MyD88-independent Signaling Pathway Research Articles

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.

TIRAP, TRAM, and Toll-Like Receptors: The Untold Story.

Toll-like receptors (TLRs) are the most studied receptors among the pattern recognition receptors (PRRs). They act as microbial sensors, playing major roles in the regulation of the innate immune system. TLRs mediate their cellular functions through the activation of MyD88-dependent or MyD88-independent signaling pathways. Myd88, or myeloid differentiation primary response 88, is a cytosolic adaptor protein essential for the induction of proinflammatory cytokines by all TLRs except TLR3. While the crucial role of Myd88 is well described, the contribution of other adaptors in mediating TLR signaling and function has been underestimated. In this review, we highlight important results demonstrating that TIRAP and TRAM adaptors are also required for full signaling activity and responses induced by most TLRs.

P38 MAPK activation by Toxoplasma gondii secretory effector protein GRA24: Role in cytokine regulation and induction of protective immunity

Abstract The apicomplexan Toxoplasma gondii induces strong protective immunity in part dependent upon recognition by Toll-like receptors (TLR) 11 and 12 operating in conjunction with MyD88 in the murine host. However, these TLR are not present in humans, prompting us to investigate MyD88-independent signaling pathways of resistance. We show here that parasite dense granule protein GRA24 induces p38 MAPK activation and subsequent IL-12 production in bone marrow derived mouse macrophages (BMDM). Furthermore, Toxoplasma triggers two waves of p38 MAPK phosphorylation in MyD88+/+ and MyD88−/− BMDM. Using the uracil auxotrophic Type I T. gondii strain cps1-1, we demonstrate that the late response does not require active parasite proliferation, but strictly depends upon GRA24. By i. p. inoculation with cps1-1 and cps1-1:Δgra24, we identified unique subsets of chemokines and cytokines that were up and down regulated by GRA24. We demonstrate that cps1-1 triggers a strong host-protective GRA24-dependent Th1 response in the absence of MyD88. To gain deeper insight into genes regulated by GRA24, we employed qPCR to evaluate expression of transcripts for 84 cytokine and chemokine genes in BMDM infected with GRA24-positive compared to GRA24-negative parasites. Our results identify a specific signature of genes regulated by this dense granule protein. In conjunction, using qPCR we evaluated the impact of GRA24 on a panel of macrophage long noncoding RNA (lncRNA) regulated by Toxoplasma infection. Taken together, our data identify GRA24 as a major mediator of p38 MAPK activation, induction of IL-12 and other immune mediators, as well as protective immunity that operates independently of the TLR/MyD88 cascade.

Sexually Dimorphic Role of Toll-like Receptor 4 (TLR4) in High Molecular Weight Hyaluronan (HMWH)-induced Anti-hyperalgesia

High molecular weight hyaluronan (HMWH), a prominent component of the extracellular matrix binds to and signals via multiple receptors, including cluster of differentiation 44 (CD44) and toll-like receptor 4 (TLR4). We tested the hypothesis that, in the setting of inflammation, HMWH acts at TLR4 to attenuate hyperalgesia. We found that the attenuation of prostaglandin E2 (PGE2)-induced hyperalgesia by HMWH was attenuated by a TLR4 antagonist (NBP2-26245), but only in male and ovariectomized female rats. In this study we sought to evaluated the role of the TLR4 signaling pathway in anti-hyperalgesia induced by HMWH in male rats. Decreasing expression of TLR4 in nociceptors, by intrathecal administration of an oligodeoxynucleotide (ODN) antisense to TLR4 mRNA, also attenuated HMWH-induced anti-hyperalgesia, in male and ovariectomized female rats. Estrogen replacement in ovariectomized females reconstituted the gonad-intact phenotype. The administration of an inhibitor of myeloid differentiation factor 88 (MyD88), a TLR4 second messenger, attenuated HMWH-induced anti-hyperalgesia, while an inhibitor of the MyD88-independent TLR4 signaling pathway did not. Since it has previously been shown that HMWH-induced anti-hyperalgesia is also mediated, in part by CD44 we evaluated the effect of the combination of ODN antisense to TLR4 and CD44 mRNA. This treatment completely reversed HMWH-induced anti-hyperalgesia in male rats. Our results demonstrate a sex hormone-dependent, sexually dimorphic involvement of TLR4 in HMWH-induced anti-hyperalgesia, that is MyD88 dependent. PerspectiveThe role of TLR4 in anti-hyperalgesia induced by HMWH is a sexually dimorphic, TLR4 dependent inhibition of inflammatory hyperalgesia that provides a novel molecular target for the treatment of inflammatory pain.

