Immunotherapy is an emerging strategy that awakens the intrinsic immune system for cancer treatment. Generally, successful immunotherapy of malignant tumours relies on the effective production of tumour-associated antigens and their lymph node delivery, antigen processing and presentation for T-cell activation, and the dismantling of the immunosuppressive tumour microenvironment. Toll-like receptor (TLR) agonists are potent stimulants in cancer immunotherapy, which can directly activate antigen-presenting cells (APCs) and further induce T cell activation for antitumour immune response and convert immunosuppressive tumour microenvironment to an immunogenic one for cooperative tumour ablation. However, TLR agonists for effective cancer immunotherapy have encountered essential challenges, such as insufficient immune activation and systemic side effects. In recent years, nano-immunomodulators with TLR agonists have been employed for tumour- and/or lymph node-targeted immune activation to improve the antitumour immune response and alleviate their systemic toxicities, providing a promising strategy for enhanced cancer immunotherapy. Herein, we introduce the recent progress in developing various TLR nano-immunomodulators for cancer immunotherapy via APC activation and tumour microenvironment remodelling. Upon elucidating the rational design principles of nano-immunomodulators, we elucidate the advancement of TLR nanoagonists to break the barriers in effective and safe Toll-like receptor stimulation for potent cancer immunotherapy.

The responses of N/TERT-1 and N/TERT-2G keratinocyte cell lines to oxidative stress and immune challenges were investigated to assess their suitability for dermatological testing. The cell lines were exposed to various stimuli, including PAMPs, DAMPs, H₂O₂, and menadione, to assess cytokine production, oxidative stress markers, cell viability, apoptosis, and membrane integrity. IL-1α, IL-6, IL-8, TNF-α, and TGF-β levels significantly increased in N/TERT-1 cells following exposure to LPS, while N/TERT-2G cells remained unaffected. Both cell lines showed increased production of IL-1α, IL-1β, TNF-α, IL-6, and IL-8 in response to dsDNA and LMW and HMW Poly I:C, although TGF-β significantly decreased only in N/TERT-1 cells. In response to H₂O₂, a dose-dependent increase in cytokine levels was observed in N/TERT-2G, whereas N/TERT-1 did not exhibit a clear dose-dependent response. Markers of oxidative stress, including SOD and GSH, displayed similar patterns in both cell lines, with N/TERT-2G showing slightly higher sensitivity. Lipid peroxidation and mitochondrial membrane potential fluctuations were more pronounced in N/TERT-2G, suggesting greater oxidative stress sensitivity. The baseline GSH levels were higher in N/TERT-1 cells, which may contribute towards the enhanced resilience to oxidative stress. Despite decreased viability in MTT assays following H₂O₂ exposure, the lack of significant changes in cleaved Caspase-3 levels indicated that apoptosis was not the primary mechanism of cell death. These findings highlight the distinct characteristics of N/TERT-1 and N/TERT-2G cells, with N/TERT-1 showing higher baseline resilience to oxidative stress and N/TERT-2G displaying greater sensitivity, particularly to H₂O₂. The study underscores the importance of selecting the appropriate cell line for specific research applications in skin biology and disease modelling, considering the differences in their responses to oxidative and immune challenges.

Microglia play a critical role in maintaining central nervous system (CNS) homeostasis and display remarkable plasticity in their response to inflammatory stimuli. However, the specific signaling profiles that microglia adopt during such challenges remain incompletely understood. Traditional transcriptomic approaches provide valuable insights, but fail to capture dynamic post-translational changes. In this study, we utilized time-resolved single-cell mass cytometry (CyTOF) to measure distinct signaling pathways activated in microglia upon exposure to bacterial and viral mimetics-lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (Poly(I:C)), respectively. Furthermore, we evaluated the immunomodulatory role of astrocytes on microglial signaling in mixed cultures. Microglia or mixed cultures derived from neonatal mice were treated with LPS or Poly(I:C) for 48 h. Cultures were stained with a panel of 33 metal-conjugated antibodies targeting signaling and identity markers. High-dimensional clustering analysis was used to identify emergent signaling modules. We found that LPS treatment led to more robust early activation of pp38, pERK, pRSK, and pCREB compared to Poly(I:C). Despite these differences, both LPS and Poly(I:C) upregulated the classical reactivity markers CD40 and CD86 at later time points. Strikingly, the presence of astrocytes significantly blunted microglial responses to both stimuli, particularly dampening CD40 upregulation. Our studies demonstrate that single-cell mass cytometry effectively captures the dynamic signaling landscape of microglia under pro-inflammatory conditions. This approach may pave the way for targeted therapeutic investigations of various neuroinflammatory disorders. Moreover, our findings underscore the necessity of considering cellular context, such as astrocyte presence, in interpreting microglial behavior during inflammation.

Systemic inflammatory conditions are classically characterized by an acute hyperinflammatory phase, followed by a late immunosuppressive phase that elevates the susceptibility to secondary infections. Comprehensive mechanistic understanding of these phases is largely lacking. To address this gap, we leveraged a controlled, human in vivo model of lipopolysaccharide (LPS)-induced systemic inflammation encompassing both phases. Single-cell RNA sequencing during the acute hyperinflammatory phase identified an inflammatory CD163+SLC39A8+CALR+ monocyte-like subset (infMono) at 4 h post-LPS administration. The late immunosuppressive phase was characterized by diminished expression of type I interferon (IFN)-responsive genes in monocytes, impaired myelopoiesis and a pronounced attenuation of the immune response on a secondary LPS challenge 1 week after the first. The infMono gene program and impaired myelopoiesis were also detected in patient cohorts with bacterial sepsis and coronavirus disease. IFNβ treatment restored type-I IFN responses and proinflammatory cytokine production and induced monocyte maturation, suggesting a potential treatment option for immunosuppression.

