MyD88-dependent Toll-like Receptor Signaling Research Articles

IKK2 modulates the innate immune response of Zhikong scallop (Chlamys farreri) to bacterial and viral stimuli through the MyD88-dependent Toll-like receptor signaling pathway

Members of the inhibitor of nuclear factor kappa-B kinase (IKK) family are pivotal components of the innate immune signaling pathway responsible for regulating immune and inflammatory responses. Nevertheless, the various roles that IKKs play in the physiology of invertebrates, particularly marine mollusks, remain largely unexplored. In this study, an IKK gene was cloned from the Zhikong scallop, Chlamys farreri, and denoted as CfIKK2. CfIKK2 belongs to the TANK-binding kinase 1 (TBK1)/IKKε family. Real-time quantitative reverse transcription-polymerase chain reaction analysis showed that CfIKK2 was expressed in various scallop tissues and that its transcript levels substantially increased under different pathogenic stress conditions (including lipopolysaccharide, peptidoglycan, poly(I:C), acute viral necrosis virus, and Vibrio challenge). Co-immunoprecipitation experiments revealed that CfIKK2 interacts with MyD88 (a crucial adaptor in the Toll-like receptor signaling pathway) proteins through its C-terminal TBK1 coiled-coil domain 1. Moreover, CfIKK2 has been shown to interact with itself. In HEK293T cells, CfIKK2 overexpression resulted in an augmentation of mitogen-activated protein kinase phosphorylation. Furthermore, overexpression of CfIKK2 resulted in the activation of interferon reporter genes. In summary, our findings offer valuable insights into the mechanisms through which CfIKK2 responds to stress generated by pathogen-associated molecular patterns, participates in Toll-like receptor signaling, and ultimately activates some of the key cytokines involved in innate immunity in C. farreri. As a critical signaling molecule and immunoregulatory factor, CfIKK2 plays a vital role in the innate immune system of the Zhikong scallop.

The podocytes’ inflammatory responses in experimental GN are independent of canonical MYD88-dependent toll-like receptor signaling

Podocytes form the kidney filtration barrier and continuously adjust to external stimuli to preserve their integrity even in the presence of inflammation. It was suggested that canonical toll-like receptor signaling, mediated by the adaptor protein MYD88, plays a crucial role in initiating inflammatory responses in glomerulonephritis (GN). We explored the influence of podocyte-intrinsic MYD88 by challenging wild-type (WT) and podocyte-specific Myd88 knockout (MyD88pko) mice, with a model of experimental GN (nephrotoxic nephritis, NTN). Next-generation sequencing revealed a robust upregulation of inflammatory pathways and changes in cytoskeletal and cell adhesion proteins in sorted podocytes from WT mice during disease. Unchallenged MyD88pko mice were healthy and showed no proteinuria, normal kidney function and lacked morphological changes. During NTN, MyD88pko exhibited a transient increase in proteinuria in comparison to littermates, while histological damage, podocyte ultrastructure in STED imaging and frequencies of infiltrating immune cells by flow cytometry were unchanged. MYD88-deficiency led to subtle changes in the podocyte transcriptome, without a significant impact on the overall podocyte response to inflammation, presumably through MYD88-independent signaling pathways. In conclusion, our study reveals a comprehensive analysis of podocyte adaptation to an inflammatory environment on the transcriptome level, while MYD88-deficiency had only limited impact on the course of GN suggesting additional signaling through MYD88-independent signaling.

The Modification of H3K4me3 Enhanced the Expression of CgTLR3 in Hemocytes to Increase CgIL17-1 Production in the Immune Priming of Crassostrea gigas.