Heme Induced Platelet Mitochondrial Oxidant Production Regulates Thrombospondin-1 Release from Platelets

Background: Sickle Cell Disease (SCD) patients develop chronic vasculopathy leading to conditions such as pulmonary hypertension (PH), a major cause of morbidity and mortality. Vasculopathy is tightly associated with hemolysis in SCD. We and others have shown that in SCD, hemolysis activates platelets, and activated platelets are implicated in vasculopathy through their release of vasoactive molecules, such as thrombosponding-1 (TSP1), from storage granules. In SCD, free heme is accumulated in the circulation as a consequence of oxidative degradation of hemoglobin secondary to intravascular hemolysis. Under homeostatic conditions, albumin, haptoglobin, hemopexin, and high- and low-density lipoproteins clear plasma heme from the circulation. However excessive hemolysis can overwhelm these clearance mechanisms, resulting in elevated levels of free heme, which can lead to platelet activation, aggregation and thrombus formation. The role of activated platelets in these thrombotic events is well studied, but the mechanisms by which heme induces platelet activation and platelet-dependent signaling to the vessel wall remain unclear. We previously showed that hemolysis induces platelet mitochondrial reactive oxygen species (mtROS) production, but the mechanism by which heme induces mtROS production and downstream consequences are less clear. We hypothesize that heme induces platelet mtROS through Toll-like Receptor 4 (TLR4) signaling, which regulates platelet activation and granule secretion. Methods: Washed platelets were prepared from whole blood collected from healthy human volunteers in 0.32% sodium citrate anticoagulant (n=6). Platelets were treated with heme (2.5µM) in presence or absence of TLR4 neutralizing antibody, IRAK1/4 inhibitor, TBK1/IKKε inhibitor, MitoTEMPO or DS16570511 (Mitochondrial calcium uniporter inhibitor). Platelet activation was measured using flow cytometry by staining for CD62P (P-selectin) on the platelet surface and the active confirmation of GPIIb/IIIa. Platelet mtROS was measured by fluorescence spectroscopy using MitoSOX Red. The granule secretion function of platelets was measured by assessing TSP-1 and Platelet Factor 4 (PF4) levels in platelet releasates by ELISA. Results: We found that heme activates platelets and induces mtROS production, which can be mitigated by TLR4 neutralizing antibody, a molecule that makes platelet surface TLR4 unavailable to heme. In heme-treated platelets, pharmacological inhibition of the interleukin-1 receptor kinase-4 (IRAK4), a key kinase recruited by the TLR4 adaptor protein MyD88, had no significant effect on platelet activation. In contrast, inhibition of tank binding kinase-1 (TBK1), a TLR4 effector molecule independent of MyD88 signaling, significantly attenuated heme-mediated platelet activation and mtROS production. Scavenging mtROS with MitoTEMPO did not alter platelet activation, instead it decreased the TSP1 release from platelets, indicating heme induced platelet mtROS tightly regulates the granule secretion function of platelets. In addition, blocking mitochondrial calcium uptake by Mitochondrial calcium uniporter (MCU) inhibitor significantly decreased mtROS production and TSP-1 release from heme treated platelets. Conclusion: Our data demonstrate that heme activates human platelets and induces mtROS production via a TLR4 -MyD88-independent signaling pathway. In addition, heme also induces mitochondrial calcium uptake that enhances mtROS production in platelets. Heme induced platelet mtROS tightly regulates platelet granule secretion function. Ongoing studies are further elucidating this pathway and determining the role of mtROS in heme-dependent platelet function. These studies will provide a mechanistic link between hemolysis and thrombosis, as well as platelet-vascular wall signaling. These studies suggest that platelet mtROS may potentially be a novel therapeutic target to prevent thrombotic events and vascular dysfunction in hemolytic disorders including SCD. Disclosures No relevant conflicts of interest to declare.