Intestinal IgA, produced by local intestinal B cells, is thought to play a major role in protection against intestinal infections. Rotavirus, a well-characterized intestinal virus, induces a rapid viral-specific intestinal IgA response that occurs in the absence of T cells. Previous work has indicated that dendritic cells facilitate the early IgA response to rotavirus. To determine whether the early Peyer's patch B cell activation associated with rotavirus infection in mice requires dendritic cells, we depleted dendritic cells and assessed B cell activation. Depletion of CD11c+ cells in vivo prior to infection resulted in a complete abrogation of Peyer's patch B cell activation. With the use of in vitro cell-based assays, CD11c+, but not T or CD11b+ cells, was shown to be essential for rotavirus-induced activation of B cells. Investigation of several pathways of B cell activation revealed that dendritic cell expression of MyD88 and signaling through the type I interferon receptor were critical for the ability of the virus to induce B cell activation. These findings indicate that CD11c+ dendritic cells can modulate B cell responses to viruses through toll-like receptor and type I interferon signaling pathways.IMPORTANCEDendritic cells are key mediators of immune responses in the intestine. They can capture and process rotavirus antigens and present these antigens to B cells, which produce critical IgA antibody that is essential for clearance of rotavirus infection and protection from reinfection. In the work presented here, we demonstrate that dendritic cell expression of MyD88, a key component of pattern recognition pathways, and not classical IgA pathway molecules such as BAFF and APRIL, is critical for the ability of the dendritic cell to induce the activation of B cells. Our findings emphasize the important role that dendritic cells play in initiating and regulating immune responses including T cell-independent B cell activation. A consideration of the role of dendritic cells in B cell activation and antibody production is an important feature in the development of therapeutic and preventive modalities to combat intestinal viral infections.

Human papillomavirus (HPV) is a major etiological agent of both malignant and benign lesions, with high-risk types, such as HPV16 and HPV18, being strongly linked to cervical cancer, while low-risk types like HPV11 are associated with benign conditions. While viral proteins such as E6 and E7 are well-established regulators of immune evasion, the role of E1 in modulating the host antiviral responses remains insufficiently characterized. This study investigates the immunomodulatory functions of HPV16 and HPV11 E1 in suppressing innate antiviral immune signaling pathways. Through a combination of RT-qPCR and luciferase reporter assays, we demonstrate that E1 suppresses the production of interferons and interferon-stimulated genes triggered by viral infections and the activation of RIG-I/MDA5-MAVS, TLR3-TRIF, cGAS-STING, and JAK-STAT pathways. Co-immunoprecipitation assays reveal that E1 interacts directly with key signaling molecules within these pathways. E1 also impairs TBK1 and IRF3 phosphorylation and obstructs the nuclear translocation of IRF3, thereby broadly suppressing IFN responses. Additionally, E1 disrupts the JAK-STAT pathway by binding STAT1, which prevents the assembly and nuclear localization of the ISGF3 complex containing STAT1, STAT2, and IRF9, thereby further diminishing antiviral response. These findings establish E1 as a pivotal regulator of immune evasion and suggest its potential as a novel therapeutic target to enhance antiviral immunity in HPV-associated diseases.

The contribution of innate immunity to clearance of viral infections of the liver, in particular sensing via Toll-like receptor 3 (TLR3), is incompletely understood. We aimed to identify the factors contributing to the TLR3 response in hepatocytes via CRISPR/Cas9 screening.

A genome-wide CRISPR/Cas9 screen on the TLR3 pathway was performed in two liver-derived cell lines, followed by siRNA knockdown validation. SiRNA knockdown and indisulam treatment were used to study the role of RNA-binding motif protein 39 (RBM39) in innate immunity upon poly(I:C) or cytokine treatment and viral infections. Transcriptome, proteome, and alternative splicing were studied via RNA sequencing and mass spectrometry upon depletion of RBM39.

Our CRISPR/Cas9 screen identified RBM39, which is highly expressed in hepatocytes, as an important regulator of the TLR3 pathway. Knockdown of RBM39 or treatment with indisulam, an aryl sulfonamide drug targeting RBM39 for proteasomal degradation, strongly reduced the induction of interferon-stimulated genes (ISGs) in response to double-stranded RNA (dsRNA) or viral infections. RNA sequencing (seq) and mass spectrometry identified that transcription and/or splicing of the key pathway components IRF3, RIG-I, and MDA5 were affected by RBM39 depletion, along with multiple other cellular processes identified previously. RBM39 knockdown further restrained type I and type III IFN pathways by reducing the expression of individual receptor subunits and STAT1/2. The function of RBM39 was furthermore not restricted to hepatocytes.

We identified RBM39 as a regulatory factor of cell intrinsic innate immune signaling. Depletion of RBM39 impaired TLR3, RIG-I/MDA5, and IFN responses by affecting the basal expression of key pathway components.

Orf virus (ORFV) is the type species of Parapoxvirus of the Poxviridae family that induces cutaneous pustular skin lesions in sheep and goats, and causes zoonotic infections in humans. Pattern recognition receptors (PRRs) sense pathogen-associated molecular patterns (PAMPs), leading to the triggering of the innate immune response through multiple signalling pathways involving type I interferons (IFNs). The major PAMPs generated during viral infection are nucleic acids, which are the most important molecules that are recognized by the host. The induction of type l IFNs leads to activation of the Janus kinase (JAK)-signal transducer activator of transcription (STAT) pathway, which results in the induction of hundreds of interferon-stimulated genes (ISGs), many of which encode proteins that have antiviral roles in eliminating virus infection and create an antiviral state. Genetic and functional analyses have revealed that ORFV, as found for other poxviruses, has evolved multiple immunomodulatory genes and strategies that manipulate the innate immune sensing response.

