Pathogen-associated Molecular Patterns Research Articles

MicroRNA-630: A promising avenue for alleviating inflammation in diabetic kidney disease

Diabetic kidney disease (DKD) is one of the complications of diabetes, affecting millions of people worldwide. The relentless progression of this condition can lead to kidney failure, requiring life-altering interventions such as dialysis or transplants. Accumulating evidence suggests that immunologic and inflammatory elements play an important role in initiating and perpetuating the damage inflicted on renal tissues, exacerbating the decline in organ function. Toll-like receptors (TLRs) are a family of receptors that play a role in the activation of the innate immune system by the recognition of pathogen-associated molecular patterns. Recent data from in vitro and in vivo studies have highlighted the critical role of TLRs, mainly TLR2 and TLR4, in the pathogenesis of DKD. In the diabetic milieu, these TLRs recognize diabetic-associated molecular signals, triggering a proinflammatory cascade that initiates and perpetuates inflammation and fibrogenesis in the diabetic kidney. Emerging non-traditional strategies targeting TLR signaling with potential therapeutic implications in DKD have been pro-posed. One of these approaches is the use of microRNAs, small non-coding RNAs that can regulate gene expression. This editorial comments on the results of this approach carried out in a rat model of diabetes by Wu et al, published in this issue of the World Journal of Diabetes . The results of the experimental study by Wu et al shows that microRNA-630 decreased levels compared to non-diabetic rats. Additionally, microRNA-630 exerted anti-inflammatory effects in the kidneys of diabetic rats through the modulation of TLR4. These findings indicate that the microRNA-630/TLR4 axis might represent a pathological mechanism of DKD and a potential therapeutic target capable of curbing the destructive inflammation characteristic of DKD.

Ro60—Roles in RNA Processing, Inflammation, and Rheumatic Autoimmune Diseases

The Ro60/SSA2 autoantigen is an RNA-binding protein and a core component of nucleocytoplasmic ribonucleoprotein (RNP) complexes. Ro60 is essential in RNA metabolism, cell stress response pathways, and cellular homeostasis. It stabilises and mediates the quality control and cellular distribution of small RNAs, including YRNAs (for the ‘y’ in ‘cytoplasmic’), retroelement transcripts, and misfolded RNAs. Ro60 transcriptional dysregulation or loss of function can result in the generation and release of RNA fragments from YRNAs and other small RNAs. Small RNA fragments can instigate an inflammatory cascade through endosomal toll-like receptors (TLRs) and cytoplasmic RNA sensors, which typically sense pathogen-associated molecular patterns, and mount the first line of defence against invading pathogens. However, the recognition of host-originating RNA moieties from Ro60 RNP complexes can activate inflammatory response pathways and compromise self-tolerance. Autoreactive B cells may produce antibodies targeting extracellular Ro60 RNP complexes. Ro60 autoantibodies serve as diagnostic markers for various autoimmune diseases, including Sjögren’s disease (SjD) and systemic lupus erythematosus (SLE), and they may also act as predictive markers for anti-drug antibody responses among rheumatic patients. Understanding Ro60’s structure, function, and role in self-tolerance can enhance our understanding of the underlying molecular mechanisms of autoimmune conditions.

The Development of a Highly Potent and Selective Human Toll-like Receptor 2 Agonist: Synthesis and Biological Evaluation of CaLGL-1 and Its Derivatives.

Toll-like receptor 2 (TLR2) plays a crucial role in detecting microbial pathogen-associated molecular patterns, offering potential applications as an adjuvant for vaccines and antitumor therapies. Here, we present the gram-scale synthesis of CaLGL-1 and its derivatives, natural products known for activating mouse TLR2 (EC50 = 3.2 μM). This synthesis involves a streamlined six-step reaction sequence utilizing oxidant-promoted acetalization, effectively preserving the acid-sensitive glycosidic bond for maintaining the compounds' functional integrity. Our structure-activity relationship studies identified R-7d as a potent human TLR2 activator. It demonstrated subnanomolar activity (EC50 = 116 pM) in human THP-1 cells, comparable to that of diprovocim (EC50 = 110 pM). Experiments revealed that R-7d enhances NF-kB promoter activation through TLR2/TLR1 heterodimers rather than TLR2/TLR6. The discovery of R-7d as a robust human TLR2 agonist opens up new possibilities for combination therapies.