Increasing evidence confirms that histone modification plays a critical role in preserving long-term immunological memory. Immune priming is a novel form of immunological memory recently verified in invertebrates. Toll-like receptor (TLR) signaling and cytokines have been reported to be involved in the immune priming of the Pacific oyster Crassostrea gigas. In the present study, the expression of Toll-like receptor 3 (CgTLR3), myeloid differentiation factor 88-2 (CgMyd88-2) and interleukin 17-1 (CgIL17-1) was found to be elevated in the hemocytes of C. gigas at 6 h after the secondary stimulation with Vibrio splendidus, which was significantly higher than that at 6 h after the primary stimulation (p < 0.05). A significant increase in histone H3 lysine 4 trimethylation (H3K4me3) enrichment was detected in the promoter region of the CgTLR3 gene at 7 d after the primary stimulation with inactivated V. splendidus (p < 0.05). After the treatment with a histone methyltransferase inhibitor (5'-methylthioadenosine, MTA), the level of H3K4me3 at the promoter of the CgTLR3 gene decreased significantly at 7 d after the primary stimulation with inactivated V. splendidus (p < 0.05), and the expression of CgTLR3, CgMyD88-2 and CgIL17-1 was significantly repressed at 6 h after the secondary stimulation with V. splendidus (p < 0.05). Conversely, the treatment with monomethyl fumarate (MEF, an inhibitor of histone demethylases) resulted in a significant increase in H3K4me3 enrichment levels at the CgTLR3 promoter at 7 d after the primary stimulation (p < 0.05), and the expression of CgTLR3, CgMyD88-2 and CgIL17-1 was observed to increase significantly at 6 h after the secondary stimulation (p < 0.05). These results suggested that H3K4me3 regulated MyD88-dependent TLR signaling in the hemocytes of C. gigas, which defined the role of histone modifications in invertebrate immune priming.

Five-hub genes identify potential mechanisms for the progression of asthma to lung cancer.

Previous studies have shown that asthma is a risk factor for lung cancer, while the mechanisms involved remain unclear. We attempted to further explore the association between asthma and non-small cell lung cancer (NSCLC) via bioinformatics analysis. We obtained GSE143303 and GSE18842 from the GEO database. Lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) groups were downloaded from the TCGA database. Based on the results of differentially expressed genes (DEGs) between asthma and NSCLC, we determined common DEGs by constructing a Venn diagram. Enrichment analysis was used to explore the common pathways of asthma and NSCLC. A protein-protein interaction (PPI) network was constructed to screen hub genes. KM survival analysis was performed to screen prognostic genes in the LUAD and LUSC groups. A Cox model was constructed based on hub genes and validated internally and externally. Tumor Immune Estimation Resource (TIMER) was used to evaluate the association of prognostic gene models with the tumor microenvironment (TME) and immune cell infiltration. Nomogram model was constructed by combining prognostic genes and clinical features. 114 common DEGs were obtained based on asthma and NSCLC data, and enrichment analysis showed that significant enrichment pathways mainly focused on inflammatory pathways. Screening of 5 hub genes as a key prognostic gene model for asthma progression to LUAD, and internal and external validation led to consistent conclusions. In addition, the risk score of the 5 hub genes could be used as a tool to assess the TME and immune cell infiltration. The nomogram model constructed by combining the 5 hub genes with clinical features was accurate for LUAD. Five-hub genes enrich our understanding of the potential mechanisms by which asthma contributes to the increased risk of lung cancer.

Identification of potential biomarkers and pathogenesis in neutrophil-predominant severe asthma: A comprehensive bioinformatics analysis.

Background:Airway neutrophilia has been associated with asthma severity and asthma exacerbations. This study attempted to identify biomarkers, pathogenesis, and therapeutic molecular targets for severe asthma in neutrophils using bioinformatics analysis.Methods:Fifteen healthy controls and 3 patients with neutrophilic severe asthma were screened from the Gene Expression Omnibus (GEO) database. Based on the analysis of differentially expressed genes (DEGs), functional and pathway enrichment analyses, gene set enrichment analysis, protein–protein interaction network construction, and analysis were performed. Moreover, small-molecule drug candidates have also been identified.Results:Three hundred and three upregulated and 59 downregulated genes were identified. Gene ontology function enrichment analyses were primarily related to inflammatory response, immune response, leukocyte migration, neutrophil chemotaxis, mitogen-activated protein kinase cascade, Jun N-terminal kinase cascade, I-kappaB kinase/nuclear factor-κB, and MyD88-dependent toll-like receptor signaling pathway. Pathway enrichment analyses and gene set enrichment analysis were mainly involved in cytokine-cytokine receptor interaction, the TNF signaling pathway, leukocyte transendothelial migration, and the NOD-like receptor signaling pathway. Furthermore, 1 important module and 10 hub genes (CXCL8, TLR2, CXCL1, ICAM1, CXCR4, FPR2, SELL, PTEN, TREM1, and LEP) were identified in the protein–protein interaction network. Moreover, indoprofen, mimosine, STOCK1N-35874, trapidil, iloprost, aminoglutethimide, ajmaline, levobunolol, ethionamide, cefaclor, dimenhydrinate, and bethanechol are potential drugs for the treatment of neutrophil-predominant severe asthma.Conclusion:This study identified potential biomarkers, pathogenesis, and therapeutic molecular targets for neutrophil-predominant severe asthma.