Lipopolysaccharide-induced DC-SIGN/TLR4 crosstalk activates NLRP3 inflammasomes via MyD88-independent signaling in gastric epithelial cells

Abnormal pattern recognition receptor (PRR) signaling plays an important role in gastric mucosal damage caused by stomach microbiota; however, the underlying molecular mechanisms remain obscure. Here, we show that DC-SIGN, a surface phenotype marker of dendritic cells, is overexpressed in gastric epithelial cells facing LPS stimulation. NLRP3 expression in gastric epithelial cells are significantly increased and related to the degree of LPS stimulation. Furthermore, DC-SIGN could interact with TLR4, promote NLRP3 and related genes expression via MyD88-independent signaling pathway and regulate the secretion of IL-1β and IL-18 in gastric epithelial cells. The results of flow cytometry analysis show that DC-SIGN primarily mediates Th1 differentiation when co-cultured with gastric epithelial cells. These results reveal that LPS-induced DC-SIGN expression modulates NLRP3 inflammasomes formation via MyD88-independent TLR4 signaling in gastric epithelial cell, and induces a Th1-predominant host immune response,these findings may indicate a new function of DC-SIGN in non-immune cells, and elucidate the diversity role of gastric epithelial cells in mechanism of immune damage caused by microbial flora.

Ferric Heme Induces Platelet Activation and mtROS Production through a MyD88-independent TLR4 Signaling Pathway

Ferric Heme Induces Platelet Activation and mtROS Production through a MyD88-independent TLR4 Signaling Pathway

Lipopolysaccharide enhances DNA-induced IFN-β expression and autophagy by upregulating cGAS expression in A549 cells.

Cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) is a newly identified cytosolic DNA sensor, but its function in lung epithelial cells is relatively unknown. In the present study, the effects of lipopolysaccharide (LPS) on the expression and function of cGAS in the A549 lung epithelial cell line was investigated. The cells were treated with LPS at different concentrations (e.g., 100, 200, 400 and 800 ng/ml), and the cGAS expression levels were examined via western blot analysis and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The cells were pretreated with LPS, followed by E. coli DNA transfection using Lipofectamine® 3000. After 24 h, interferon (IFN)-β production was measured using ELISA and the expression of the autophagic markers, microtubule-associated proteins 1A/1B light chain 3 and sequestosome-1, were determined using western blot analysis. The cells were also pretreated with either a toll-like receptor (TLR) 4 inhibitor, a serine/threonine-protein kinase TBK1 (TBK1) inhibitor or an nuclear factor (NF)-κB inhibitor, followed by LPS treatment, and the cGAS expression levels were examined via western blot analysis and RT-qPCR. The result showed that LPS treatment upregulated cGAS expression in a dose-dependent manner. E. coli DNA treatment could induce IFN-β production and autophagy via cGAS, which was enhanced by LPS pretreatment. The effect of LPS on cGAS expression was suppressed by treatment with a TLR4 inhibitor, a TBK1 inhibitor and an NF-κB inhibitor. In conclusion, LPS enhances DNA-induced IFN-β production and autophagy by upregulating cGAS expression through the myeloid differentiation primary response protein MyD88-independent TLR4 signaling pathway in A549 cells.

The inhibition of RANKL expression in fibroblasts attenuate CoCr particles induced aseptic prosthesis loosening via the MyD88-independent TLR signaling pathway

Periprosthetic osteolysis and aseptic loosening are mainly caused by wear particles (Ps) that are generated from friction interfaces. However, the mechanisms underlying the development of aseptic loosening remain unclear. Therefore, we aimed toclarify how the myeloid differentiation factor 88 (MyD88)-independent Toll-like receptor (TLR) signaling pathway mediates cobalt and chromium (CoCr)-Ps-induced osteolysis. We quantified the expression levels of TLRs, MyD88, RANKL, and inflammatory factors in patients experiencing aseptic loosening after primary total hip arthroplasty (THA) with metal-on-metal (MoM) bearings and hip osteoarthritis (hOA). We observed the in vitro and in vivo levels of RANKL, TLRs, and MyD88 in fibroblasts challenged with CoCr Ps by applying shMyD88 interference lentivirus vectors to block the MyD88-independent TLR pathway. The levels of TLRs, MyD88, RANKL, and inflammatory factors in the revision THA (rTHA) with MoM group were higher than those in the hOA group. Our data collectively revealed that inhibiting MyD88 expression could reduce osteoclastogenesis in vitro and CoCr-Ps-induced osteolysis in vivo. Our findings suggested that osteoclastogenesis is promoted by the CoCr-Ps-induced expression of RANKL in fibroblasts and that MyD88 is a potential target in the treatment of wear Ps-induced osteolysis.