Inflammation is one defense mechanism of the body that has multiple origins, ranging from physical agents to infectious agents including viruses and bacteria. The resolution of inflammation has emerged as a critical endogenous process that protects host tissues from prolonged or excessive inflammation, which can become chronic. Failure of the inflammation resolution is a key pathological mechanism that drives the progression of numerous inflammatory diseases. Owing to the various side effects of currently available drugs to control inflammation, novel therapeutic agents that can prevent or suppress inflammation are needed. Supersulfides are highly reactive and biologically potent molecules that function as antioxidants, redox regulators, and modulators of cell signaling. The catenation state of individual sulfur atoms endows supersulfides with unique biological activities. Great strides have recently been made in achieving a molecular understanding of these sulfur species, which participate in various physiological and pathological pathways. This review mainly focuses on the anti-inflammatory effects of supersulfides. The review starts with an overview of supersulfide biology and highlights the roles of supersulfides in both immune and inflammatory responses. The various donors used to generate supersulfides are assessed as research tools and potential therapeutic agents. Deeper understanding of the molecular and cellular bases of supersulfide-driven biology can help guide the development of innovative therapeutic strategies to prevent and treat diseases associated with various immune and inflammatory responses.

ARNAX is a synthetic nucleotide-based Toll-like receptor 3 (TLR3) ligand that specifically stimulates the TLR3/TIR domain-containing adaptor molecule 1 (TICAM-1) pathway without activating inflammatory responses. ARNAX activates cellular immunity via cross-presentation; hence, its practical application has been demonstrated in cancer immunotherapy. Given the importance of cellular immunity in virus infections, ARNAX is expected to be a more effective vaccine adjuvant for virus infections than alum, an adjuvant approved for human use that mainly enhances humoral immunity. In the present study, the trimeric recombinant spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was prepared as a vaccine antigen and formulated with ARNAX. When T-cell and neutralizing antibody responses were evaluated in immunized mice, antigen formulated with ARNAX generated significantly larger numbers of antigenspecific CD4+ and CD8+ T cells, as well as higher titers of neutralizing antibodies, compared to antigen alone or antigen formulated with alum. In experiments where immunized mice were challenged with a SARS-CoV-2 mouse-adapted virus derived from the ancestral strain, immunization with antigen formulated with ARNAX reduced virus titers in the lungs at 3 days post-infection to a much greater extent than did immunization with either antigen alone or that formulated with alum. These results show that ARNAX potently enhances the levels of both cellular and humoral immunity above those seen with alum, providing significantly greater viral clearing responses. Thus, ARNAX may act as a useful adjuvant for prophylactic vaccines, particularly for viral infectious diseases.

Cellular immunity is a critical immunological defense system against virus infections. However, aluminum salts, the most widely used adjuvant for vaccines for human use, do not promote strong cellular immunity. To prepare for the next pandemic of viral origin, the development of Th1-type adjuvants with low adverse reactions that induce cellular immunity is necessary. ARNAX is a TLR3 agonist consisting of DNA-RNA hybrid nucleic acid, which is expected to be an adjuvant that induces cellular immunity. The present study using a coronavirus disease 2019 mouse model demonstrated that ARNAX potently induces cellular immunity in addition to humoral immunity with minimal induction of inflammatory cytokines. Therefore, ARNAX has the potential to be used as a potent and welltolerated adjuvant for vaccines against pandemic viruses emerging in the future.

A dysregulated nucleotide metabolism has been implicated in the pathogenesis of diabetic retinopathy (DR). RNA sequencing datasets, GSE102485, GSE60436, and GSE165784, were downloaded from the GEO database. The differentially expressed genes (DEGs) between the DR and controls overlapped with nucleotide metabolism-related genes (NM-RGs), resulting in the differentially expressed NM-RGs (DE-NMRGs). Next, the core genes were identified by the five algorithms of the CytoHubba plugin. Receiver Operating Characteristic (ROC) curves and gene expression analysis were utilized to confirm the biomarkers. Then, the correlations between biomarker expression and the immune-related module were analyzed. The miRNA and transcription factor (TF) predictions, biomarker-targeting drugs, and molecular docking were implemented separately. The interaction between each subcluster of DR was elucidated through single-cell RNA (scRNA) analysis. Moreover, RT-PCR was applied to verify the expression of the biomarkers. In GSE102485, 48 DE-NMRGs were identified via the intersection of 1359 DEGs and 882 NM-RGs. Using the CytoHubba plugin, HMOX1, TLR4, and ACE were selected as core genes. As per the GSVA result, the interferon alpha response, IL6_JAK_STAT3 signaling, and apoptosis were activated in the DR group. The TF prediction identified TLR4 and HMOX1 as potential target genes of USF2. In conclusion, ACE and HMOX1 were possible diagnostic biomarkers related to nucleotide metabolism in DR.