Alteration of β-glucan in the emerging fungal pathogen Candida auris leads to immune evasion and increased virulence

Candida auris is an emerging pathogenic yeast that has been categorized as a global public health threat and a critical priority among fungal pathogens. Despite this, the immune response against C. auris infection is still not well understood. Hosts fight Candida infections through the immune system that recognizes pathogen-associated molecular patterns such as β-glucan, mannan, and chitin on the fungal cell wall. In this study, levels of β-glucan and mannan exposures in C. auris grown under different physiologically relevant stimuli were quantified by flow cytometry-based analysis. Lactate, hypoxia, and sublethal concentration of fluconazole trigger a decrease in surface β-glucan while low pH triggers an increase in β-glucan. There is no inverse pattern between exposure levels of β-glucan and mannan in the cell wall architecture among the three clades. To determine the effect of cell wall remodeling on the immune response, a phagocytosis assay was performed, followed by quantification of released cytokines by ELISA. Lactate-induced decrease in β-glucan leads to reduced uptake of C. auris by PMA-differentiated THP-1 and RAW 264.7 macrophages. Furthermore, reduced production of CCL3/MIP-1⍺ but not TNF-⍺ and IL-10 were observed. An in vivo infection analysis using silkworms reveals that a reduction in β-glucan triggers an increase in the virulence of C. auris. This study demonstrates that β-glucan alteration occurs in C. auris and serves as an escape mechanism from immune cells leading to increased virulence.

DAMP-ing IBD: Extinguish the Fire and Prevent Smoldering.

In inflammatory bowel diseases (IBD), the most promising therapies targeting cytokines or immune cell trafficking demonstrate around 40% efficacy. As IBD is a multifactorial inflammation of the intestinal tract, a single-target approach is unlikely to solve this problem, necessitating an alternative strategy that addresses its variability. One approach often overlooked by the pharmaceutically driven therapeutic options is toaddress the impact of environmental factors. This is somewhat surprising considering that IBD is increasingly viewed as a condition heavily influenced by such factors, including diet, stress, and environmental pollution-often referred to as the "Western lifestyle". In IBD, intestinal responses result from a complex interplay among the genetic background of the patient, molecules, cells, and the local inflammatory microenvironment where danger- and microbe-associated molecular patterns (D/MAMPs) provide an adjuvant-rich environment. Through activating DAMP receptors, this array of pro-inflammatory factors can stimulate, for example, the NLRP3 inflammasome-a major amplifier of the inflammatory response in IBD, and various immune cells via non-specific bystander activation of myeloid cells (e.g., macrophages) and lymphocytes (e.g., tissue-resident memory T cells). Current single-target biological treatment approaches can dampen the immune response, but without reducing exposure to environmental factors of IBD, e.g., by changing diet (reducing ultra-processed foods), the adjuvant-rich landscape is never resolved and continues to drive intestinal mucosal dysregulation. Thus, such treatment approaches are not enough to put out the inflammatory fire. The resultant smoldering, low-grade inflammation diminishes physiological resilience of the intestinal (micro)environment, perpetuating the state of chronic disease. Therefore, our hypothesis posits that successful interventions for IBD must address the complexity of the disease by simultaneously targeting all modifiable aspects: innate immunity cytokines and microbiota, adaptive immunity cells and cytokines, and factors that relate to the (micro)environment. Thus the disease can be comprehensively treated across the nano-, meso-, and microscales, rather than with a focus on single targets. A broader perspective on IBD treatment that also includes options to adapt the DAMPing (micro)environment is warranted.

A NAC transcription factor MNAC3-centered regulatory network negatively modulates rice immunity against blast disease.