MyD88 in antigen-presenting cells is not required for CD4+ T-cell responses during peptide nanofiber vaccination.

Self-assembled peptide nanofibers raise significant antibody and T cell responses without adjuvants, but the mechanism by which they achieve this has not been fully elucidated. Myeloid differentiation primary response gene 88 (MyD88) previously has been shown to be critical for the antibody response to antigens presented by peptide nanofibers. The present study sought to determine the cell subset in which MyD88 is essential for T cell responses. Mice deficient in MyD88 or CD11c+ cells had severely attenuated T cell responses. However, mice lacking MyD88 in only CD11c+ cells remained capable of internalizing, processing, and presenting nanofiber-derived epitopes to stimulate T cell responses. The necessity of inflammasome pathway was ruled out. Using adoptive transfer models where MyD88 was eliminated in CD4+ T cells or in the host, we observed that deficiency only in T cells or only in the host had no impact on the T cell response to nanofiber vaccines. Therefore, knocking out MyD88 in either antigen presenting cells (APCs) or CD4 T cells could not compromise the CD4 T cell responses, suggesting that self-assembled peptide nanofibers trigger redundant MyD88-dependent and MyD88-independent signaling pathways in APCs and T cells. Similar redundancy has been observed for other adjuvants, and this is discussed.

Capsular Polysaccharide is a Main Component of Mycoplasma ovipneumoniae in the Pathogen-Induced Toll-Like Receptor-Mediated Inflammatory Responses in Sheep Airway Epithelial Cells.

Mycoplasma ovipneumoniae (M. ovipneumoniae) is characterized as an etiological agent of primary atypical pneumonia that specifically infects sheep and goat. In an attempt to better understand the pathogen-host interaction between the invading M. ovipneumoniae and airway epithelial cells, we investigated the host inflammatory responses against capsular polysaccharide (designated as CPS) of M. ovipneumoniae using sheep bronchial epithelial cells cultured in an air-liquid interface (ALI) model. Results showed that CPS derived from M. ovipneumoniae could activate toll-like receptor- (TLR-) mediated inflammatory responses, along with an elevated expression of nuclear factor kappa B (NF-κB), activator protein-1 (AP-1), and interferon regulatory factor 3 (IRF3) as well as various inflammatory-associated mediators, representatively including proinflammatory cytokines, such as IL1β, TNFα, and IL8, and anti-inflammatory cytokines such as IL10 and TGFβ of TLR signaling cascade. Mechanistically, the CPS-induced inflammation was TLR initiated and was mediated by activations of both MyD88-dependent and MyD88-independent signaling pathways. Of importance, a blockage of CPS with specific antibody led a significant reduction of M. ovipneumoniae-induced inflammatory responses in sheep bronchial epithelial cells. These results suggested that CPS is a key virulent component of M. ovipneumoniae, which may play a crucial role in the inflammatory response induced by M. ovipneumoniae infections.

Abstract 3047: Mitochondrial DNA copy variation and TFAM expression in astrocytoma