Target molecular-based neuroactivity screening of Panax ginseng through network pharmacology integrated molecular docking

Panax ginseng is a medicinal herb that has therapeutic effects against neuronal damage. The main goal of the present study is to investigate the therapeutic potential of P. ginseng in treating neuronal damage through in silico analysis. The literature review was conducted to create a ingredients-target database for P. ginseng. The database was essential in identifying the targets related to neuronal damage, which was followed by the construction of protein-protein interaction network by the STITCH database. The enrichment analysis was performed using Cytoscape (ClueGO) software. The molecular docking analysis was ran using AutoDock and Discovery Studio. The literature search revealed the presence of 66 compounds in P. ginseng, which subsequently resulted in the retrieval of 102 potential targets of these compounds. They were screened for their association with neuronal damage. Three proteins, namely, Glutathione S-transferase P1, HSP90, and Mitogen-activated protein kinases (MAPK8), were found to be in prime interaction with the relative compounds. Subsequently, 91 Gene ontology terms were found, of which four genes were associated with neuronal damage. Molecular docking analysis also revealed good binding affinities of various compounds of P. ginseng to the selected target, MAPK8. In conclusion, the biological processes linked to the therapeutic action of P. ginseng involve the positive regulation of JUN kinase activity, chaperone-mediated protein folding, and MyD88-dependent toll-like receptor signaling pathway.

The transcriptional profiling identifies hub genes in immune subsets of patients with Behçet's syndrome.

Behçet's syndrome (BS) is a variable vessel vasculitis characterised by heterogeneity of organ manifestations. Antigen-presenting cells, such as macrophages and T cells, play critical roles in their immunopathology. This study aimed to identify hub genes and biological processes in patients with BS. We downloaded expression profiles, GSE61399, containing CD14+ monocytes and CD4+T cells between BS and healthy controls from the Gene Expression Omnibus (GEO). We screened the differential expression genes (DEGs) by the GEO2R. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed. Protein-protein interaction (PPI) network and core genes were analysed by the Search Tool for the Retrieval of Interacting Genes (STRING) and Cytoscape with Molecular Complex Detection (MCODE) plug-in tools. We identified 102 DEGs in CD14+ monocytes and 48 in CD4+ T cells. In monocytes, the gene enrichment was mainly involved in type I interferon signaling pathway, defense response to virus, cell chemotaxis, granulocyte chemotaxis, granulocyte migration, leukocyte chemotaxis, and neutrophil chemotaxis. The changed genes in CD4+ cells were enriched in MyD88-dependent toll-like receptor signaling pathway, positive regulation of innate immune response, and IL1B production. In combination with PPI and Markov Cluster Algorithm (MCL), we defined three driving protein-protein modules, IL1B, CCL2, CCL4, CXCL2, CCL20, CXCL3, TLR6, CD83, IFIT3, and THBD as a set of hub genes in CD14+ monocytes, associated with inflammation and thrombosis; CD300LF, CLEC5A, DMXL2, MS4A14, TMEM176A in CD4+ T cells. Our findings provide novel insights into the immune subsets related to the biological process in BS, which could contribute to identifying potential biomarkers and novel treatment strategies for BS.

MyD88-dependent TLR signaling oppositely regulates hematopoietic progenitor and stem cell formation in the embryo.

Hemogenic endothelial (HE) cells in the dorsal aorta undergo an endothelial-to-hematopoietic transition (EHT) to form multipotent progenitors, lympho-myeloid biased progenitors (LMPs), pre-hematopoietic stem cells (pre-HSCs) and adult-repopulating HSCs. These briefly accumulate in intra-arterial hematopoietic clusters (IAHCs) before being released into the circulation. It is generally assumed that the number of IAHC cells correlates with the number of HSCs. Here, we show that changes in the number of IAHC cells, LMPs and HSCs can be uncoupled. Mutations impairing MyD88-dependent toll-like receptor (TLR) signaling decreased the number of IAHC cells and LMPs, but increased the number of HSCs in the aorta-gonad-mesonephros region of mouse embryos. TLR4-deficient embryos generated normal numbers of HE cells, but IAHC cell proliferation decreased. Loss of MyD88-dependent TLR signaling in innate immune myeloid cells had no effect on IAHC cell numbers. Instead, TLR4 deletion in endothelial cells (ECs) recapitulated the phenotype observed with germline deletion, demonstrating that MyD88-dependent TLR signaling in ECs and/or in IAHCs regulates the numbers of LMPs and HSCs.