Toll-like receptor 4 (TLR4) signaling at the plasma membrane and in endosomes results in distinct contributions to inflammation and host defence. Current understanding indicates that endocytosis of cell surface-activated TLR4 is required to enable subsequent signaling from endosomes. Contrary to this prevailing model, our data show that endosomal TLR4 signaling is not reliant on cell surface-expressed TLR4 or ligand-induced TLR4 endocytosis. Moreover, previously recognized requirements for the accessory molecule CD14 in TLR4 endocytosis and endosomal signaling are likely attributable to CD14 binding as well as trafficking and transferring lipopolysaccharide (LPS) to TLR4 at different subcellular localizations. TLR4 endocytosis requires the TLR4 intracellular signaling domain, contributions by phospholipase C gamma 2, spleen tyrosine kinase, E1/E2 ubiquitination enzymes, but not canonical TLR signaling adaptors and cascades. Thus, our study identifies independently operating TLR4 signaling modes that control TLR4 endocytosis, pro-inflammatory cell surface-derived, as well as endosomal TLR4 signaling. This revised understanding of how TLR4 functions within cells might be harnessed to selectively amplify or restrict TLR4 activation for the development of adjuvants, vaccines and therapeutics.

Chimeric antigen receptor-T (CAR-T) cell therapy is a cellular immunotherapy that has emerged in recent years, and increasing studies showed that therapeutic resistance to CAR-T cell therapy presents in colorectal cancer patients. Lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria, is known to preserve a high concentration in the colon. Whether LPS is a contributing factor to the development of resistance in colorectal cancer cells against CAR-T cell therapy remains unclear. For in vivo experiments, colorectal cancer cells COLO205 were pretreated with LPS for 24 h and then were injected into nude mice through the tail vein, followed by CAR-T cells transplantation one day later. Later, the number of tumors in the lung tissues of the mice was observed. The in vitro experiments were performed on COLO205 cells, which were treated with LPS for 24 h. The effect of LPS on the stemness of COLO205 and SW620 cells was observed by using the colony formation assay and spheroidization experiments. The effect of LPS on the expression of stemness-related genes, including CD44, SOX2, and NANOG, was observed by qRT-PCR assay, Western blotting assay, and immunofluorescence staining. Inhibitors of TGF-β and the MYD88 inhibitor were used to study the mechanisms by which LPS induces the stemness enhancement and resistance to CAR-T cell therapy of COLO205 cells. The correlation between MYD88 and TGFB1, as well as the correlation between TGFB1 and stemness-related genes was analyzed using the TCGA database. Both the in vivo assay of nude mice and the in vitro assay showed that LPS pretreatment could induce resistance to CAR-T cell therapy of colorectal cancer cells. LPS could enhance COLO205 and SW620 cells stemness presented by upregulation of CD44, SOX2, and NANOG. The reverse interfering assay using the TGF-β inhibitor indicated that the autosecretion of TGF-β induced by LPS played a critical role in the stemness enhancement of colorectal cancer cells. The TCGA database analysis revealed a strong positive correlation between MYD88 and TGFB1. Additionally, TGFB1 has been found to upregulate the expression of genes associated with stemness. Further mechanism studies showed that the TLR4/MYD88 pathway medicates LPS-induced TGF-β expression. Our results suggested that LPS-induced resistance to CAR-T cell therapy of colorectal cancer cells through stemness enhancement. TLR4/MYD88 signal pathway-dependent TGF-β expression was involved in stemness enhancement and CAR-T cell therapy resistance. In conclusion, our findings help us to understand the underlying mechanisms of CAR-T cell therapy resistance and indicate that inhibitors of TGF-β and MYD88 are promising targeting candidates to promote a therapeutic effect of CAR-T cell therapy in colorectal cancer in the clinic.

Autoimmune diseases are typically characterized by aberrant activation of immune system that leads to excessive inflammatory reactions and tissue damage. Nevertheless, precise targeted and efficient therapies are limited. Thus, studies into novel therapeutic targets for the management of autoimmune diseases are urgently needed. Radical S-adenosyl methionine domain-containing 2 (RSAD2) is an interferon-stimulated gene (ISG) renowned for the antiviral properties of the protein it encodes, named viperin. An increasing number of studies have underscored the new roles of RSAD2/viperin in immunomodulation and mitochondrial metabolism. Previous studies have shown that there is a complex interplay between RSAD2/vipeirn and mitochondria and that binding of the iron-sulfur (Fe-S) cluster is necessary for the involvement of viperin in mitochondrial metabolism. Viperin influences the proliferation and development of immune cells as well as inflammation via different signaling pathways. However, the function of RSAD2/viperin varies in different studies and a comprehensive overview of this emerging theme is lacking. This review will describe the characteristics of RSAD2/viperin, decipher its function in immunometabolic processes, and clarify the crosstalk between RSAD2/viperin and mitochondria. Furthermore, we emphasize the crucial roles of RSAD2 in autoimmune diseases and its potential application value.

Receptors are crucial for converting chemical and environmental signals into cellular responses, making them prime targets in drug discovery, with about 70% of drugs targeting these receptors. Biased signaling, or functional selectivity, has revolutionized drug development by enabling precise modulation of receptor signaling pathways. This concept is more firmly established in G protein-coupled receptor and has now been applied to other receptor types, including ion channels, receptor tyrosine kinases, and nuclear receptors. Advances in structural biology have further refined our understanding of biased signaling. This targeted approach enhances therapeutic efficacy and potentially reduces side effects. Numerous biased drugs have been developed and approved as therapeutics to treat various diseases, demonstrating their significant therapeutic potential. This review provides a comprehensive overview of biased signaling in drug discovery and disease treatment, highlighting recent advancements and exploring the therapeutic potential of these innovative modulators across various diseases.