Abstract Mitochondrial dysfunction plays a role in determining the phenotype through bioenergetic depletion and increased production of ROS in several diseases, including cancer. We have previously demonstrated a relevant reduction of mitochondrial DNA (mtDNA) copy number in astrocytoma of different grades of malignancy, predominantly in grade IV-glioblastoma (GBM). We observed a stepwise increase of TFAM in parallel to the increase of malignancy, and TFAM expression was higher in GBM patients with overall survival longer than 24 months than less than 12 months. TFAM is codified in the nucleus and transported into mitochondria, where is essential for mtDNA replication, repair and protection by forming nucleoid complexes. To further understand the role of TFAM in astrocytoma progression we performed TFAM immunohistochestry in human astrocytoma samples, confocal analysis in GBM cell lines, in vitro functional assays after silencing TFAM by siRNA in two GBM cell lines (U87MG, A172), and high throughput RNA-Seq in Illumina platform for network analysis by Metacore. TFAM protein was detected in cytoplasm in granular pattern of low grade astrocytomas, and also in nuclei of malignant atsrocytomas. Such localization was also confirmed by confocal analysis in two GBM cell lines. TFAM-siRNA transfection decreased TFAM expression level >80% in D2, which persisted up to D7 with evident decrease of mtDNA copy number after 4 days, and maintained until 7 days. Interestingly, a decrease in migration but not in proliferation was observed in both cell lines after TFAM knockdown. Comparison of RNA-Seq of TFAM-siRNA to NTC- siRNA, with 20 million of reads in each experiment performed in duplicate, resulted in 1000 genes differentially expressed and among them 315 were detected in both cell lines. These genes were in pathways related to cell adhesion (integrin-mediated: p = 5.79e-5, histamine H1 receptor signaling in the interruption of cell barrier integrity: p = 3.98e-5, chemokines-mediated: p = 1.22e-5) and ECM remodeling (p = 9.17e-5), supporting the results of in vitro migration assay. 82 genes were downregulated together with TFAM in both cell lines. These genes were in pathways related to immune response IL15 signaling via JAK-STAT cascade involving MyD88-independent toll-like receptor signaling pathway involving ADAM17 and STAT2 (p = 1.89e-5), regulation of cytoskeleton proteins (p = 1.65e-2), hypoxia-induced EMT in cancer and fibrosis (p = 3.37e-2), polyamine and arginine metabolism (p = 2.73e-2, p = 4.65e-2) and as expected, regulation of apoptotic process (p = 9.50e-21). The role of TFAM in cytoskeleton and ECM remodeling may worth additional studies. Also, TFAM in extracellular space, as released by necrotic cells, may potentiate mitochondrial N-formyl peptide-induced secretion of chemokines and activate inflammatory responses, which may open another strategy to further understand TFAM role in tumorigeniesis. Citation Format: Suely K. Marie, Roseli Silva, Antonio Lerario, Miyuki Uno, Sueli Mieko Oba-Shinjo. Mitochondrial DNA copy variation and TFAM expression in astrocytoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3047. doi:10.1158/1538-7445.AM2015-3047

Mycoplasma ovipneumoniae induces inflammatory response in sheep airway epithelial cells via a MyD88-dependent TLR signaling pathway

Mycoplasma ovipneumoniae (M. ovipneumoniae) is a bacterium that specifically infects sheep and goat and causes ovine infectious pleuropneumonia. In an effort to understand the pathogen–host interaction between the M. ovipneumoniae and airway epithelial cells, we investigated the host inflammatory response using a primary air–liquid interface (ALI) epithelial culture model generated from bronchial epithelial cells of Ningxia Tan sheep (Ovis aries). The ALI culture of sheep bronchial epithelial cells showed a fully differentiated epithelium comprising distinct epithelial types, including the basal, ciliated and goblet cells. Exposure of ALI cultures to M. ovipneumoniae led to increased expression of Toll-like receptors (TLRs), and components of the myeloid differentiation factor 88 (MyD88)-dependent TLR signaling pathway, including the MyD88, TNF receptor-associated factor 6 (TRAF6), IL-1 receptor-associated kinases (IRAKs) and nuclear factor-kappa B (NF-κB), as well as subsequent pro-inflammatory cytokines in the epithelial cells. Of interest, infection with M. ovipneumoniae failed to induce the expression of TANK-binding kinase 1 (TBK1), TRAF3 and interferon regulatory factor 3 (IRF3), key components of the MyD88-independent signaling pathway. These results suggest that the MyD88-dependent TLR pathway may play a crucial role in sheep airway epithelial cells in response to M. ovipneumoniae infection, which also indicate that the ALI culture system may be a reliable model for investigating pathogen–host interactions between M. ovipneumoniae and airway epithelial cells.

76: Regulation of activins and the activin-binding protein, follistatin, by Toll-like receptor ligands in the adult mouse

