430077, China
| | - Xu Liu
- Hubei Province Key Laboratory of Allergy and Immunology, School of Basic Medical Sciences, Wuhan University, Wuhan, Hubei 430071, China
| | - Minghong Xie
- Hubei Province Key Laboratory of Allergy and Immunology, School of Basic Medical Sciences, Wuhan University, Wuhan, Hubei 430071, China
| | - Mingjia Liu
- Hubei Province Key Laboratory of Allergy and Immunology, School of Basic Medical Sciences, Wuhan University, Wuhan, Hubei 430071, China
| | - Mengling Ye
- Hubei Province Key Laboratory of Allergy and Immunology, School of Basic Medical Sciences, Wuhan University, Wuhan, Hubei 430071, China
| | - Mingxuan Li
- Hubei Province Key Laboratory of Allergy and Immunology, School of Basic Medical Sciences, Wuhan University, Wuhan, Hubei 430071, China
| | - Xian-Ming Chen
- Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, NE 68178; and
| | - Xiaoqing Li
- Center for Stem Cell Research and Application, Union Hospital, Tongji Medical School, Huazhong University of Science and Technology, Wuhan, Hubei 430022, China
| | - Rui Zhou
- Hubei Province Key Laboratory of Allergy and Immunology, School of Basic Medical Sciences, Wuhan University, Wuhan, Hubei 430071, China;
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48
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Santana AL, Oldenburg DG, Kirillov V, Malik L, Dong Q, Sinayev R, Marcu KB, White DW, Krug LT. RTA Occupancy of the Origin of Lytic Replication during Murine Gammaherpesvirus 68 Reactivation from B Cell Latency. Pathogens 2017; 6:pathogens6010009. [PMID: 28212352 PMCID: PMC5371897 DOI: 10.3390/pathogens6010009] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2016] [Accepted: 02/10/2017] [Indexed: 02/06/2023] Open
Abstract
RTA, the viral Replication and Transcription Activator, is essential for rhadinovirus lytic gene expression upon de novo infection and reactivation from latency. Lipopolysaccharide (LPS)/toll-like receptor (TLR)4 engagement enhances rhadinovirus reactivation. We developed two new systems to examine the interaction of RTA with host NF-kappaB (NF-κB) signaling during murine gammaherpesvirus 68 (MHV68) infection: a latent B cell line (HE-RIT) inducible for RTA-Flag expression and virus reactivation; and a recombinant virus (MHV68-RTA-Bio) that enabled in vivo biotinylation of RTA in BirA transgenic mice. LPS acted as a second stimulus to drive virus reactivation from latency in the context of induced expression of RTA-Flag. ORF6, the gene encoding the single-stranded DNA binding protein, was one of many viral genes that were directly responsive to RTA induction; expression was further increased upon treatment with LPS. However, NF-κB sites in the promoter of ORF6 did not influence RTA transactivation in response to LPS in HE-RIT cells. We found no evidence for RTA occupancy of the minimal RTA-responsive region of the ORF6 promoter, yet RTA was found to complex with a portion of the right origin of lytic replication (oriLyt-R) that contains predicted RTA recognition elements. RTA occupancy of select regions of the MHV-68 genome was also evaluated in our novel in vivo RTA biotinylation system. Streptavidin isolation of RTA-Bio confirmed complex formation with oriLyt-R in LPS-treated primary splenocytes from BirA mice infected with MHV68 RTA-Bio. We demonstrate the utility of reactivation-inducible B cells coupled with in vivo RTA biotinylation for mechanistic investigations of the interplay of host signaling with RTA.
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Affiliation(s)
- Alexis L Santana
- The Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York, NY 10016, USA.
- Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794, USA.
| | | | - Varvara Kirillov
- Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794, USA.
| | - Laraib Malik
- Department of Computer Science, Stony Brook University, Stony Brook, NY 11794, USA.
| | - Qiwen Dong
- Program in Molecular and Cellular Biology, Stony Brook University, Stony Brook, NY 11794, USA.
| | - Roman Sinayev
- Applied Mathematics and Statistics, Stony Brook University, Stony Brook, NY 11794, USA.
| | - Kenneth B Marcu
- Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794, USA.
- Biomedical Research Foundation Academy of Athens (BRFAA), Athens 115 27, Greece.
- Biochemistry and Cell Biology Dept., Stony Brook University, Stony Brook, NY 11794, USA.
- Department of Pathology, Health Sciences Center, Stony Brook University, Stony Brook, NY 11794, USA.
| | | | - Laurie T Krug
- Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794, USA.