The hybrid offspring of barbel chub Squaliobarbus curriculus and grass carp Ctenopharyngodon idella exhibit stronger resistance to the grass carp reovirus (GCRV) infection than grass carp. Toll-like receptors (TLRs) play indispensable roles in the antiviral immunity of fish. In this study, the structures and antiviral immune functions of barbel chub TLR19 (ScTLR19) and grass carp TLR19 (CiTLR19) were compared. The amino acid sequence of ScTLR19 shared high similarity (97.4%) and identity (94.0%) with that of CiTLR19, and a phylogenetic tree revealed the close evolutionary relationship between ScTLR19 and CiTLR19. Protein domain composition analyses showed that ScTLR19 possessed an additional leucine-rich repeat (designated as LRR9) located at amino acid positions 654-677 in the extracellular region, which was absent in CiTLR19. Multiple sequence alignment and three-dimensional structure comparison also indicated that the extracellular regions of ScTLR19 and CiTLR19 exhibited greater differences compared to their intracellular regions. Molecular docking revealed that the extracellular region of ScTLR19 (docking score = -512.31) showed a stronger tendency for binding with polyI:C, compared to the extracellular region of CiTLR19 (docking score = -474.90). Replacing LRR9 in ScTLR19 with the corresponding amino acid sequence from CiTLR19 reduced the binding activity of ScTLR19 to polyI:C, as confirmed by an ELISA. Moreover, overexpression experiments suggested that ScTLR19 could regulate both the IRF3-TRIF and IRF3-MyD88 signaling pathways during GCRV infection, while CiTLR19 only regulated the IRF3-MyD88 signaling pathway. Importantly, replacing LRR9 in ScTLR19 with the corresponding amino acid sequence from CiTLR19 altered the expression regulation on IRF3, MyD88, and TRIF during GCRV infection. These findings collectively reveal the structural and functional differences between ScTLR19 and CiTLR19, and they may provide data to support a deeper understanding of the molecular mechanisms underlying the differences in GCRV resistance between barbel chub and grass carp, as well as the genetic basis for the heterosis of GCRV resistance in their hybrid offspring.

SUMMARYHuman coronaviruses cause a range of respiratory diseases, from the common cold (HCoV-229E, HCoV-NL63, HCoV-OC43, and SARS-CoV-2) to lethal pneumonia (SARS-CoV, SARS-CoV-2, and MERS-CoV). Coronavirus interactions with host innate immune antiviral responses are an important determinant of disease outcome. This review compares the host's innate response to different human coronaviruses. Host antiviral defenses discussed in this review include frontline defenses against respiratory viruses in the nasal epithelium, early sensing of viral infection by innate immune effectors, double-stranded RNA and stress-induced antiviral pathways, and viral antagonism of innate immune responses conferred by conserved coronavirus nonstructural proteins and genus-specific accessory proteins. The common cold coronaviruses HCoV-229E and -NL63 induce robust interferon signaling and related innate immune pathways, SARS-CoV and SARS-CoV-2 induce intermediate levels of activation, and MERS-CoV shuts down these pathways almost completely.

The lung as an organ that is fully exposed to the external environment for extended periods, comes into contact with numerous inhaled microorganisms. Lung macrophages are crucial for maintaining lung immunity and operate primarily through signaling pathways such as toll-like receptor 4 and nuclear factor-κB pathways. These macrophages constitute a diverse population with significant plasticity, exhibiting different phenotypes and functions on the basis of their origin, tissue residence, and environmental factors. During lung homeostasis, they are involved in the clearance of inhaled particles, cellular remnants, and even participate in metabolic processes. In disease states, lung macrophages transition from the inflammatory M1 phenotype to the anti-inflammatory M2 phenotype. These distinct phenotypes have varying transcriptional profiles and serve different functions, from combating pathogens to repairing inflammation-induced damage. However, macrophages can also exacerbate lung injury during prolonged inflammation or exposure to antigens. In this review, we delve into the diverse roles of pulmonary macrophages the realms in homeostasis, pneumonia, tuberculosis, and lung tumors.

Investigating innate immunity and its signaling transduction is essential to understand inflammation and host defence mechanisms. Toll-like receptors (TLRs), an evolutionarily ancient group of pattern recognition receptors, are crucial for detecting microbial components and initiating immune responses. This review summarizes the mechanisms and outcomes of TLR-mediated signaling, focusing on motifs shared with other immunological pathways, which enhances our understanding of the innate immune system. TLRs recognize molecular patterns in microbial invaders, activate innate immunity and promote antigen-specific adaptive immunity, and each of them triggers unique downstream signaling patterns. Recent advances have highlighted the importance of supramolecular organizing centers (SMOCs) in TLR signaling, ensuring precise cellular responses and pathogen detection. Furthermore, this review illuminates how TLR pathways coordinate metabolism and gene regulation, contributing to adaptive immunity and providing novel insights for next-generation therapeutic strategies. Ongoing studies hold promise for novel treatments against infectious diseases, autoimmune conditions, and cancers.

Nucleic acids from both self- and non-self-sources act as vital danger signals that trigger immune responses. Critical illnesses such as acute respiratory distress syndrome, sepsis, trauma and ischemia lead to the aberrant cytosolic accumulation and massive release of nucleic acids that are detected by antiviral innate immune receptors in the endosome or cytosol. Activation of receptors for deoxyribonucleic acids and ribonucleic acids triggers inflammation, a major contributor to morbidity and mortality in critically ill patients. In the past decade, there has been growing recognition of the therapeutic potential of targeting nucleic acid sensing in critical care. This review summarizes current knowledge of nucleic acid sensing in acute respiratory distress syndrome, sepsis, trauma and ischemia. Given the extensive research on nucleic acid sensing in common pathological conditions like cancer, autoimmune disorders, metabolic disorders and aging, we provide a comprehensive summary of nucleic acid sensing beyond critical illness to offer insights that may inform its role in critical conditions. Additionally, we discuss potential therapeutic strategies that specifically target nucleic acid sensing. By examining nucleic acid sources, sensor activation and function, as well as the impact of regulating these pathways across various acute diseases, we highlight the driving role of nucleic acid sensing in critical illness.