NAC transcription factors (TFs) are pivotal in plant immunity against diverse pathogens. Here, we report the functional and regulatory network of MNAC3, a novel NAC TF, in rice immunity. MNAC3, a transcriptional activator, negatively modulates rice immunity against blast and bacterial leaf blight diseases and pathogen-associated molecular pattern (PAMP)-triggered immune responses. MNAC3 binds to a CACG cis-element and activates the transcription of immune-negative target genes OsINO80, OsJAZ10, and OsJAZ11. The negative function of MNAC3 in rice immunity depends on its transcription of downstream genes such as OsINO80 and OsJAZ10. MNAC3 interacts with immunity-related OsPP2C41 (a protein phosphatase), ONAC066 (a NAC TF), and OsDjA6 (a DnaJ chaperone). ONAC066 and OsPP2C41 attenuate MNAC3 transcriptional activity, while OsDjA6 promotes it. Phosphorylation of MNAC3 at S163 is critical for its negative functions in rice immunity. OsPP2C41, which plays positive roles in rice blast resistance and chitin-triggered immune responses, dephosphorylates MNAC3, suppressing its transcriptional activity on the target genes OsINO80, OsJAZ10, and OsJAZ11 and promoting the translocation of MNAC3 from nucleus to cytoplasm. These results establish a MNAC3-centered regulatory network in which OsPP2C41 dephosphorylates MNAC3, attenuating its transcriptional activity on downstream immune-negative target genes in rice. Together, these findings deepen our understanding of molecular mechanisms in rice immunity and offer a novel strategy for genetic improvement of rice disease resistance.

Molluscan pleiotropic FADD involved in innate immune signaling and induces apoptosis

Fas-associated protein with death domain (FADD) was initially identified as a crucial adaptor protein in the apoptotic pathway mediated by death receptor (DR). Subsequently, many studies have confirmed that FADD plays a vital role in innate immunity and inflammatory responses in animals. However, the function of this pleiotropic molecule in mollusk species has not been well explored. In this study, we successfully verified the gene sequence of FADD in the Zhikong scallop (Chlamys farreri) and designated it as CfFADD. The CfFADD protein contains a conserved death effector and death domains. Phylogenetic analysis showed that CfFADD is a novel addition to the molluscan FADD family with a close evolutionary relationship with molluscan FADD subfamily proteins. CfFADD mRNA expression in various scallop tissues was significantly induced by challenge with pathogen-associated molecular patterns (lipopolysaccharide, peptidoglycan, and poly(I:C)), suggesting its role in innate immunity in scallops. Co-immunoprecipitation showed that CfFADD interacted with the scallop DR (tumor necrosis factor receptor) and a signaling molecule involved in the Toll-like receptor pathway (interleukin-1 receptor-associated kinase), confirming that CfFADD may be involved in DR-mediated apoptosis and innate immune signaling pathways. Further studies showed that CfFADD interacted with CfCaspase-8 and activated caspase-3. HEK293T cells exhibited distinct apoptotic features after transfection with a CfFADD-expression plasmid, suggesting a functional DR-FADD-caspase apoptotic pathway in scallops. Overexpression of CfFADD led to a significant dose-dependent activation of interferon β and nuclear factor-κB reporter genes, demonstrating the key role of CfFADD in innate immunity. In summary, our research has confirmed the critical roles of CfFADD in innate immunity and apoptosis and provides valuable information for developing comparative immunology theories.

Immunostimulatory and immunoadjuvant capacities of soluble Rhamnan-type Ulva oligosaccharides

Algae polysaccharides were discovered to act as pathogen-associated molecular patterns (PAMPs) to trigger immune responses, while bioactivities of oligosaccharides have not been thoroughly explored. Ulva oligosaccharides from Ulva prolifera were obtained using PL25 family ulvan lyase and evaluated for cell viability and phagocytic activity. Ulva oligosaccharides with 542 Da (OUP), consisting of tetrasaccharide (∆UA-RhaS-Xyl-RhaS) and disaccharides (∆UA-RhaS), exhibited the most pronounced phagocytic activities. Besides, OUP could effectively stimulate the expressions of proinflammatory cytokines and chemokines like TNF-α, IL-1β, IL-6, IFN-γ and CXCL1 in primary macrophages and subsequently promote the proliferation and activation of T and B lymphocytes in vitro. Transcriptome analysis also gave rise to the systemic proinflammatory responses triggered by OUP in macrophages, such as activation of multiple PRRs-related signaling pathways and downstream JAK-STAT and NF-κB signaling. Furthermore, in an in vivo mouse OVA immunization model, OUP upregulated the specific anti-OVA IgG antibody production in vivo, pointing to its immunoadjuvant capacity. OUP increases cell proliferation and secretion of IL-4 and IFN-γ of splenocytes in response to OVA stimulation ex vivo, thereby enhancing both Th1 and Th2 immune responses. Collectively, all this data suggests that OUP could function as the immune stimulator to enhance host immune response to infections.

