TIR-domain-containing Adapter-inducing Interferon-β Research Articles

Comparison of HAV and HCV infections in vivo and in vitro reveals distinct patterns of innate immune evasion and activation

Hepatitis A virus (HAV) infections are considered not to trigger innate immunity invivo, in contrast to hepatitis C virus (HCV). This lack of induction has been imputed to strong interference by HAV proteases 3CD and 3ABC. We aimed to elucidate the mechanisms of immune activation and counteraction by HAV and HCV invivo and invitro. Albumin-urokinase-type plasminogen activator/severe combined immunodeficiency (Alb/uPA-SCID) mice with humanised livers were infected with HAV and HCV. Hepatic cell culture models were used to assess HAV and HCV sensing by Toll-like receptor 3 and retinoic acid-inducible gene I/melanoma differentiation-associated protein 5 (RIG-I/MDA5), respectively. Cleavage of the adaptor proteins TIR-domain-containing adapter-inducing interferon-β (TRIF) and mitochondrial antiviral-signalling protein (MAVS) was analysed by transient and stable expression of HAV and HCV proteases and virus infection. We detected similar levels of interferon-stimulated gene induction in hepatocytes of HAV- and HCV-infected mice with humanised liver. In cell culture, HAV induced interferon-stimulated genes exclusively upon MDA5 sensing and depended on LGP2 (laboratory of genetics and physiology 2). TRIF and MAVS were only partially cleaved by HAV 3ABC and 3CD, not sufficiently to abrogate signalling. In contrast, HCV NS3-4A efficiently degraded MAVS, as previously reported, whereas TRIF cleavage was not detected. HAV induces an innate immune response in hepatocytes via MDA5/LGP2, with limited control of both pathways by proteolytic cleavage. HCV activates Toll-like receptor 3 and lacks TRIF cleavage, suggesting that this pathway mainly contributes to HCV-induced antiviral responses in hepatocytes. Our results shed new light on the induction of innate immunity and counteraction by HAV and HCV. Understanding the mechanisms that determine the differential outcomes of HAV and HCV infections is crucial for the development of effective therapies. Our study provides insights into the interplay between these viruses and the host innate immune response invitro and invivo, shedding light on previously controversial or only partially investigated aspects. This knowledge could tailor the development of new strategies to combat HCV persistence, as well as improve our understanding of the factors underlying successful HAV clearance.

Inhibition of HMGB1 Attenuates Spinal Cord Edema by Reducing the Expression of Na+-K+-Cl- Cotransporter-1 and Na+/H+ Exchanger-1 in Both Astrocytes and Endothelial Cells After Spinal Cord Injury in Rats.

Sodium/water transport through Na+-K+-Cl- cotransporter-1 (NKCC1) and sodium/hydrogen exchanger-1 (NHE1) in both astrocytes and endothelial cells is critical to cytotoxic and ionic edema following spinal cord injury (SCI). High-mobility group box-1 (HMGB1) promotes spinal cord edema after SCI. Accordingly, we sought to identify both the role of HMGB1 and the mechanism of its effect on NKCC1 and NHE1 expression in astrocytes and endothelial cells as well as the role of the regulation of spinal cord edema after SCI. An SCI model was generated in adult female rats using a heavy falling object, and an in vitro oxygen-glucose deprivation/reoxygenation (OGD/R) model was generated in rat spinal cord astrocytes and microvascular endothelial cells. The inhibition of HMGB1 reduced NKCC1 and NHE1 expression in the spinal cord of SCI rats, in cultured spinal cord astrocytes, and in cultured microvascular endothelial cells. The effects of HMGB1 on NKCC1 and NHE1 expression were mediated-at least in part-by activation of the Toll-like receptor 4 (TLR4)-Toll/interleukin-1 receptor domain-containing adapter inducing interferon-β (TRIF)-nuclear factor-kappa B (NF-κB) signaling pathway. The inhibition of NKCC1 or NHE1 decreased the spinal cord water content in rats following SCI, increased the Na+ concentration in the medium of cultured astrocytes after OGD/R, and reduced the astrocytic cell volume and AQP4 expression. These results imply that HMGB1 inhibition results in a reduction in NKCC1 and NHE1 expression in both astrocytes and microvascular endothelial cells and thus decreases spinal cord edema after SCI in rats and that these effects occur through the HMGB1-TLR4-TRIF-NF-κB signaling pathway.