The LPS-TLR4 immune response is a critical mechanism in the body's defense against Gram-negative bacterial infections, yet its dysregulation can lead to severe inflammatory diseases. Lipopolysaccharide (LPS), a pivotal pathogen-associated molecular pattern (PAMP) on the surface of gram-negative bacteria, is recognized by Toll-like receptor 4 (TLR4), initiating a complex cascade of immune responses. This review delves into the intricate molecular structures and protein-protein interactions that underpin the LPS-TLR4 signaling pathway, offering a comprehensive analysis of both extracellular recognition and intracellular signal transduction. We explore the roles of key molecules such as LBP, CD14, MD-2, and TLR4 in the initial recognition of LPS, followed by the downstream signaling pathways mediated by MyD88-dependent and MyD88-independent mechanisms. The MyD88-dependent pathway primarily activates NF-κB and AP-1, leading to macrophage M1 polarization and the release of pro-inflammatory cytokines, while the MyD88-independent pathway triggers IRF activation and type-I interferon production. By elucidating the structural basis and functional interactions of these signaling molecules, this review not only enhances our understanding of the LPS-TLR4 immune response but also highlights its implications in both infectious and non-infectious diseases. Our findings underscore the potential of targeting this pathway for therapeutic interventions, offering new avenues for the treatment of inflammatory and immune-related disorders.

This study primarily aims to elucidate the underlying mechanism of Tenascin-C in neuroinflammation and microglia polarization in a mouse model of subarachnoid hemorrhage (SAH).

The subarachnoid hemorrhage model was constructed by injecting blood into the anterior chiasmatic cistern and stimulating primary microglia with hemoglobin in vitro. Then, Imatinib mesylate was used to inhibit Tenascin-C. Through neurological function scoring, brain edema, primary cell extraction, immunofluorescence staining, CCK8, Tunel staining, Elisa, Western blot and other methods, the potential mechanism of Tenascin-C induced microglia cell polarization was explored.

The results of this study observed that the expression of Tenascin-C was up-regulated after subarachnoid hemorrhage. Inhibiting the increase of Tenascin-C by imatinib could significantly ameliorate neuroinflammation, neuronal apoptosis, blood brain barrier disruption and brain edema. When the level of Tenascin-C decreased, the numbers of TLR4 positive, MyD88 positive and NF-κB positive microglial cells decreased accordingly. Moreover, after subarachnoid hemorrhage, the number of microglial cells positive for M1-type markers increased significantly. After imatinib inhibited Tenascin-C, the number of M1-type microglial cells decreased and the number of M2-type microglial cells increased significantly.

In summary, the elevated level of Tenascin-C after subarachnoid hemorrhage induces the activation of microglia, releasing a large number of inflammatory factors and aggravating early brain injury.

Alveolar macrophages (AMs) are lung-resident myeloid cells and airway sentinels for inhaled pathogens and environmental particles. While AMs can be highly inflammatory in response to respiratory viruses, they do not mount proinflammatory responses to all airborne pathogens. For example, we previously showed that AMs fail to mount a robust proinflammatory response to Mycobacterium tuberculosis. Here, we address this discrepancy by investigating the capacity of murine AMs for direct innate immune sensing, using LPS as a model. Use of LPS-coated fluorescent beads enabled us to distinguish between directly exposed and bystander cells to measure transcriptional responses, by RNA-sequencing after cell sorting, and cytokine responses, by flow cytometry. We find that AMs have decreased proinflammatory responses to low-dose LPS compared to other macrophage types (bone marrow-derived macrophages, peritoneal macrophages), as measured by TNF, IL-6, Ifnb, and Ifit3. The reduced response to low-dose LPS correlates with minimal TLR4 and CD14 surface expression, despite sufficient internal expression of TLR4. We also find that AMs do not produce IL-10 in response to a variety of stimuli due to low expression of the transcription factor c-Maf, while exogenous c-Maf expression restores IL-10 production in AMs. Lastly, we show that lack of IL-10 enables type I IFN enhancement of AM responses to LPS. Overall, we demonstrate AMs have a cell-intrinsic hyporesponsiveness to LPS, which makes them uniquely tolerant to low-dose exposure. Regulation of AM innate responses by distinct CD14, c-Maf, and IL-10 expression patterns has important implications for both respiratory infections and environmental airborne exposures.

RNA-sensing TLRs are strategically positioned in the endolysosome to detect incoming nonself RNA. RNase T2 plays a critical role in processing long, structured RNA into short oligoribonucleotides that engage TLR7 or TLR8. In addition to its positive regulatory role, RNase T2 also restricts RNA recognition through unknown mechanisms, as patients deficient in RNase T2 suffer from neuroinflammation. Consistent with this, mice lacking RNase T2 exhibit interferon-dependent neuroinflammation, impaired hematopoiesis, and splenomegaly. However, the mechanism by which RNase T2 deficiency unleashes inflammation in vivo remains unknown. Here, we report that the inflammatory phenotype found in Rnaset2-/- mice is completely reversed in the absence of TLR13, suggesting aberrant accumulation of an RNA ligand for this receptor. Interestingly, this TLR13-driven inflammatory phenotype is also fully present in germ-free mice, suggesting a role for RNase T2 in limiting erroneous TLR13 activation by an as yet unidentified endogenous ligand. These results establish TLR13 as a potential self-sensor that is kept in check by RNase T2.