Modeling Host-Pathogen Interactions in C. elegans: Lessons Learned from Pseudomonas aeruginosa Infection.

Infections, such as that by the multiresistant opportunistic bacterial pathogen Pseudomonas aeruginosa, may pose a serious health risk, especially on vulnerable patient populations. The nematode Caenorhabditis elegans provides a simple organismal model to investigate both pathogenic mechanisms and the emerging role of innate immunity in host protection. Here, we review the virulence and infection strategies of P. aeruginosa and host defenses of C. elegans. We summarize the recognition mechanisms of patterns of pathogenesis, including novel pathogen-associated molecular patterns and surveillance immunity of translation, mitochondria, and lysosome-related organelles. We also review the regulation of antimicrobial and behavioral defenses by the worm's neuroendocrine system. We focus on how discoveries in this rich field align with well-characterized evolutionary conserved protective pathways, as well as on potential crossovers to human pathogenesis and innate immune responses.

Zebrafish use conserved CLR and TLR signaling pathways to respond to fungal PAMPs in zymosan.

Pattern recognition receptors (PRRs) such as C-type lectin receptors (CLRs) and Toll-like receptors (TLRs) are used by hosts to recognize pathogen-associated molecular patterns (PAMPs) in microorganisms and to initiate innate immune responses. While PRRs exist across invertebrate and vertebrate species, the functional homology of many of these receptors is still unclear. In this study, we investigate the innate immune response of zebrafish larvae to zymosan, a β-glucan-containing particle derived from fungal cell walls. Macrophages and neutrophils robustly respond to zymosan and are required for zymosan-induced activation of the NF-κB transcription factor. Full activation of NF-κB in response to zymosan depends on Card9/Syk and Myd88, conserved CLR and TLR adaptor proteins, respectively. Two putative CLRs, Clec4c and Sclra, are both required for maximal sensing of zymosan and NF-κB activation. Altogether, we identify conserved PRRs and PRR signaling pathways in larval zebrafish that promote recognition of fungal PAMPs. These results inform modeling of human fungal infections in zebrafish and increase our knowledge of the evolution and conservation of PRR pathways in vertebrates.

TRPM8 affects relative “cooling and heating” of subcellular organelles in microglia in a context-dependent manner

Thermoregulation and thermal homeostasis at the cellular and sub-cellular organelle level are poorly understood events. In this work, we used BV2, a microglial cell line, and a series of thermo-sensitive subcellular organelle-specific probes to analyze the relative changes in the spatio-temporal temperatures of different sub-cellular organelles, both qualitatively and quantitatively. These methodologies allowed us to understand the thermal relationship of different subcellular organelles also. We modulated BV2 cells by pharmacological application of activator or inhibitor of TRPM8 ion channel (a cold-sensitive ion channel) and/or by treating the cells with LPS, a molecule that induces pathogen-associated molecular patterns (PAMPs) signaling. We demonstrate that the temperatures of individual organelles remain variable within a physiological range, yet vary in different conditions. We also demonstrate that treating BV2 cells by TRPM8 modulators and/or LPS alters the organelle temperatures in a specific and context-dependent manner. We show that TRPM8 modulation and/or LPS can alter the relationship of mitochondrial membrane potential to mitochondrial temperature. Our work suggests that mitochondrial temperature positively influences ER temperature and negatively influences Golgi temperature. Golgi temperature positively influences membrane temperature. This understanding of thermal relationship may be crucial for dissecting cellular structures, function, and stress signaling and may be relevant for different diseases.

Structural characterization of ligand binding and pH-specific enzymatic activity of mouse Acidic Mammalian Chitinase.

Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high-resolution crystal structures of mAMCase in complex with oligomeric GlcNAcn, the building block of chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation.