MyD88 and not TRIF knockout is sufficient to abolish LPS-induced inflammatory responses in bone-derived macrophages.

Macrophages play an important role in the response to infection and/or repair of injury in tissues. To examine the NF-κB pathway in response to an inflammatory stimulus, we used wild-type bone-marrow-derived macrophages (BMDMs) or BMDMs with knockout (KO) of myeloid differentiation primary response 88 (MyD88) and/or Toll/interleukin-1 receptor domain-containing adapter-inducing interferon-β (TRIF) via CRISPR/Cas9. Following treatment of BMDMs with lipopolysaccharide (LPS) to induce an inflammatory response, translational signalling of NF-κB was quantified via immunoblot and cytokines were measured. Our findings reveal that MyD88 KO, but not TRIF KO, decreased LPS-induced NF-κB signalling, and 10% expression of basal MyD88 expression was sufficient to partially rescue the abolished inflammatory cytokine secretion observed upon MyD88 KO.

Lindenane-Type Sesquiterpene Dimers Mitigate Lipopolysaccharide-Induced Inflammation by Inhibiting Toll-Like Receptor Signaling.

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns and trigger an inflammatory response via the myeloid differential factor 88 (MyD88)-dependent and toll-interleukin-1 receptor domain-containing adapter-inducing interferon-β (TRIF)-dependent pathways. Lindenane type sesquiterpene dimers (LSDs) are characteristic metabolites of plants belonging to the genus Sarcandra (Chloranthaceae). The aim of this study was to evaluate the potential anti-inflammatory effects of the LSDs shizukaol D (1) and sarcandrolide E (2) on lipopolysaccharides (LPS)-stimulated RAW264.7 macrophages in vitro, and explore the underlying mechanisms. Both LSDs neutralized the LPS-induced morphological changes and production of nitric oxide (NO), as determined by CCK-8 assay and Griess assay, respectively. Furthermore, shizukaol D (1) and sarcandrolide E (2) downregulated interferon β (IFNβ), tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) mRNA levels as measured by reverse transcription polymerase chain reaction (RT-PCR), and inhibited the phosphorylation of nuclear factor kappa B p65 (p65), nuclear factor kappa-Bα (IκBα), Jun N-terminal kinase (JNK), extracellular regulated kinase (ERK), mitogen-activated protein kinase p38 (p38), MyD88, IL-1RI-associated protein kinase 1 (IRAK1), and transforming growth factor-β-activated kinase 1 (TAK1) proteins in the Western blotting assay. In conclusion, LSDs can alleviate the inflammatory response by inhibiting the TLR/MyD88 signalling pathway.

Melatonin decreases IRF-3 protein expression in the gastrocnemius muscle, reduces IL-1β and LPS plasma concentrations, and improves the lipid profile in rats with apical periodontitis fed on a high-fat diet.

To evaluate the effects of melatonin (MEL) on the expression of toll-like receptor-4 (TLR4); myeloid differentiation primary response protein-88 (MyD88); TIR-domain-containing adapter-inducing interferon-β (TRIF); IFN regulatory-factor-3 (IRF-3); nuclear factor kappa-B (NF-κB); plasma concentrations of interleukin-1β (IL-1β) and lipopolysaccharide (LPS); and lipid profile of rats with apical periodontitis (AP) fed on a high-fat diet (HFD). Eighty 60-day-old rats were divided into eight groups: control, AP, HFD, HFDAP, CNMEL, APMEL, HFDMEL and HFDAPMEL. HFD groups were fed on a HFD for 107days. On day 7, experimental AP was induced in the AP groups, and after 70days, MEL (5mg/kg) was administered to the MEL groups for 30days. Plasma concentrations of LPS and IL-1β were analyzed using enzyme-linked immunosorbent assay, and the lipid profile was analyzed using biochemical tests. The expression of proteins involved in the TLR4 pathway (TLR4, MyD88, TRIF, IRF-3 and NF-κB) in the gastrocnemius muscle (GM) was evaluated using western blotting and qRT-PCR. Treatment with MEL decreased IRF-3 protein expression in GM and IL-1β plasma concentration in the APMEL and HFDMEL groups. Reduction in LPS plasma concentration was reported only in the HFDMEL group. Additionally, a decrease in LDL and an increase in HDL were observed in the HFDMEL and HFDAPMEL groups. Treatment with MEL exhibited anti-inflammatory and anti-hyperlipidemic effects attributed to HFD and AP by reducing the plasma concentrations of IL-1β and LPS in addition to reducing IRF-3 protein expression in the GM, which is associated with the production of inflammatory cytokines.