Lysosomal stress due to the accumulation of nucleic acids (NAs) activates endosomal TLRs in macrophages. Here, we show that lysosomal RNA stress, caused by the lack of RNase T2, induces macrophage accumulation in multiple organs such as the spleen and liver through TLR13 activation by microbiota-derived ribosomal RNAs. TLR13 triggered emergency myelopoiesis, increasing the number of myeloid progenitors in the bone marrow and spleen. Splenic macrophages continued to proliferate and mature into macrophages expressing the anti-inflammatory cytokine IL-10. In the liver, TLR13 activated monocytes/macrophages to proliferate and mature into monocyte-derived KCs (moKCs), in which, the liver X receptor (LXR) was activated. In accumulated moKCs, tissue clearance genes such as MerTK, AXL, and apoptosis inhibitor of macrophage (AIM) were highly expressed, while TLR-dependent production of proinflammatory cytokines was impaired. Consequently, Rnaset2-/- mice were resistant to acute liver injuries elicited by acetaminophen (APAP) and LPS with D-galactosamine. These findings suggest that TLR13 activated by lysosomal RNA stress promotes the replenishment of tissue-protective Kupffer cells.

Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS) are increasingly linked to mitochondrial dysfunction and neuroinflammation. Central to this link are mitochondrial damage-associated molecular patterns (mtDAMPs), including mitochondrial DNA, ATP, and reactive oxygen species, released during mitochondrial stress or damage. These mtDAMPs activate inflammatory pathways, such as the NLRP3 inflammasome and cGAS-STING, contributing to the progression of neurodegenerative diseases. This review delves into the mechanisms by which mtDAMPs drive neuroinflammation and discusses potential therapeutic strategies targeting these pathways to mitigate neurodegeneration. Additionally, it explores the cross-talk between mitochondria and the immune system, highlighting the complex interplay that exacerbates neuronal damage. Understanding the role of mtDAMPs could pave the way for novel treatments aimed at modulating neuroinflammation and slowing disease progression, ultimately improving patient outcome.

Non-alcoholic steatohepatitis (NASH), an advanced manifestation of non-alcoholic fatty liver disease (NAFLD), is characterized by hepatocyte injury, inflammation, and fibrosis. Saturated fatty acids (SFAs) have emerged as key contributors to hepatocyte lipotoxicity and disease progression. Toll-like receptor 4 (TLR4) acts as a sentinel for diverse ligands, including lipopolysaccharide (LPS) and endogenous molecules like palmitic acid (PA)-induced ceramide (CER) accumulation, promoting hepatocyte demise. However, the intricate mechanisms underlying TLR4's modulation of ceramide metabolism and their concerted effect on SFA-mediated hepatotoxicity remain elusive.

A NASH mouse model with liver-specific TLR4 knockdown was established through palm oil feeding and AAV2/8 tail vein injection. Histological and biochemical assessments were conducted to evaluate the mice's condition and liver damage extent. Liquid chromatography-mass spectrometry (LC-MS) was employed to quantify ceramide levels in liver tissues, offering insights into NASH mechanisms.

The PO-fed model exhibited elevated serum ALT, AST, and liver TG levels, enhancing lipid accumulation and hepatocellular damage. TLR4 knock-down reduced liver mass and the liver-to-body weight ratio, signifying a decreased hepatic burden. Histopathological evaluations revealed substantial improvement in hepatic steatosis in TLR4-silenced PO-fed mice, with diminished lipid droplets and inflammatory infiltrates. LC-MS analysis showed a marked decrease in long-chain ceramides (C14, C16, C20) in TLR4-knockdown PO-fed mice. Furthermore, expression of MyD88, SPTLC1, SPTLC2, and inflammatory markers IL-1β, IL-6, TNF-α were significantly attenuated.

SFAs activate the TLR4 signaling pathway via MyD88, fostering ceramide de novo synthesis, which exacerbates hepatocyte lipotoxicity and accelerates NASH progression.

Mammalian injury responses are predominantly characterized by fibrosis and scarring rather than functional regeneration. This limited regenerative capacity in mammals could reflect a loss of proregeneration programs or active suppression by genes functioning akin to tumor suppressors. To uncover programs governing regeneration in mammals, we screened transcripts in human participants following laser rejuvenation treatment and compared them with mice with enhanced wound-induced hair neogenesis (WIHN), a rare example of mammalian organogenesis. We found that Rnasel-/- mice exhibit an increased regenerative capacity, with elevated WIHN through enhanced IL-36α. Consistent with RNase L's known role to stimulate caspase-1, we found that pharmacologic inhibition of caspases promoted regeneration in an IL-36-dependent manner in multiple epithelial tissues. We identified a negative feedback loop, where RNase L-activated caspase-1 restrains the proregenerative dsRNA-TLR3 signaling cascade through the cleavage of toll-like adaptor protein TRIF. Through integrated single-cell RNA-seq and spatial transcriptomic profiling, we confirmed OAS & IL-36 genes to be highly expressed at the site of wounding and elevated in Rnasel-/- mouse wounds. This work suggests that RNase L functions as a regeneration repressor gene, in a functional trade off that tempers immune hyperactivation during viral infection at the cost of inhibiting regeneration.

Toll-like receptors (TLRs) are protein structures belonging to the pattern recognition receptors family. TLRs have the great potential that can directly recognize the specific molecular structures on the surface of pathogens, damaged senescent cells and apoptotic host cells. Available evidence suggests that TLRs have crucial roles in maintaining tissue homeostasis through control of the inflammatory and tissue repair responses during injury. TLRs are the player of first line of defense against different microbes and activate the signaling cascades which help to induce the immune system and inflammatory responses by affecting various signaling pathways, including nuclear factor-κB (NF-κB), interferon regulatory factors, and mitogen-activated protein kinases (MAPKs). TLRs have been identified to be over-expressed in different types of cancers and play an important role in control of health and management of diseases. The current review provides updated knowledge on the implication of TLRs in growth and management of cancers including prostate cancer.