Transcriptome-based protein-protein interaction analysis reveals immune gene network elucidating white body immunity mechanisms in response to LPS stimulation in Amphioctopus fangsiao

One of the major challenges in the aquaculture of marine organisms is the susceptibility to pathogens. Immune cells defend bacterial invasion by recognizing pathogen-associated molecular patterns (PAMPs) such as Lipopolysaccharides (LPS) in Gram-negative bacteria. The white body are key organs for immune response and hematopoiesis, and play an important role in understanding the antimicrobial biological response in gold-ringed octopus (Amphioctopus fangsiao). In the present research, transcriptome sequencing and bioinformatics analysis were conducted on A. fangsiao white body tissue after 6 h and 24 h of LPS stimulation. The results showed 2,029 and 73 differentially expressed genes (DEGs) at the 6- and 24-hour time points, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were beneficial in determining immune-related terms and signaling pathways. Subsequently, a protein-protein interaction (PPI) network was constructed and 20 hub genes, such as AKT3, MAPK14, and PIK3CA, were identified. These genes are involved in a variety of signaling pathways, including the Hippo signaling pathway, chemokine signaling pathway, and leukocyte trans endothelial migration. Using qRT-PCR, the accuracy of the 20 hub genes was verified. The present study provides a substantial theoretical foundation for the research on A. fangsiao white body immunity, enhancing the current understanding of cephalopods' innate immunity.

Uncovering the impact of alcohol on internal organs and reproductive health: Exploring TLR4/NF‐kB and CYP2E1/ROS/Nrf2 pathways

AbstractThis review delves into the detrimental impact of alcohol consumption on internal organs and reproductive health, elucidating the underlying mechanisms involving the Toll‐like receptor 4 (TLR4)/Nuclear factor kappa light chain enhancer of activated B cells (NF‐kB) pathway and the Cytochrome P450 2E1 (CYP2E1)/reactive oxygen species (ROS)/nuclear factor erythroid 2‐related factor 2 (Nrf2) pathways. The TLR4/NF‐kB pathway, crucial for inflammatory and immune responses, triggers the production of pro‐inflammatory agents and type‐1 interferon, disrupting the balance between inflammatory and antioxidant responses when tissues are chronically exposed to alcohol. Alcohol‐induced dysbiosis in gut microbes heightens gut wall permeability to pathogen‐associated molecular patterns (PAMPs), leading to liver cell infection and subsequent inflammation. Concurrently, CYP2E1‐mediated alcohol metabolism generates ROS, causing oxidative stress and damaging cells, lipids, proteins, and deoxyribonucleic acid (DNA). To counteract this inflammatory imbalance, Nrf2 regulates gene expression, inhibiting inflammatory progression and promoting antioxidant responses. Excessive alcohol intake results in elevated liver enzymes (ADH, CYP2E1, and catalase), ROS, NADH, acetaldehyde, and acetate, leading to damage in vital organs such as the heart, brain, and lungs. Moreover, alcohol negatively affects reproductive health by inhibiting the hypothalamic–pituitary‐gonadal axis, causing infertility in both men and women. These findings underscore the profound health concerns associated with alcohol‐induced damage, emphasizing the need for public awareness regarding the intricate interplay between immune responses and the multi‐organ impacts of alcohol consumption.

Comparative RNA-seq analysis of Arabidopsis thaliana response to AtPep1 and flg22, reveals the identification of PP2-B13 and ACLP1 as new members in pattern-triggered immunity.

In this research, a high-throughput RNA sequencing-based transcriptome analysis technique (RNA-Seq) was used to evaluate differentially expressed genes (DEGs) in the wild type Arabidopsis seedlings in response to AtPep1, a well-known peptide representing an endogenous damage-associated molecular pattern (DAMP), and flg22, a well-known microbe-associated molecular pattern (MAMP). We compared and dissected the global transcriptional landscape of Arabidopsis thaliana in response to AtPep1 and flg22 and could identify shared and unique DEGs in response to these elicitors. We found that while a remarkable number of flg22 up-regulated genes were also induced by AtPep1, 256 genes were exclusively up-regulated in response to flg22, and 328 were exclusively up-regulated in response to AtPep1. Furthermore, among down-regulated DEGs upon flg22 treatment, 107 genes were exclusively down-regulated by flg22 treatment, while 411 genes were exclusively down-regulated by AtPep1. We found a number of hitherto overlooked genes to be induced upon treatment with either flg22 or with AtPep1, indicating their possible involvement general pathways in innate immunity. Here, we characterized two of them, namely PP2-B13 and ACLP1. pp2-b13 and aclp1 mutants showed increased susceptibility to infection by the virulent pathogen Pseudomonas syringae DC3000 and its mutant Pst DC3000 hrcC (lacking the type III secretion system), as evidenced by increased proliferation of the two pathogens in planta. Further, we present evidence that the aclp1 mutant is deficient in ethylene production upon flg22 treatment, while the pp2-b13 mutant is deficient in the production of reactive oxygen species (ROS). The results from this research provide new information for a better understanding of the immune system in Arabidopsis.