Oxybaphus himalaicus Mitigates Lipopolysaccharide-Induced Acute Kidney Injury by Inhibiting TLR4/MD2 Complex Formation.

Acute kidney injury (AKI) is described as the abrupt decrease in kidney function always accompanied by inflammation. The roots of Oxybaphus himalaicus Edgew. have long been used in Tibetan folk medicine for the treatment of nephritis. Nevertheless, modern pharmacological studies, especially about the underlying mechanism of O. himalaicus medications, are still lacking. Here, in lipopolysaccharide (LPS)-induced RAW264.7 macrophages, the O. himalaicus extract (OE) showed significant anti-inflammatory activity with the dose dependently reducing the LPS-stimulated release of nitric oxide and the mRNA level and protein expression of inflammatory cytokines and reversed the activation of nuclear factor kappa B (NF-κB). Co-immunoprecipitation assay indicated that OE inhibited Toll-like receptor 4/myeloid differentiation factor 2 (TLR4/MD2) complex formation and further suppressed both myeloid differentiation factor 88 (MyD88)-dependent and TIR-domain-containing adapter-inducing interferon-β (TRIF)-dependent cascades activation. In addition, OE could restrain NADPH oxidase 2 (NOX2) endocytosis by blocking TLR4/MD2 complex formation to prevent reactive oxygen species production. In LPS-induced AKI mice, OE treatment mitigated renal injury and inflammatory infiltration by inhibiting TLR4/MD2 complex formation. UPLC-MS/MS analysis tentatively identified 41 components in OE. Our results indicated that OE presented significant anti-inflammatory activity by inhibiting TLR4/MD2 complex formation, which alleviated LPS-induced AKI in mice.

Polyguanine alleviated autoimmune hepatitis through regulation of macrophage receptor with collagenous structure and TLR4-TRIF-NF-κB signalling.

Autoimmune hepatitis (AIH) is a progressive and chronic inflammatory disease in the liver. MARCO is a surface receptor of macrophage involving in tissue inflammation and immune disorders. Moreover, polyguanine (PolyG) is considered to bind to macrophage receptor with collagenous structure (MARCO). However, the role of MARCO and PolyG in the development and treatment of AIH still remains unclear. Therefore, this study explores the expression of MARCO and therapeutic activity of PolyG in both S100-induced AIH in mouse and Lipopolysaccharide (LPS)-treated macrophage (RAW264.7 cells). Moreover, there were significant increases in inflammatory factors and MARCO, as well as decrease in I-kappa-B-alpha (Ik-B) in the liver of AIH mice and LPS-induced cells. However, PolyG treatment significantly reversed the elevation of inflammatory cytokins, MARCO and reduction of Ik-B. In addition, PolyG treatment could downregulate the expression of Toll-like receptor 4 (TLR4) and TIR-domain-containing adaptor inducing interferon-β (TRIF), decrease macrophage M1 polarization and increase macrophage M2 polarization. When hepatocytes were co-cultured with different treatment of macrophages, similar expression profile of inflammatory cytokines was observed in hepatocytes. The research revealed that MARCO expression was elevated in AIH mice. PolyG treatment and inhibition of MARCO significantly reduced inflammatory cytokines expression in the liver as well as hepatocytes and macrophages. Therefore, MARCO could be a target for the treatment of AIH.

Inhibition of TRIF-Dependent Inflammation Decelerates Afterload-Induced Myocardial Remodeling.