Apostichopus japonicus matrix metalloproteinase-16 might act as a pattern recognition receptor

Matrix metalloproteinases (MMPs) are an important family of proteinases involved in various physiological processes and associated with the immune response. However, the role of MMPs in the immune response remains unclear. To explore the possible role of MMPs in innate immunity, this study selected the MMP-16 gene encoding peptidoglycan (PGN) binding domain identified in the sea cucumber Apostichopus japonicus (named AjMMP-16, GenBank accession No. AQT26486) for microbial polysaccharide-induced transcriptional expression analysis by quantitative real-time PCR, correlation analysis with nine representative genes from A. japonicus immune pathways in microbial polysaccharide-induced transcriptional expression by using Pearson's correlation test, and prokaryotic recombinant expression. Next, its recombinant protein was employed for microbial polysaccharide-binding analysis with ELISA and bacterial binding analysis with the indirect immunofluorescence method. The results showed that AjMMP-16 was significantly induced by diaminopimelic acid (DAP)-type PGN, lipopolysaccharide, mannan, and β-1,3-glucan and was closely correlated with myeloid differentiation factor 88 (MyD88) in microbial polysaccharide-induced transcriptional expression. In addition, recombinant AjMMP-16 bound to lysine-type PGN, DAP-type PGN, lipopolysaccharide, mannan, β-1,3-glucan, Vibrio splendidus, Pseudoalteromonas nigrifaciens, Shewanella baltica, Bacillus cereus, Escherichia coli, and Staphylococcus aureus. These results suggest that AjMMP-16 might act as a pattern recognition receptor in innate immunity and play an important role in initiating the MyD88-dependent Toll-like receptor signaling pathway.

Characterization and expression analysis of key genes of pathogen-associated molecular pattern-triggered Toll-like receptor 5 signaling in the lined seahorse responses to bacterial and parasitic infection

The innate immune system plays an important role in the immune responses of fish, particularly in immunodeficient seahorses, which are vulnerable to infection. In this study, we investigated the evolutionary adaptation of the Toll-like receptor 5 signaling in the lined seahorse (Hippocampus erectus) and the physiological and molecular responses of seahorse to bacterial and parasitic tapeworm infections. Four sites under positive selection among three genes, hsp70, tlr5, and tak1, showed Bayes Empirical Bayes values >0.95. The respiratory rate of H. erectus increased after being challenged with Vibrio and Scalithrium. Stress-induced expression of hsp60 and hsp70, which can bind PAMP molecules and modulate PAMP-induced Toll-like receptor (TLR) signaling, was significantly upregulated in immune organs such as the gill, intestine, kidney, and liver. Additionally, the MyD88-dependent TLR signaling pathway of H. erectus was activated in response to both bacterial and parasitic infections. The expression profiles of TLR signaling molecules revealed that these molecules mediate immune processes and act against various pathogens. These results improve our understanding of the functional role of key genes involved in PAMP-mediated signaling and provide theoretical support for the prevention and control of diseases in cultured seahorses.

Treating Autoimmune Inflammatory Diseases with an siERN1-Nanoprodrug That Mediates Macrophage Polarization and Blocks Toll-like Receptor Signaling.

The clinical application of small interfering RNA (siRNA) drugs provides promising opportunities to develop treatment strategies for autoimmune inflammatory diseases. In this study, siRNAs targeting the endoplasmic reticulum to nucleus signaling 1 (ERN1) gene (siERN1) were screened. Two cationic polymers, polyethylenimine (PEI) and poly(β-amino amine) (PBAA), which can improve the efficiency of the siRNA transfection, were used as siERN1 delivery carriers. They were implemented to construct a nanodrug delivery system with macrophage-targeting ability and dual responsiveness for the treatment of autoimmune inflammatory diseases. In terms of the mechanism, siERN1 can regulate the intracellular calcium ion concentration by interfering with the function of inositol 1,4,5-trisphosphate receptor 1/3 (IP3R1/3) and thus inducing M2 polarization of macrophages. Furthermore, siERN1-nanoprodrug [FA (folic acid)-PEG-R(RKKRRQRRR)-NPs(ss-PBAA-PEI)@siERN1] acts as a conductor of macrophage polarization by controlling the calcium ion concentration and is an inhibitor of MyD88-dependent Toll-like receptor signaling. The results revealed that the FA-PEG-R-NPs@siERN1 has universal biocompatibility, long-term drug release responsiveness, superior targeting properties, and therapeutic effects in mouse collagen-induced arthritis and inflammatory bowel disease models. In conclusion, this study reveals a potential strategy to treat autoimmune inflammatory disorders.