Two structurally different oomycete lipophilic microbe-associated molecular patterns induce distinctive plant immune responses.

Plants recognize a variety of external signals and induce appropriate mechanisms to increase their tolerance to biotic and abiotic stresses. Precise recognition of attacking pathogens and induction of effective resistance mechanisms are critical functions for plant survival. Some molecular patterns unique to a certain group of microbes, microbe-associated molecular patterns (MAMPs), are sensed by plant cells as nonself molecules via pattern recognition receptors. While MAMPs of bacterial and fungal origin have been identified, reports on oomycete MAMPs are relatively limited. This study aimed to identify MAMPs from an oomycete pathogen Phytophthora infestans, the causal agent of potato late blight. Using reactive oxygen species (ROS) production and phytoalexin production in potato (Solanum tuberosum) as markers, two structurally different groups of elicitors, namely ceramides and diacylglycerols, were identified. P. infestans ceramides (Pi-Cer A, B, and D) induced ROS production, while diacylglycerol (Pi-DAG A and B), containing eicosapentaenoic acid (EPA) as a substructure, induced phytoalexins production in potato. The molecular patterns in Pi-Cers and Pi-DAGs essential for defense induction were identified as 9-methyl-4,8-sphingadienine (9Me-Spd) and 5,8,11,14-tetraene-type fatty acid (5,8,11,14-TEFA), respectively. These structures are not found in plants, but in oomycetes and fungi, indicating that they are microbe molecular patterns recognized by plants. When Arabidopsis (Arabidopsis thaliana) was treated with Pi-Cer D and EPA, partially overlapping but different sets of genes were induced. Furthermore, expression of some genes is upregulated only after the simultaneous treatment with Pi-Cer D and EPA, indicating that plants combine the signals from simultaneously recognized MAMPs to adapt their defense response to pathogens.

Blockade of endolysosomal acidification suppresses TLR3-mediated proinflammatory signaling in airway epithelial cells

BackgroundEndolysosomal compartments are acidic and contain low pH-dependent proteases, and these conditions are exploited by respiratory viruses, such as SARS-CoV-2 and influenza virus, for escaping into the cytosol. Moreover, endolysosomes contain various pattern recognition receptors (PRRs), which respond to virus-derived pathogen-associated molecular patterns (PAMPs) by production of pro-inflammatory cytokines/chemokines. However, excessive pro-inflammatory responses can lead to a potentially lethal cytokine storm. ObjectivesHere we investigated the endosomal PRR expression profile in primary human small airway epithelial cells (HSAECs), and whether blockade of endolysosomal acidification affects their cytokine/chemokine production after challenge with virus-derived stimulants. MethodsHSAECs were exposed to stimulants mimicking virus-derived PAMPs, either in the absence or presence of compounds causing blockade of endolysosomal acidification, followed by measurement of cytokine expression and release. ResultsWe show that toll-like receptor 3 (TLR3) is the major endosomal PRR expressed by HSAECs, and that TLR3 expression is strongly induced by TLR3 agonists, but not by a range of other PRR agonists. We also demonstrate that TLR3 engagement with its agonists elicits a robust pro-inflammatory cytokine/chemokine response, which is profoundly suppressed through blockade of endolysosomal acidification, by bafilomycin A1, monensin, or niclosamide. Using TLR3 reporter cells, it was confirmed that TLR3 signaling is strongly induced by Poly(I:C) and that blockade of endolysosomal acidification efficiently blocked TLR3 signaling. Finally, we show that blockade of endolysosomal acidification causes a reduction in the levels of TLR3 mRNA and protein. ConclusionThese findings show that blockade of endolysosomal acidification suppresses TLR3-dependent cytokine and chemokine production in HSAECs. Clinical implicationThese findings may be exploited for therapeutic strategies aiming to ameliorate the cytokine storm in response to respiratory virus infection.