Pressure-overload-induced cardiac hypertrophy represents one cause of the development of heart failure. The aim of this study is to characterize the influence of the TIR-domain-containing adapter-inducing interferon-β (TRIF) during afterload-induced myocardial remodeling. After trans-aortic constriction (TAC), cardiac pressure overload leads to an early increase in MyD88- (Myeloid differentiation primary response gene 88) and TRIF-dependent cytokines. The maximum cytokine expression appeared within the first week and decreased to its control level within five weeks. While cardiomyocyte hypertrophy was comparable, the myocardial accumulation of the inflammatory cells was lower in TRIF−/−mice. At d7, TRIF deficiency reduced transcription factors and TRIF-dependent cytokines. Through the modulation of the TGF-β-signaling pathway and anti-fibrotic microRNAs, TRIF was involved in the development of interstitial fibrosis. The absence of TRIF was associated with a decreased expression of proapoptotic proteins. In echocardiography and working heart analyses, TRIF deficiency slowed left-ventricular wall thickening, myocardial hypertrophy, and reduces the ejection fraction. In summary, TRIF is an important adapter protein for the release of inflammatory cytokines and the accumulation of inflammatory cells in the early stage of maladaptive cardiac remodeling. TRIF is involved in the development of cardiac fibrosis by modulating inflammatory and fibrotic signal transduction pathways.

A genome-wide search of Toll/Interleukin-1 receptor (TIR) domain-containing adapter molecule (TICAM) and their evolutionary divergence from other TIR domain containing proteins

Toll/Interleukin-1 receptor (TIR) domains are cytoplasmic domain that mediates receptor signalling. These domains are present in proteins like Toll-like receptors (TLR), its signaling adaptors and Interleukins, that form a major part of the immune system. These TIR domain containing signaling adaptors binds to the TLRs and interacts with their TIR domains for downstream signaling. We have examined the evolutionary divergence across the tree of life of two of these TIR domain containing adaptor molecules (TICAM) i.e., TIR domain-containing adapter-inducing interferon-β (TRIF/TICAM1) and TIR domain containing adaptor molecule2 (TRAM/TICAM2), by using computational approaches. We studied their orthologs, domain architecture, conserved motifs, and amino acid variations. Our study also adds a timeframe to infer the duplication of TICAM protein from Leptocardii and later divergence into TICAM1/TRIF and TICAM2/TRAM. More evidence of TRIF proteins was seen, but the absence of conserved co-existing domains such as TRIF-NTD, TIR, and RHIM domains in distant relatives hints on diversification and adaptation to different biological functions. TRAM was lost in Actinopteri and has conserved domain architecture of TIR across species except in Aves. An additional isoform of TRAM, TAG (TRAM adaptor with the GOLD domain), could be identified in species in the Mesozoic era. Finally, the Hypothesis based Likelihood ratio test was applied to look for selection pressure amongst orthologues of TRIF and TRAM to search for positively selected sites. These residues were mostly seen in the non-structural region of the proteins. Overall, this study unravels evolutionary information on the adaptors TRAM and TRIF and how well they had duplicated to perform diverse functions by changes in their domain architecture across lineages.

Effects of acupuncture on microglial polarization and the TLR4/TRIF/MyD88 pathway in a rat model of traumatic brain injury.

Neuroinflammation caused by traumatic brain injury (TBI) can lead to neurological deficits. Acupuncture can inhibit neuroinflammation and promote nerve repair; however, the specific mechanism is still unclear. The purpose of this study was to explore whether acupuncture could modulate the M1 and M2 phenotypic polarization of microglia in a rat model of TBI via the toll-like receptor 4 (TLR4)/intracellular toll-interleukin-1 receptor (TIR) domain-containing adaptor inducing interferon-β (TRIF)/myeloid differentiation factor 88 (MyD88) pathway. A total of 90 adult male Sprague-Dawley (SD) rats, SPF grade, were randomly divided into a normal group, model group and acupuncture group. Each group was further divided into three subgroups (first, third, and fifth day groups) according to the treatment time (n = 10 rats/subgroup). We used the modified neurological severity score (mNSS) method to quantify neurological deficits before and after modeling. We used Nissl staining to observe the pathological changes in brain tissue, flow cytometry to detect the proportion of M1 and M2 polarized microglia in the injured area on the first, third and fifth day, and co-immunoprecipitation (Co-IP) to examine TLR4/TRIF/MyD88 expression in microglia on the first, third and fifth day, as well as expression of the amount of binding of TLR4 with TRIF and MyD88. Compared to the model group, mNSS in the acupuncture group gradually decreased and pathological morphology improved. The proportion of CD11b/CD86 positive cells was decreased, while that of CD11b/CD206 was increased in the acupuncture group. Expression of IP TLR4, IP TRIF and IP MyD88 also decreased in the acupuncture group. The results of this study demonstrate that one of the mechanisms through which acupuncture mitigates neuroinflammation and promotes nerve repair in TBI rats may be inhibition of M1 phenotypic polarization and promotion of M2 phenotypic polarization through inhibition of the TLR4/TRIF/MyD88 signaling pathway.