The activins, A and B, are members of the transforming growth factor- β cytokine family, and are regulators of inflammation, immunity and fibrosis. Production of activin A and the activin-binding protein, follistatin, increases during inflammation, although aspects of this regulation require clarification and activin B has not been studied in inflammation, previously. Adult male mice were injected with Toll-like receptor (TLR) ligands, acting though MyD88-dependent (TLR2, 4, 7 and 9) and MyD88-independent (TLR3, 4) signalling pathways. Activation of TLR4 by lipopolysaccharide (LPS) was also examined in MyD88-deficient mice, and in mice pre-treated with cycloheximide and actinomycin D. Activin A, B and follistatin were measured in serum by specific immunoasssays, and mRNA expression was measured in the liver by qRT-PCR. Activin A secretion, but not mRNA expression, was stimulated via activation of TLR2, 3, 4 and 7, reaching a peak in the serum around 2–3 h. By contrast, all ligands stimulated activin B at both protein and mRNA level. Compared with activin A, activin B in serum increased more slowly (3–5 h), but reached much higher maximum concentrations. Follistatin was stimulated at the mRNA level by TLR2, 3, 4 and 7. In the absence of MyD88, induction of activin A and follistatin by LPS was prevented, but activin B production was unaffected. While production of activin A was inhibited by cycloheximide, but not by actinomycin D, activin B was inhibited to a similar degree by both compounds. These data indicate that viral and bacterial ligands, acting through both MyD88-dependent and MyD88-independent signaling pathways, stimulate production of activin A, B and follistatin. These data further indicate that activin B, like follistatin, but unlike activin A, is primarily regulated at the transcriptional level during inflammation. These studies suggest that delayed production of activin B may complement the more rapid release of activin A during inflammatory responses.

TLR2 signaling may play a key role in the probiotic modulation of intestinal microbiota in grouper Epinephelus coioides

Abstract In homothermic animals, toll-like receptors (TLRs) participate in detecting microbial-associated molecular patterns (MAMPs) of probiotics for activation of intestinal mucosal immunity and modulation of gut microbiota. This study evaluated the possible roles of TLRs in fish intestinal mucosal immune response to probiotic Psychrobacter sp. SE6 and microbiota modulation by examining the dominant microbial community and expression of TLR genes and other genes involved in TLR-mediated signaling within the intestine of grouper Epinephelus coioides. The fish were fed for 60 days with three different diets: control (without probiotic), diets T1 and T2 supplemented with 1.0 × 108 cells g− 1 viable and heat-inactivated Psychrobacter sp. SE6, respectively. Upregulated expression of TLR2 and TLR5, adaptor MyD88 and cytokines (IL-1β, IL-8 and TGF-β1) was observed in fish fed the viable SE6, while elevated expression of TLR2, but not MyD88 and cytokines, was observed in fish fed the heat-inactivated SE6, which suggested that MyD88-independent TLR2 signaling pathway may be involved in the probiotic recognition in E. coioides. The induced activation of intestinal mucosal immunity, especially the enhanced expression of antibacterial epinecidin-1 and IgM, was consistent with the intestinal microbial data showing that several bacteria were suppressed to undetectable level by both the viable and heat-inactivated SE6. These findings lay the foundation for future studies on the molecular interactions between the probiotics, gut microbiota and mucosal immunity in fish.

FRI0026 Interleuikin-32 induces tnfalpha and type i interferon via proteinase 3 - protease activated receptor 2-tir-domain-containing adapter-inducing interferon-beta axis

BackgroundInterleukin (IL)-32 is known to exert adujvant effects on innate immune response and has an important role in inflammatory disease, such as rheumatoid arthritis [1]. However, receptors and downstream signaling...

MyD88, a central innate immune adaptor, is dispensable for spontaneous autoimmune demyelination in a mouse model of multiple sclerosis (P4077)

Abstract Multiple sclerosis (MS) is an autoimmune disease that is mediated by myelin-reactive T cells resulting in CNS demyelination. Mice that are transgenic for a myelin proteolipid protein (PLP)-specific T cell receptor (TCR) spontaneously develop experimental autoimmune encephalomyelitis (EAE), the animal model of MS. MyD88 is the key adaptor protein in Toll-like receptor (TLR), IL-1R and IL-18R signaling, resulting in the activation of innate immune cells, including DCs. Although, MyD88-signaling is critical for T cell responses against microbial agents, it is unknown whether it is required for priming of myelin-reactive T cells in spontaneous EAE or MS. Here, we investigated the requirement of MyD88-signaling for the pathogenesis of spontaneous EAE in PLP TCR transgenic mice that genetically lacked MyD88. We demonstrate that MyD88 is dispensable for the development of spontaneous EAE and autoimmune demyelination in these mice. MyD88-independent EAE was associated with activated DCs that showed superior ability to promote PLP-specific Th1 and Th17 responses in vitro compared to DCs from healthy counterparts. In addition, we provide evidence indicating that the MyD88-independent TRIF signaling pathway was induced in EAE-diseased mice. Taken together, our data suggest that MyD88-independent innate immune signaling critically contributes to priming of myelin-reactive T cells and initiation of spontaneous EAE in our MS model.