The role of the cGAS-STING signaling pathway in viral infections, inflammatory and autoimmune diseases.

Pattern recognition receptors are an essential part of the immune system, which detect pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) and help shape both innate and adaptive immune responses. When dsDNA is present, cyclic GMP-AMP Synthase (cGAS) produces a second messenger called cyclic GMP-AMP (cGAMP), which then triggers an adaptor protein called STING, and eventually activates the expression of type I interferon (IFN) and pro-inflammatory cytokines in immune cells. The cGAS-STING signaling pathway has been receiving a lot of attention lately as a key immune-surveillance mediator. In this review, we summarize the present circumstances of the cGAS-STING signaling pathway in viral infections and inflammatory diseases, as well as autoimmune diseases. Modulation of the cGAS-STING signaling pathway provides potential strategies for treating viral infections, inflammatory diseases, and autoimmune diseases.

Impact of secreted glucanases upon the cell surface and fitness of Candida albicans during colonisation and infection

Host recognition of the pathogen-associated molecular pattern (PAMP), β-1,3-glucan, plays a major role in antifungal immunity. β-1,3-glucan is an essential component of the inner cell wall of the opportunistic pathogen Candida albicans. Most β-1,3-glucan is shielded by the outer cell wall layer of mannan fibrils, but some can become exposed at the cell surface. In response to host signals such as lactate, C. albicans shaves the exposed β-1,3-glucan from its cell surface, thereby reducing the ability of innate immune cells to recognise and kill the fungus. We have used sets of barcoded xog1 and eng1 mutants to compare the impacts of the secreted β-glucanases Xog1 and Eng1 upon C. albicans in vitro and in vivo. Flow cytometry of Fc-dectin-1-stained strains revealed that Eng1 plays the greater role in lactate-induced β-1,3-glucan masking. Transmission electron microscopy and stress assays showed that neither Eng1 nor Xog1 are essential for cell wall maintenance, but the inactivation of either enzyme compromised fungal adhesion to gut and vaginal epithelial cells. Competitive barcode sequencing suggested that neither Eng1 nor Xog1 strongly influence C. albicans fitness during systemic infection or vaginal colonisation in mice. However, the deletion of XOG1 enhanced C. albicans fitness during gut colonisation. We conclude that both Eng1 and Xog1 exert subtle effects on the C. albicans cell surface that influence fungal adhesion to host cells and that affect fungal colonisation in certain host niches.

TLR21 is involved in the NF-κB and IFN-β pathways in largemouth bass (Micropterus salmoides) and interacts with TRIF but not with the Myd88 adaptor

Toll-like receptors (TLRs) are pattern recognition receptors that trigger host immune responses against various pathogens by detecting evolutionarily conserved pathogen-associated molecular patterns (PAMPs). TLR21 is a member of the Toll-like receptor family, and emerging data suggest that it recognizes unmethylated CpG DNA and is considered a functional homologue of mammalian TLR9. However, little is known regarding the role of TLR21 in the fish immune response. In the present study, we isolated the cDNA sequence of TLR21 from the largemouth bass (Micropterus salmoides) and termed it MsTLR21. The MsTLR21 gene contained an open reading frame (ORF) of 2931 bp and encodes a polypeptide of 976 amino acids. The predicted MsTLR21 protein has two conserved domains, a conserved leucine-rich repeats (LRR) domain and a C-terminal Toll-interleukin (IL) receptor (TIR) domain, similar to those of other fish and mammals. In healthy largemouth bass, the TLR21 transcript was broadly expressed in all the examined tissues, with the highest expression levels in the gills. After challenge with Nocardia seriolae and polyinosinic polycytidylic acid (Poly[I:C]), the expression of TLR21 mRNA was upregulated or downregulated in all tissues tested. Overexpression of TLR21 in 293T cells showed that it has a positive regulatory effect on nuclear factor-kappaB (NF-κB) and interferons-β (IFN-β) activity. Subcellular localisation analysis showed that TLR21 was expressed in the cytoplasm. We performed pull-down assays and determined that TLR21 did not interact with myeloid differentiation primary response gene 88 (Myd88); however, it interacted with TIR domain-containing adaptor inducing interferon-β (TRIF). Taken together, these findings suggest that MsTLR21 plays important roles in TLR/IL-1R signalling pathways and the immune response to pathogen invasion.