MiR-30e-5p attenuates neuronal deficit and inflammation of rats with intracerebral hemorrhage by regulating TLR4.

microRNAs (miRNAs or miRs) have been reported to regulate the pathology of intracerebral hemorrhage (ICH). Therefore, the present study aimed to investigate the function of miR-30e-5p in rats with ICH with specific focus on Toll-like receptor (TLR)4. In the present study, collagenase type IV was used for the establishment of the ICH model in rats, prior to which the rats were injected with miR-30e-5p mimic or miR-30e-5p mimic + pcDNA3.1-TLR4 plasmid. The expression levels of miR-30e-5p and TLR4 were then measured using reverse transcription-quantitative PCR and western blotting. The potential interaction between miR-30e-5p and TLR4 was tested using the MicroRNA Target Prediction Database and dual-luciferase reporter and RNA immunoprecipitation assay. In addition, the concentration of TNF-α, IL-6 and IL-1β was measured using ELISA. The protein expression levels of TLR4/myeloid differentiation factor 88 (MyD88)/TIR-domain-containing adapter-inducing interferon-β (TRIF) signaling-associated molecules were measured by western blotting. Following induction of ICH, miR-30e-5p expression was downregulated, while TLR4 expression was upregulated. By contrast, injection with miR-30e-5p mimic rescued neuronal function while suppressing neuronal inflammation in rats following ICH; these effects were reversed by co-overexpression of TLR4. Furthermore, overexpression of miR-30e-5p inactivated TLR4/MyD88/TRIF signaling in rats with ICH; this was also reversed by overexpression of TLR4. Taken together, these results suggested that overexpression of miR-30e-5p exerted a protective role against neuronal deficit and inflammation caused by ICH in rats by targeting TLR4 and inactivating TLR4/MyD88/TRIF signaling.

The RHIM of the Immune Adaptor Protein TRIF Forms Hybrid Amyloids with Other Necroptosis-Associated Proteins.

TIR-domain-containing adapter-inducing interferon-β (TRIF) is an innate immune protein that serves as an adaptor for multiple cellular signalling outcomes in the context of infection. TRIF is activated via ligation of Toll-like receptors 3 and 4. One outcome of TRIF-directed signalling is the activation of the programmed cell death pathway necroptosis, which is governed by interactions between proteins that contain a RIP Homotypic Interaction Motif (RHIM). TRIF contains a RHIM sequence and can interact with receptor interacting protein kinases 1 (RIPK1) and 3 (RIPK3) to initiate necroptosis. Here, we demonstrate that the RHIM of TRIF is amyloidogenic and supports the formation of homomeric TRIF-containing fibrils. We show that the core tetrad sequence within the RHIM governs the supramolecular organisation of TRIF amyloid assemblies, although the stable amyloid core of TRIF amyloid fibrils comprises a much larger region than the conserved RHIM only. We provide evidence that RHIMs of TRIF, RIPK1 and RIPK3 interact directly to form heteromeric structures and that these TRIF-containing hetero-assemblies display altered and emergent properties that likely underlie necroptosis signalling in response to Toll-like receptor activation.

ZBP1 is a crucial regulator for tumor necroptosis during tumor development.