Network Pharmacology Analysis of the Mechanisms of Compound Herba Sarcandrae (Fufang Zhongjiefeng) Aerosol in Chronic Pharyngitis Treatment.

PurposeThis study aimed to investigate the molecular mechanisms of compound herba Sarcandrae aerosol, also known as the Fufang Zhongjiefeng (FFZJF) aerosol, in treating chronic pharyngitis (CP) using network pharmacology and in vivo experimental approaches.MethodsActive compounds and putative targets of five herbs in FFZJF were identified from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, Chemistry Database, and Swiss Target Prediction databases. The therapeutic targets of CP were obtained from OMIM, Durgbank, DisGeNT, and GAD databases. The active compounds-target networks were constructed using Cytoscape 3.6.1. The overlapping targets of FFZJF active compounds and CP targets were further analyzed using the String database to construct protein–protein interaction (PPI) network. KEGG pathway and Gene Ontology enrichment analysis was performed using the Database for Annotation, Visualization, and Integrated Discovery. The predicted targets and pathways were validated in a group A β-hemolytic streptococcus-induced rat CP model.ResultsThere were 45 active compounds identified from FFZJF and 11 potential protein targets identified for CP treatment. PPI network demonstrated that IL6, PTGS2, TLR-4, and TNF may serve as the key targets of FFZJF for the treatment of CP. The main functional pathways involving these key targets include cytokine secretion, inflammatory response, MyD88-dependent toll-like receptor signaling pathway, toll-like receptor signaling pathway, TNF signaling pathway, and NF-κB signaling pathway. In a rat CP model, the elevation of serum TNF-α, IL1β, and IL6 levels, as well as the upregulation of TLR-4, MyD88, NF-κB P65 in the pharyngeal mucosal tissues could be effectively reduced by FFZJF treatment in a dose-dependent manner.ConclusionThrough a network pharmacology approach and animal study, we predicted and validated the active compounds of FFZJF and their potential targets for CP treatment. The results suggest that FFZJF can markedly alleviate GAS-induced chronic pharyngitis by modulating the TLR-4/MyD88/NF-κB signaling pathways.

Hippo Pathway Counter-Regulates Innate Immunity in Hepatitis B Virus Infection.

Whether hepatitis B virus (HBV) activates or represses innate immunity continues to be debated. Toll-like receptor (TLR) 2 has been identified to recognize HBV particles in human hepatocytes. The Hippo pathway, known for growth control, is suggested to play a vital role in immune regulation. Here, molecular interactions between HBV-triggered TLR signaling and the Hippo pathway were comprehensively investigated. Reanalysis of GSE69590 data, in which human hepatocytes have been treated with cell culture-derived HBV particles, identified changes in Hippo and NF-κB signaling. Immunocytochemical staining and western blotting revealed time-dependent nuclear translocation of YAP and NF-κB in HBV-exposed primary human and murine hepatocytes (PMH). Analysis of PMH isolated from MyD88- or IRAK4-deficient mice and the inhibition of TLR2 and MST1/2 in vitro confirmed the relation between TLR2 and Hippo signaling in HBV-induced immunity. Loss and gain of function experiments implied that Hippo-downstream effector YAP directly regulated IκBα expression. Functional investigations confirmed the regulation of Nfkbia promoter activity by the YAP/TEAD4 transcription factor complex. Administration of TLR ligands to mice highlighted the relevance of the TLR2-MyD88-IRAK4-Hippo axis in hepatic immunity. Interestingly, reanalysis of gene expression pattern in liver biopsies of patients chronically infected with HBV (GSE83148, GSE65359) indicated an activation of TLR2 and however, an MST1-dominated Hippo control in the immune clearance phase of patients with chronic HBV infection. We demonstrated that MyD88-dependent TLR signaling activates NF-κB and Hippo signaling, with YAP prompting the IκBα-mediated negative feedback, alongside NF-κB. Imbalance between immune induction and Hippo activation may have implications for the safety of novel HBV cure strategies interfering with pathogen recognition receptors.