Mouse pyrin and HIN domain family member 1 (pyhin1) protein positively regulates LPS-induced IFN-β and NO production in macrophages

The pyrin and HIN-domain (PYHIN) family member1 (pyhin1) is a member of PYHIN proteins and involved in transcriptional regulation of genes important for cell cycle control, differentiation and apoptosis. The regulatory action of mouse pyhin1 on LPS-induced inflammatory response was examined. LPS augmented the pyhin1 mRNA expression in murine RAW 264.7 macrophage cells and peritoneal macrophages. The augmentation of pyhin1 mRNA expression was abolished by parthenolide, a NF-κB inhibitor. Silencing of pyhin1 with small interfering RNA reduced the production of IFN-β and NO. However, pyhin1 silencing did not affect the production of TNF-α, IL-6, IL-10 and prostaglandin E2. Reduced IFN-β production by pyhin1 silencing caused inactivation of STAT1 and reduced expression of IRF1. Pyhin1 silencing inhibited the expression of TRAF6, TBK1 and TRIF, which trigger IFN-β production in the MyD88-independent pathway. However, pyhin1 silencing did not affect the expression of MyD88, IRAK4 and several mitogen-activated protein kinases in the MyD88-dependent pathway. Taken together, mouse pyhin1 was suggested to be a NF-κB-responsible gene in response to LPS and positively regulate LPS-induced IFN-β and NO production through up-regulating the MyD88-independent signaling pathway.

Phylogeny of Toll-Like Receptor Signaling: Adapting the Innate Response

The Toll-like receptors represent a largely evolutionarily conserved pathogen recognition machinery responsible for recognition of bacterial, fungal, protozoan, and viral pathogen associated microbial patterns and initiation of inflammatory response. Structurally the Toll-like receptors are comprised of an extracellular leucine rich repeat domain and a cytoplasmic Toll/Interleukin 1 receptor domain. Recognition takes place in the extracellular domain where as the cytoplasmic domain triggers a complex signal network required to sustain appropriate immune response. Signal transduction is regulated by the recruitment of different intracellular adaptors. The Toll-like receptors can be grouped depending on the usage of the adaptor, MyD88, into MyD88-dependent and MyD88 independent subsets. Herein, we present a unique phylogenetic analysis of domain regions of these receptors and their cognate signaling adaptor molecules. Although previously unclear from the phylogeny of full length receptors, these analyses indicate a separate evolutionary origin for the MyD88-dependent and MyD88-independent signaling pathway and provide evidence of a common ancestor for the vertebrate and invertebrate orthologs of the adaptor molecule MyD88. Together these observations suggest a very ancient origin of the MyD88-dependent pathway Additionally we show that early duplications gave rise to several adaptor molecule families. In some cases there is also strong pattern of parallel duplication between adaptor molecules and their corresponding TLR. Our results further support the hypothesis that phylogeny of specific domains involved in signaling pathway can shed light on key processes that link innate to adaptive immune response.

Hantaan Virus Triggers TLR4-Dependent Innate Immune Responses

The innate immune response induced by Hantavirus is responsible for endothelial cell dysfunction and viral pathogenicity. Recent studies demonstrate that TLR4 expression is upregulated and mediates the secretion of several cytokines in Hantaan virus (HTNV)-infected endothelial cells. To examine viral interactions with host endothelial cells and characterize the innate antiviral responses associated with Toll-like receptors, we selected TLR4 as the target molecule to investigate anti-hantavirus immunity. TLR4 mRNA-silenced EVC-304 (EVC-304 TLR4-) cells and EVC-304 cells were used to investigate signaling molecules downstream of TLR4. The expression of the adaptor protein TRIF was higher in HTNV-infected EVC-304 cells than in EVC-304 TLR4- cells. However, there was no apparent difference in the expression of MyD88 in either cell line. The transcription factors for NF-κB and IRF-3 were translocated from the cytoplasm into the nucleus in HTNV-infected EVC-304 cells, but not in HTNV-infected EVC-304 TLR4- cells. Our results demonstrate that TLR4 may play an important role in the antiviral immunity of the host against HTNV infection through an MyD88-independent signaling pathway.