Abstract Tumor necrosis happens commonly in advanced solid tumors. We reported that necroptosis plays a major role in tumor necrosis. By studying TNF-induced necroptosis, it is now known that receptor interacting protein kinase 1 and 3 (RIPK1 and RIPK3) and the mixed lineage kinase domain-like (MLKL) constitute the core of the necroptosis machinery. Two other proteins, Z-DNA-binding protein 1 (ZBP1) and TIR-domain-containing adapter-inducing interferon-β (TRIF) are known to function upstream of RIPK3 and interact with RIPK3 to mediate necroptosis in response to viral infection or TLR signaling respectively. Our study shows that it is the ZBP1, and not RIPK1 nor TRIF, that mediates tumor necroptosis during tumor development in preclinical cancer models. We found that ZBP1 expression is dramatically elevated in the later stages of both the MVT-1 breast cancer model and the MMTV-PyMT, a genetically engineered mouse model (GEMM) of breast cancer, when necrosis happens. Next, we generated CRISPR ZBP1 knockout (ZBP1 KO) MVT-1 cells to examine whether ZBP1 is needed for tumor necroptosis during MVT-1 mammary tumor development. ZBP1 deletion blocks tumor necroptosis during primary tumor development, more importantly, we observed decreased lung metastasis comparing to wildtype. We showed that glucose deprivation (GD) triggers ZBP1-depedent necroptosis in tumor cells. GD causes mitochondrial DNA (mtDNA) release to the cytoplasm and the binding of mtDNA to ZBP1 to activate MLKL in a BCL-2 family protein, NOXA-dependent manner. Therefore, our study reveals ZBP1 as the key regulator of tumor necroptosis and provides a potential drug target for controlling tumor metastasis.

Inhibitory effects of luteolin on TLR3-mediated inflammation caused by TAK/NF-κB signaling in human corneal fibroblasts.

To study the role of luteolin (LUT) in the expression of toll-like receptors 3 (TLR3) ligand polyI:C stimulated inflammatory factors in human corneal fibroblasts (HCFs). HCFs cells were cultivated with or without LUT or polyI:C. The expression levels of interleukin (IL)-6, IL-8, monocyte chemotactic protein-1 (MCP-1), vascular cell adhesion molecule (VCAM)-1, as well as intercellular adhesion molecule (ICAM)-1 were measured using enzyme-linked immunosorbent assay (ELISA), immunoblotting or reverse transcription-quantitative polymerase chain reaction (PCR) analyses. Immunoblotting was used to assess toll-interleukin-1 receptor-domain-containing adapter-inducing interferon-β (TRIF), TLR3, transforming growth factor-b-activated kinase 1 (TAK1), tumor necrosis factor receptor-associated factor 6 (TRAF6), the transcription factor AP-1, as well as transcription factor nuclear factor (NF-κB)-inhibitory protein IκB-α degradation and phosphorylation. Immunofluorescence assays were used to localize the cellular location of the p65 subunit of NF-κB. Corneal fibroblasts exposed to polyI:C demonstrated decreased VCAM-1, ICAM-1, MCP-1, IL-6, and IL-8 expression levels upon exposure to LUT in a time-dependent and concentration-dependent manner. LUT was observed to suppress polyI:C-triggered expression of TLR3, the translocation of NF-κB p65 into cell nuclei, as well as the phosphorylation of TAK, c-Jun, and IκB-α, while no impact on the expression levels of TRIF and TRAF6 were observed. LUT suppress the expression of proinflammatory adhesion molecules, chemokines, and cytokines in polyI:C exposed HCFs. These effects are likely mediated through TAK/NF-κB signal attenuation. Therefore, LUT is a candidate molecule that can prevent the TLR3-mediated inflammation response associated with corneal viral infection.

Rare catastrophes and evolutionary legacies: human germline gene variants in MLKL and the necroptosis signalling pathway.

Programmed cell death has long been characterised as a key player in the development of human disease. Necroptosis is a lytic form of programmed cell death that is universally mediated by the effector protein mixed lineage kinase domain-like (MLKL), a pseudokinase. MLKL's activating kinase, receptor interacting protein kinase 3 (RIPK3), is itself activated within context specific scaffolds of receptor interacting protein kinase 1 (RIPK1), Z-DNA Binding Protein-1 (ZBP1) or TIR domain-containing adaptor inducing interferon-β (TRIF). These core necroptosis modulating proteins have been comprehensively revealed as potent drivers and suppressors of disease in inbred mouse strains. However, their roles in human disease within the ‘real world’ of diverse genetic backgrounds, natural infection and environmental challenges remains less well understood. Over 20 unique disease-associated human germline gene variants in this core necroptotic machinery have been reported in the literature and human clinico-genetics databases like ClinVar to date. In this review, we provide an overview of these human gene variants, with an emphasis on those encoding MLKL. These experiments of nature have the potential to not only enrich our understanding of the basic biology of necroptosis, but offer important population level insights into which clinical indications stand to benefit most from necroptosis-targeted drugs.