High‐Altitude Exposure and Sustained Hypoxia Increase Expression of Inflammatory Genes and Enhance the Peripheral Blood Inflammatory Response to LPS

Transcriptional responses to hypoxia and inflammatory stimuli are linked via hypoxia inducible factor (HIF) and NF-kB crosstalk. Tissues experience hypoxia during infection or inflammatory states due to increased metabolic demand of immune cells, or impaired oxygen delivery by occluded blood supply or fluid buildup. However, while the evolutionarily conserved inflammatory responses to oxygen limitation are necessary for modulating the physiological responses to hypoxic stress, during chronic and/or systemic hypoxia, the crosstalk between these two pathways can become maladaptive. For example, hypoxia-induced inflammation may contribute to the development of Acute and Chronic Mountain Sickness. We hypothesized that acute sustained hypoxia exposure induces a systemic inflammatory response and changes sensitivity to subsequent inflammatory stimuli. To test this hypothesis, we used a high-altitude exposure model (1-3 days). We compared the RNA profiles in whole blood collected in the morning during fasting at sea level, and after 1 night and 3 nights at high altitude (3800m elevation) in 14 healthy sojourners (4 women, 10 men). RNA sequencing was coupled with Nanostring direct digital detection of 255 inflammatory pathway genes. 127 genes were significantly upregulated, and 140 genes were significantly downregulated with a fold change threshold > 1 and adjusted p-value < 0.05 on the first day at high altitude. Of all significant genes, 13 were also detected as significantly upregulated via Nanostring: HMGB1, TGFBR1, HMGN1, DDIT3, TLR8, MAPK1, TLR1, IFIT1, IFI44, MAPK8, HMGB2, TLR5, IFIT2 (adj. p < 0.05 for all). Among the significant gene ontology terms for all differentially expressed genes with adj p < 0.05 were: toll-like receptor signaling pathway (p<0.0001), MyD88-dependent toll-like receptor signaling pathway (p=0.012), interleukin-1-mediated signaling pathway (p=0.003), and I-kappaB kinase/NF-kappaB signaling (p<<0.0001). To test if upregulation of toll-like receptor signaling pathways increased sensitivity of peripheral blood mononuclear cells to inflammatory stimuli, we cultured whole blood and isolated monocytes in the presence of normoxia (18% O2, 5% CO2 at sea level) or hypoxia (1% O2, 5% CO2 at sea level) and with or without a lipopolysaccharide (LPS) stimulus for 24 hours. Both LPS (p<<0.0001) and hypoxia (p=0.01) independently increased TNF-a expression in whole blood and there was a significant interaction between hypoxia and LPS (p=0.002) in which hypoxia augmented TNF-a expression in response to LPS. TNF-a expression was higher in monocyte cultures exposed to LPS (p<<0.0001) and showed a nonsignificant trend to be higher in the presence of hypoxia, but there was no interaction. In conclusion, our results indicate that acute sustained hypoxia exposure may exacerbate the response to subsequent inflammatory stimuli. This may be particularly important in the context of pulmonary diseases if hypoxemia exacerbates the inflammatory response to viral or bacterial infection.

Talin1 controls dendritic cell activation by regulating TLR complex assembly and signaling.

Talin critically controls integrin-dependent cell migration, but its regulatory role in skin dendritic cells (DCs) during inflammatory responses has not been investigated. Here, we show that talin1 regulates not only integrin-dependent Langerhans cell (LC) migration, but also MyD88-dependent Toll-like receptor (TLR)-stimulated DC activation. Talin1-deficient LCs failed to exit the epidermis, resulting in reduced LC migration to skin-draining lymph nodes (sdLNs) and defective skin tolerance induction, while talin1-deficient dermal DCs unexpectedly accumulated in the dermis despite their actomyosin-dependent migratory capabilities. Furthermore, talin1-deficient DCs exhibited compromised chemotaxis, NFκB activation, and proinflammatory cytokine production. Mechanistically, talin1 was required for the formation of preassembled TLR complexes in DCs at steady state via direct interaction with MyD88 and PIP5K. Local production of PIP2 by PIP5K then recruited TIRAP to the preassembled complexes, which were required for TLR signalosome assembly during DC activation. Thus, talin1 regulates MyD88-dependent TLR signaling pathways in DCs through a novel mechanism with implications for antimicrobial and inflammatory immune responses.