Influenza virus infection initiates an exaggerated inflammatory response, which may culminate in a fatal cytokine storm characterized by the excessive production of pro-inflammatory cytokines. Prior research indicates that IP-10, IL-8, and MCP-1, primarily produced by monocytes and macrophages, play a crucial role in influenza-induced inflammation. The lung injury from influenza virus infection can be mitigated by suppressing or inhibiting these cytokines through knockout, knockdown, or targeted intervention approaches. To identify the key host signaling pathways responsible for producing pro-inflammatory cytokines, we utilized a U937 cell model that secretes IP-10, IL-8, and MCP-1 in response to influenza infection. This model has been previously validated in our laboratory as an appropriate system for screening anti-inflammatory agents and potential drug targets. We conducted a screening assay employing an inhibitor library consisting of 2,138 compounds that target various known pathways and host factors. Our findings indicated that inhibitors targeting protein tyrosine kinases and mitogen-activated protein kinases demonstrated superior efficacy in suppressing cytokine production induced by influenza A virus infection compared to inhibitors aimed at other host factors. Notably, a substantial proportion of the identified hits capable of inhibiting the expression of all three cytokines in the secondary screening were classified as tyrosine kinase inhibitors. Validation experiments further established that Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathways, along with p38 MAPK and Raf-MEK-ERK pathways, are the principal regulators of pro-inflammatory cytokine expression in monocytes and macrophages. Moreover, our results suggest that TKIs present promising opportunities as novel therapeutic agents against influenza-induced pneumonia.

Immune thrombocytopenia (ITP) is characterized by a reduction in platelet counts, stemming from an autoimmune-mediated process where platelets are excessively cleared by macrophages. This enhanced phagocytosis is a cardinal pathogenic mechanism in ITP. Antigen B (AgB), a principal component of the Echinococcus granulosus cyst fluid, plays a pivotal role in safeguarding the parasite from host immune defenses by modulating macrophage activation. In this study, we explored the potential of AgB to regulate macrophage activation in the context of ITP. Our observations indicated a diminished presence of M1 macrophages and a reduced phagocytic capacity in patients infected with E. granulosus sensu stricto. We isolated AgB from E. granulosus cyst fluid (EgCF) and discovered that it could suppress the polarization of M1 macrophages and weaken their phagocytic activity via Fcγ receptors, consequently alleviating thrombocytopenia in an ITP mouse model. At the molecular level, AgB was found to suppress the activation of nuclear factor kappa B (NF-κB) and interferon regulatory factor 3 (IRF3) by impeding their nuclear translocation, leading to a reduction in the generation of inflammatory cytokines. Furthermore, AgB was shown to inhibit Toll-like receptor 4 (TLR4) endocytosis and the recycling of CD14. In aggregate, our findings uncover a novel immunomodulatory mechanism of AgB, which suppresses macrophage phagocytosis by regulating TLR4 endocytosis and the subsequent activation of NF-κB and IRF3 signaling pathways. These insights shed new light on the molecular intricacies of E. granulosus-induced immune evasion and suggest that AgB may serve as a promising therapeutic agent for ITP.

Fibroblasts play a pivotal role in key processes within the heart, particularly in cardiac remodeling that follows both ischemic and non-ischemic injury. During remodeling, fibroblasts drive fibrosis and inflammation by reorganizing the extracellular matrix and modulating the immune response, including toll-like receptor (TLR) activation, to promote tissue stabilization. Building on findings from our prior research on heart tissue from patients with advanced coronary artery disease and aortic valve disease, this study sought to explore specific effects of TLR1, TLR3, and TLR7 activation on NF-κB signaling, proinflammatory cytokine production, and γ-protocadherin expression in cardiac fibroblasts. Human cardiac fibroblasts were exposed to agonists for TLR1, TLR3, or TLR7 for 24 h, followed by an analysis of NF-κB signaling, cytokine production, and γ-protocadherin expression. The activation of these TLRs triggered distinct responses in the NF-κB signaling pathway, with TLR3 showing a stronger activation profile compared to TLR1 and TLR7, particularly in downregulating γ-protocadherin expression. These findings highlight a potential role for TLR3 in amplifying inflammatory responses and reducing γ-protocadherin levels in cardiac fibroblasts, correlating with the enhanced inflammation and lower γ-protocadherin expression observed in diseased myocardium from patients with coronary artery disease and aortic valve disease. Consequently, TLR3 represents a potential therapeutic target for modulating immune responses in cardiovascular diseases.

Interferons (IFNs) are a diverse family of cytokines secreted by various cells, including immune cells, fibroblasts, and certain viral-parasitic cells. They are classified into three types and encompass 21 subtypes based on their sources and properties. The regulatory functions of IFNs closely involve cell surface receptors and several signal transduction pathways. Initially investigated for their antiviral properties, IFNs have shown promise in combating cancer-associated viruses, making them a potent therapeutic approach. Most IFNs have been identified for their role in inhibiting cancer; however, they have also demonstrated cancer-promoting effects under specific conditions. These mechanisms primarily rely on immune regulation and cytotoxic effects, significantly impacting cancer progression. Despite widespread use of IFN-based therapies in viral-related cancers, ongoing research aims to develop more effective treatments. This review synthesizes the signal transduction pathways and regulatory capabilities of IFNs, highlighting their connections with viruses, cancers, and emerging clinical treatments.