Membrane Vesicles Derived From Clostridium botulinum and Related Clostridial Species Induce Innate Immune Responses via MyD88/TRIF Signaling in vitro.

Clostridium botulinum produces botulinum neurotoxin complexes that cause botulism. Previous studies elucidated the molecular pathogenesis of botulinum neurotoxin complexes; however, it currently remains unclear whether other components of the bacterium affect host cells. Recent studies provided insights into the role of bacterial membrane vesicles (MVs) produced by some bacterial species in host immunity and pathology. We herein examined and compared the cellular effects of MVs isolated from four strains of C. botulinum with those of closely related Clostridium sporogenes and two strains of the symbiont Clostridium scindens. MVs derived from all strains induced inflammatory cytokine expression in intestinal epithelial and macrophage cell lines. Cytokine expression was dependent on myeloid differentiation primary response (MyD) 88 and TIR-domain-containing adapter-inducing interferon-β (TRIF), essential adaptors for toll-like receptors (TLRs), and TLR1/2/4. The inhibition of actin polymerization impeded the uptake of MVs in RAW264.7 cells, however, did not reduce the induction of cytokine expression. On the other hand, the inhibition of dynamin or phosphatidylinositol-3 kinase (PI3K) suppressed the induction of cytokine expression by MVs, suggesting the importance of these factors downstream of TLR signaling. MVs also induced expression of Reg3 family antimicrobial peptides via MyD88/TRIF signaling in primary cultured mouse small intestinal epithelial cells (IECs). The present results indicate that MVs from C. botulinum and related clostridial species induce host innate immune responses.

TIR-Domain-Containing Adapter-Inducing Interferon-β (TRIF)-Dependent Antiviral Responses Protect Mice against Ross River Virus Disease.

ABSTRACTRoss River virus (RRV) is the major mosquito-borne virus in the South Pacific region. RRV infections are characterized by arthritic symptoms, which can last from several weeks to months. Type I interferon (IFN), the primary antiviral innate immune response, is able to modulate adaptive immune responses. The relationship between the protective role of type I IFN and the induction of signaling proteins that drive RRV disease pathogenesis remains poorly understood. In the present study, the role of TIR-domain-containing adapter-inducing interferon-β (TRIF), an essential signaling adaptor protein downstream of Toll-like receptor (TLR) 3, a key single-stranded RNA (ssRNA)-sensing receptor, was investigated. We found that TRIF−/− mice were highly susceptible to RRV infection, with severe disease, high viremia, and a low type I IFN response early during disease development, which suggests the TLR3-TRIF axis may engage early in response to RRV infection. The number and the activation level of CD4+ T cells, CD8+ T cells, and NK cells were reduced in TRIF−/− mice compared to those in infected wild-type (WT) mice. In addition, the number of germinal center B cells was lower in TRIF−/− mice than WT mice following RRV infection, with lower titers of IgG antibodies detected in infected TRIF−/− mice compared to WT. Interestingly, the requirement for TRIF to promote immunoglobulin class switch recombination was at the level of the local immune microenvironment rather than B cells themselves. The slower resolution of RRV disease in TRIF−/− mice was associated with persistence of the RRV genome in muscle tissue and a continuing IFN response.