Waterborne microcystin-LR exposure induced chronic inflammatory response via MyD88-dependent toll-like receptor signaling pathway in male zebrafish

Waterborne microcystin-LR (MC-LR) released by cyanobacterial blooms in eutrophic water bodies have caused serious risk to aquatic animal and human health. In the present study, we for the first time conducted a comprehensive in vivo investigation on chronic inflammatory responses and its molecular pathways of different environmental relevant levels of MC-LR (0, 0.4, 2 and 10 μg/L) in male zebrafish (Danio rerio). The results showed that chronic MC-LR exposure caused splenic inflammatory changes including the formation of melano-macrophage centers, remarkable elevation of serum tumor necrosis factor alpha (TNFα) and interleukin 1 beta (IL1β) levels as well as significant upregulated expression of MyD88-dependent toll-like receptor (TLR/MyD88) signaling pathway genes (tlr4a, myd88, erk2, p38a, il1β and tnfα). The immunohistochemical and western blot results further validated that higher MC-LR concentrations tended to enhance the MyD88 signal. Moreover, significant decreases of serum C3 levels along with splenic c3b expression in the 10 μg/L exposure group proved that chronic MC-LR exposure could ultimately decrease the innate immunity of fish. Our findings revealed that chronic exposure of MC-LR could cause chronic inflammation through TLR/MyD88 signaling pathway and subsequently induce immune disorders in male zebrafish, which also urge us to pay more attention on the potential immunotoxicity of long-term exposure to low concentration of MC-LR.

Multiple Myd88-dependent Toll-like receptor signaling pathways mediate inflammation in a mouse model of primary Sjögren’s syndrome

Abstract Primary Sjögren’s syndrome (pSS) is an autoimmune disease characterized by exocrine gland dysfunction and immune hyperactivity. The adaptor Myd88 is critical for immune function, as most TLRs utilize Myd88 for signal transduction. Previous work by our group found Myd88 is required for pSS disease. Our objective was to identify the specific Myd88-dependent TLR pathways that mediate salivary and systemic inflammation. We used the pSS mouse model NOD.B10Sn-H2b/J (NOD.B10), Myd88-deficient NOD.B10 mice (NOD.B10Myd88−/−), and C57BL/10 controls. For all experiments we used female mice that were at least 26 weeks old, the age that NOD.B10 females display clinical disease. We isolated spleens from NOD.B10 mice and C57BL/10 controls and performed RNA-sequencing. We then harvested salivary tissue or spleens from NOD.B10, NOD.B10Myd88−/−, and C57BL/10 mice. We performed qPCR and flow cytometry to assess expression of Myd88-dependent TLRs. We cultured salivary tissue from NOD.B10 and NOD.B10Myd88−/− mice and performed cytokine arrays on the supernatants. Finally, we treated salivary tissue with a TLR4 inhibitor and quantified spontaneous IL-6 secretion. We identified dysregulation of numerous TLR-related networks in pSS splenocytes. We found upregulation of Myd88 dependent-TLRs in both the spleen and salivary tissue from NOD.B10 mice. Salivary tissue from Myd88-sufficient NOD.B10 females exhibited spontaneous secretion of IL-6, MCP-1 and TNFa, and this was diminished in NOD.B10Myd88−/− mice. Finally, TLR4 blockade reduced IL-6 secretion in NOD.B10 salivary tissue. Our preliminary data suggest Myd88-dependent pathways contribute to the inflammatory landscape in pSS, and inhibition of such will likely have therapeutic utility.

Induction of TNF, CXCL8 and IL-1β in macrophages by Helicobacter pylori secreted protein HP1173 occurs via MAP-kinases, NF-κB and AP-1 signaling pathways