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49
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Tse AKW, Chen YJ, Fu XQ, Su T, Li T, Guo H, Zhu PL, Kwan HY, Cheng BCY, Cao HH, Lee SKW, Fong WF, Yu ZL. Sensitization of melanoma cells to alkylating agent-induced DNA damage and cell death via orchestrating oxidative stress and IKKβ inhibition. Redox Biol 2017; 11:562-576. [PMID: 28107677 PMCID: PMC5247288 DOI: 10.1016/j.redox.2017.01.010] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2016] [Revised: 01/11/2017] [Accepted: 01/11/2017] [Indexed: 01/22/2023] Open
Abstract
Nitrosourea represents one of the most active classes of chemotherapeutic alkylating agents for metastatic melanoma. Treatment with nitrosoureas caused severe systemic side effects which hamper its clinical use. Here, we provide pharmacological evidence that reactive oxygen species (ROS) induction and IKKβ inhibition cooperatively enhance nitrosourea-induced cytotoxicity in melanoma cells. We identified SC-514 as a ROS-inducing IKKβ inhibitor which enhanced the function of nitrosoureas. Elevated ROS level results in increased DNA crosslink efficiency triggered by nitrosoureas and IKKβ inhibition enhances DNA damage signals and sensitizes nitrosourea-induced cell death. Using xenograft mouse model, we confirm that ROS-inducing IKKβ inhibitor cooperates with nitrosourea to reduce tumor size and malignancy in vivo. Taken together, our results illustrate a new direction in nitrosourea treatment, and reveal that the combination of ROS-inducing IKKβ inhibitors with nitrosoureas can be potentially exploited for melanoma therapy.
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Affiliation(s)
- Anfernee Kai-Wing Tse
- Center for Cancer and Inflammation Research, School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong.
| | - Ying-Jie Chen
- Center for Cancer and Inflammation Research, School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong
| | - Xiu-Qiong Fu
- Center for Cancer and Inflammation Research, School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong
| | - Tao Su
- Center for Cancer and Inflammation Research, School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong
| | - Ting Li
- Center for Cancer and Inflammation Research, School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong
| | - Hui Guo
- Center for Cancer and Inflammation Research, School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong
| | - Pei-Li Zhu
- Center for Cancer and Inflammation Research, School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong
| | - Hiu-Yee Kwan
- Center for Cancer and Inflammation Research, School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong
| | - Brian Chi-Yan Cheng
- Center for Cancer and Inflammation Research, School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong
| | - Hui-Hui Cao
- Center for Cancer and Inflammation Research, School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong
| | - Sally Kin-Wah Lee
- Center for Cancer and Inflammation Research, School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong
| | - Wang-Fun Fong
- Center for Cancer and Inflammation Research, School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong
| | - Zhi-Ling Yu
- Center for Cancer and Inflammation Research, School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong.
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50
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Purushotham PM, Kim JM, Jo EK, Senthil K. Withanolides against TLR4-Activated Innate Inflammatory Signalling Pathways: A Comparative Computational and Experimental Study. Phytother Res 2016; 31:152-163. [PMID: 27859734 DOI: 10.1002/ptr.5746] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2016] [Revised: 09/23/2016] [Accepted: 10/22/2016] [Indexed: 11/12/2022]
Abstract
Innate inflammations are dominant causes of poor health and high mortality. The pathogen-associated molecular pattern and lipopolysaccharide (LPS) are sensed by immune cells through activation of toll-like receptor 4 leading to mitogen-activated protein kinases (MAPKs) and NF-κB activations. Controlled MAPK and Nf-κB inhibitors have been proposed as potential antiinflammatory drugs. Withania somnifera is an important medicinal herb with known antiinflammatory activity. In this study, the selected Withania somnifera extracts and withanolides were analysed on LPS-induced macrophages comparatively. Molecular docking analysis revealed withaferin A, withanone and withanolide A as effective withanolides against inflammatory target molecules. In experiments, withaferin A and withanone treatment had prominent suppressions on LPS-induced expression of pro-inflammatory cytokines in bone marrow-derived macrophages. Withaferin A regulated all the major four pathways (MAPKs and NF-κB) involved in innate inflammations. Similarly among the Withania extracts analysed, the in vitro propagated leaf and field grown root extracts containing high withaferin A content suppressed the inflammatory molecules through NF-κB and MAPK pathways. Withaferin A was found to be best in suppressing the activated inflammatory pathways among all the analysed withanolides. Therefore, withaferin A and extracts with high withaferin A content can be used as promising drug candidates against innate inflammations. Copyright © 2016 John Wiley & Sons, Ltd.
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Affiliation(s)
- Preethi M Purushotham
- Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam Institute for Home Science and Higher Education for Women University, Coimbatore, 641043, Tamil Nadu, India
| | - Jin-Man Kim
- Department of Pathology, College of Medicine, Chungnam National University, Daejeon, Korea
| | - Eun-Kyeong Jo
- Infection Signaling Network Research Center, College of Medicine, Chungnam National University, Daejeon, Korea
| | - Kalaiselvi Senthil
- Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam Institute for Home Science and Higher Education for Women University, Coimbatore, 641043, Tamil Nadu, India
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