DDX43 recruits TRIF or IPS-1 as an adaptor and activates the IFN-β pathway in Nile tilapia (Oreochromis niloticus)

DDX43 is one of the members of the DExD/H-box protein family, and emerging data suggest that it may play an important role in antiviral immunity across mammals. However, little is known about DDX43 in the fish immune response. In this study, we isolated the cDNA sequence of ddx43 in Nile tilapia (Oreochromis niloticus). The ddx43 gene was 2338 bp in length, contained an open reading frame (ORF) of 2064 bp and encoded a polypeptide of 687 amino acids. The predicted protein of OnDDX43 has three conserved domains, including the RNA binding domain KH, DEAD-like helicase superfamily DEXDc and C-terminal HELICc domain. In healthy Nile tilapia, the Onddx43 transcript was broadly expressed in all examined tissues, with the highest expression levels in the muscle and brain and the lowest in the liver. After challenge with Streptococcus agalactiae, lipopolysaccharides (LPS) and polyinosinic polycytidylic acid (Poly I:C), the expression level of Onddx43 mRNA was upregulated or downregulated in all of the tissues tested. Overexpression of OnDDX43 in 293 T cells showed that it has a positive regulatory effect on IFN-β. The subcellular localization showed that OnDDX43 was expressed in the cytoplasm. We performed further pull-down assays and found that OnDDX43 interacted with both interferon-β promoter stimulator1 (IPS-1) and TIR domain-containing adaptor inducing interferon-β (TRIF).

Inhibiting RGS1 attenuates secondary inflammation response and tissue degradation via the TLR/TRIF/NF-κB pathway in macrophage post spinal cord injury

Macrophage-dominated inflammation by the activation of Toll‐like receptor (TLR) pathway leads to neurological disruption after spinal cord injury (SCI). Regulator of G‐protein signaling 1 (RGS1) is reported to be a regulator in inflammation. The present study thus purposes to identify the unknown role of RGS1 mediating TLR on inflammation post SCI. A mouse model of traumatic SCI was established by a mechanical trauma at T10. The mice underwent SCI and a macrophage line activated by lipopolysaccharide (LPS) were treated with shRNA-RGS1 to elucidate the role of RGS1 in inflammatory progression. The inflammatory factors were measured, and the degree of histology and function protection were determined. The expression levels of RGS1, myeloid differentiation primary response protein 88 (Myd88), (TIR-domain-containing adaptor inducing interferon-β (TRIF), p38, metalloproteinase (MMP)‐2, and MMP‐9 were determined. RGS1 was robustly increased both in LPS-activated macrophage and SCI mice. The TLR signaling pathway-induced inflammation was suppressed by RGS1 knockdown. shRNA‐mediated silence of RGS1 was exhibited a prominent decrease in TNF‐α, IL‐1β and IL-6 via TLR/TRIF/ nuclear factor kappa-B (NF-κB) axis. Depletion of RGS1 also inhibited MMP-induced tissue degradation via MAPK-p38 pathway in SCI mice. Moreover, suppression of RGS1 improved spinal cord histology and function recovery. These findings suggest that RGS1 regulates inflammation and tissue disruption via TLR/TRIF/NF-κB signaling pathway in mice with SCI, thereby explaining a novel target that regulates macrophage inflammation post SCI.

Selenium Deficiency Causes Inflammatory Injury in the Bursa of Fabricius of Broiler Chickens by Activating the Toll-like Receptor Signaling Pathway.

The aim of our study was to observe the effect of selenium (Se) deficiency on inflammatory injury in the bursa of Fabricius of broiler chickens and to determine the role of the Toll-like receptor (TLR)/myeloid differential protein-88 (MyD88)/nuclear factor-κB (NF-κB) signaling pathway during this process. Here, we revealed that severe inflammatory injury occurred in the broiler bursa of Fabricius with Se deficiency via histopathology. Moreover, the ultrastructural pathological results showed that the nuclear, mitochondrial, endoplasmic reticulum and cytomembrane structures were damaged to varying degrees. Additionally, interleukin-2 (IL-2), interleukin-6 (IL-6), and interferon (IFN-γ) mRNA expression was markedly upregulated in the broiler bursa of Fabricius with Se deficiency. Furthermore, TLR, toll-interleukin-1 receptor domain-containing adapter-inducing interferon-β (TRIF), MyD88, and NF-κB mRNA expression was also markedly elevated in the broiler bursa of Fabricius with Se deficiency. The above results suggested that Se deficiency increases the expression of numerous proinflammatory cytokines and is probably due to the activation of the TLR/MyD88/NF-κB signaling pathway, which causes inflammatory injury in the bursa of Fabricius of broiler chickens. Our findings provide a theoretical reference for further studying the underlying mechanism of Se deficiency-induced inflammatory injury in the bursa of Fabricius of broiler chickens.