Macrophages play an important role in the generation of host immune responses against H. pylori. In this study, we investigated the effect of a functionally uncharacterized H. pylori secreted protein HP1173 on macrophage responses. In a screen of eight H. pylori strains, similar expression levels of the HP1173 protein were observed in the whole-cell extracts, however; the amount of protein released into the culture medium varied significantly among strains. Recombinant purified HP1173 (rHP1173) was found to bind to THP-1 cells that were differentiated into macrophages via phorbol-12-myristate-13-acetate (PMA) treatment. The exposure of macrophages to rHP1173 led to the production of the proinflammatory cytokines TNF and IL-1β and the chemokine CXCL8 in a dose- and time-dependent manner. Under similar conditions, rHP1173 failed to induce apoptosis in macrophages. Furthermore, rHP1173-induced expression of TNF, IL-1β and CXCL8 was observed at the level of gene transcription. Incubation of macrophages in the conditioned medium from a mutant H. pylori strain 26695 lacking HP1173 protein expression resulted in the reduced induction of TNF, CXCL8 and IL-1β, confirming the role of the endogenous protein. Intracellular signaling involving MAPKs, NF-κB and the AP-1 family of transcription factors was required for rHP1173-induced TNF, CXCL8 and IL-1β release from macrophages. The blocking of MyD88, which is an adaptor for multiple toll-like receptors (TLRs), had no effect on rHP1173-induced TNF, CXCL8 and IL-1β release from macrophages, suggesting that Myd88-dependent TLR signaling was not involved in the recognition of and responses to rHP1173. These findings provide novel insights into the potential role of HP1173 in H. pylori infection-associated disease development.

MyD88-mediated innate sensing by oral epithelial cells controls periodontal inflammation

Periodontal diseases are a class of non-resolving inflammatory diseases, initiated by a pathogenic subgingival biofilm, in a susceptible host, which if left untreated can result in soft and hard tissue destruction. Oral epithelial cells are the first line of defense against microbial infection within the oral cavity, whereby they can sense the environment through innate immune receptors including toll-like receptors (TLRs). Therefore, oral epithelial cells directly and indirectly contribute to mucosal homeostasis and inflammation, and disruption of this homeostasis or over-activation of innate immunity can result in initiation and/or exacerbation of localized inflammation as observed in periodontal diseases. Dynamics of TLR signaling outcomes are attributable to several factors including the cell type on which it engaged. Indeed, our previously published data indicates that oral epithelial cells respond in a unique manner when compared to canonical immune cells stimulated in a similar fashion. Thus, the objective of this study was to evaluate the role of oral epithelial cell innate sensing on periodontal disease, using a murine poly-microbial model in an epithelial cell specific knockout of the key TLR-signaling molecule MyD88 (B6K5Cre.MyD88plox). Following knockdown of MyD88 in the oral epithelium, mice were infected with Porphorymonas gingivalis and Aggregatibacter actinomycetemcomitans by oral lavage 4 times per week, every other week for 6 weeks. Loss of oral epithelial cell MyD88 expression resulted in exacerbated bone loss, soft tissue morphological changes, soft tissue infiltration, and soft tissue inflammation following polymicrobial oral infection. Most interestingly while less robust, loss of oral epithelial cell MyD88 also resulted in mild but statistically significant soft tissue inflammation and bone loss even in the absence of a polymicrobial infection. Together these data demonstrate that oral epithelial cell MyD88-dependent TLR signaling regulates the immunological balance within the oral cavity under conditions of health and disease.

Rational design of peptide derivatives for inhibition of MyD88-mediated toll-like receptor signaling in human peripheral blood mononuclear cells and epithelial cells exposed to Francisella tularensis.

Small molecules were developed to attenuate proinflammatory cytokines resulting from activation of MyD88-mediated toll-like receptor (TLR) signaling by Francisella tularensis. Fifty-three tripeptide derivatives were synthesized to mimic a key BB-loop region involved in toll-like/interleukin-1 receptor recognition (TIR) domain interactions. Compounds were tested for inhibition of TNF-α, IFN-γ, IL-6, and IL-1β in human peripheral blood mononuclear cells (PBMCs) and primary human bronchial epithelial cells exposed to LPS extracts from F. tularensis. From 53 compounds synthesized and tested, ten compounds were identified as effective inhibitors of F.tularensisLPS-induced cytokines. Compound stability testing in the presence of human liver microsomes and human serum resulted in the identification of tripeptide derivative 7 that was a potent, stable, and drug-like small molecule. Target corroboration using a cell-based reporter assay and competition experiments with MyD88 TIR domain protein supported that the effect of 7 was through MyD88 TIR domain interactions. Compound 7 also attenuated proinflammatory cytokines in human peripheral blood mononuclear cells and bronchial epithelial cells challenged with a live vaccine strain of F. tularensis at a multiplicity of infection of 1:5. Small molecules that target TIR domain interactions in MyD88-dependent TLR signaling represent a promising strategy toward host-directed adjunctive therapeutics for inflammation associated with biothreat agent-induced sepsis.