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

Crude extract of Camellia oleifera pomace ameliorates the progression of non-alcoholic fatty liver disease via decreasing fat accumulation, insulin resistance and inflammation.

Consumption of a high-fat diet increases fat accumulation and may further lead to inflammation and hepatic injuries. The aim of the study was to investigate the effects of Camellia oleifera seed extract (CSE) on non-alcoholic fatty liver disease (NAFLD). After a 16-week NAFLD-inducing period, rats were assigned to experimental groups fed an NAFLD diet with or without CSE. At the end of the study, we found that consuming CSE decreased the abdominal fat weight and hepatic fat accumulation and modulated circulating adipokine levels. We also found that CSE groups had lower hepatic cytochrome P450 2E1 and transforming growth factor (TGF)-β protein expressions. In addition, we found that CSE consumption may have affected the gut microbiota and reduced toll-like receptor (TLR)-4, myeloid differentiation primary response gene 88, toll/IL-1 receptor domain-containing adaptor-inducing interferon-β (TRIF) expression and proinflammatory cytokine concentrations in the liver. Our results suggest that CSE may alleviate the progression of NAFLD in rats with diet-induced steatosis through reducing fat accumulation and improving lipid metabolism and hepatic inflammation.

TIR Domain-Containing Adaptor-Inducing Interferon-β (TRIF) Participates in Antiviral Immune Responses and Hepatic Lipogenesis of Large Yellow Croaker (Larimichthys Crocea).

TIR domain-containing adaptor-inducing interferon-β (TRIF), a cytosolic adaptor protein, plays a key role in the mammalian toll-like receptor-mediated signaling pathway. However, the role of TRIF in large yellow croaker (LcTRIF) remains poorly understood. The main objective of this study was to explore the role of LcTRIF in triggering antiviral immune responses and the potential function of LcTRIF in regulating lipid metabolism. In the present study, the full-length coding sequence of TRIF from large yellow croaker was cloned and characterized. In vivo, upon poly (I:C) stimulation, the transcriptional levels of LcTRIF were rapidly elevated in immune-related tissues at the early stage of injection. In vitro, the MRNA expression of LcTRIF was significantly but not dramatically upregulated in macrophages treated with poly (I:C). Activation of LcTRIF by poly (I:C) significantly increased the transcription of genes involved in inflammatory responses, and this induction was blocked by knockdown of LcTRIF. Moreover, the ability of LcTRIF to induce inflammatory responses may partially be attributed to the promotion of mRNA expression of IFNh and NF-κB pathway genes. In addition, activation of the LcTRIF-mediated pathway inhibited the increase in hepatic stearoyl-coenzyme A (CoA) desaturase 1 induced by palmitic acid and subsequently alleviated lipid accumulation in hepatocytes. These results revealed the crucial role of LcTRIF in triggering antiviral immune responses and the unconventional metabolic function of LcTRIF in regulating hepatic lipogenesis in large yellow croaker.

Regulation of TRIF-mediated innate immune response by K27-linked polyubiquitination and deubiquitination

TIR domain-containing adaptor inducing interferon-β (TRIF) is an essential adaptor protein required for innate immune responses mediated by Toll-like receptor (TLR) 3- and TLR4. Here we identify USP19 as a negative regulator of TLR3/4-mediated signaling. USP19 deficiency increases the production of type I interferons (IFN) and proinflammatory cytokines induced by poly(I:C) or LPS in vitro and in vivo. Usp19-/- mice have more serious inflammation after poly(I:C) or LPS treatment, and are more susceptible to inflammatory damages and death following Salmonella typhimurium infection. Mechanistically, USP19 interacts with TRIF and catalyzes the removal of TRIF K27-linked polyubiquitin moieties, thereby impairing the recruitment of TRIF to TLR3/4. In addition, the RING E3 ubiquitin ligase complex Cullin-3-Rbx1-KCTD10 catalyzes K27-linked polyubiquitination of TRIF at K523, and deficiency of this complex inhibits TLR3/4-mediated innate immune signaling. Our findings thus reveal TRIF K27-linked polyubiquitination and deubiquitination as a critical regulatory mechanism of TLR3/4-mediated innate immune responses.

Liver Inflammatory Injury Initiated by DAMPs-TLR4-MyD88/TRIF-NFκB Signaling Pathway Is Involved in Monocrotaline-Induced HSOS.

Hepatic sinusoidal obstruction syndrome (HSOS) causes considerable morbidity and mortality in clinic. Up to now, the molecular mechanisms involved in the development of HSOS still remain unclear. Here, we report that hepatic inflammation initiated by damage-associated molecular patterns (DAMPs) plays a critical role in the development of HSOS. Monocrotaline (MCT) belongs to pyrrolizidine alkaloids. Monocrotaline-induced HSOS in mice and rats was evidenced by the increased serum alanine/aspartate aminotransferase (ALT/AST) activities, the elevated hepatic metalloproteinase 9 (MMP9) expression, and results from liver histological evaluation and scanning electron microscope observation. However, MCT-induced HSOS was markedly attenuated in myeloid differentiation primary response gene 88 (MyD88), TIR-domain-containing adapter-inducing interferon-β (TRIF) and toll like receptor 4 (TLR4) knock-out mice. Monocrotaline increased liver myeloperoxidase activity, serum contents of proinflammatory cytokines, hepatic aggregation of immune cells, and nuclear accumulation of nuclear factor κB (NFκB). However, these inflammatory responses induced by MCT were all diminished in MyD88, TRIF, and TLR4 knock-out mice. Monocrotaline elevated serum contents of DAMPs including high mobility group box 1 (HMGB1) and heat shock protein 60 (HSP60) both in mice and in rats. HSOS was markedly exacerbated and serum contents of HMGB1 and HSP60 were elevated in nuclear factor erythroid 2-related factor 2 (Nrf2) knock-out mice treated with MCT. Our findings indicate that hepatic inflammatory injury mediated by DAMPs-initiated TLR4-MyD88/TRIF-NFκB inflammatory signal pathway plays an important role in HSOS development.

β-Caryophyllene as a Potential Protective Agent Against Myocardial Injury: The Role of Toll-Like Receptors.

Myocardial infarction (MI) remains one of the major causes of mortality around the world. A possible mechanism involved in myocardial infarction is the engagement of Toll-like receptors (TLRs). This study was intended to discover the prospective cardioprotective actions of β-caryophyllene, a natural sesquiterpene, to ameliorate isoproterenol (ISO)-induced myocardial infarction through HSP-60/TLR/MyD88/NFκB pathway. β-Caryophyllene (100 or 200 mg/kg/day orally) was administered for 21 days then MI was induced via ISO (85 mg/kg, subcutaneous) on 20th and 21st days. The results indicated that ISO induced a significant infarcted area associated with several alterations in the electrocardiogram (ECG) and blood pressure (BP) indices and caused an increase in numerous cardiac indicators such as creatine phosphokinase (CPK), creatine kinase-myocardial bound (CK-MB), lactate dehydrogenase (LDH), and cardiac tropinine T (cTnT). In addition, ISO significantly amplified heat shock protein 60 (HSP-60) and other inflammatory markers, such as TNF-α, IL-Iβ, and NFκB, and affected TLR2 and TLR4 expression and their adaptor proteins; Myeloid differentiation primary response 88 (MYD88), and TIR-domain-containing adapter-inducing interferon-β (TRIF). On the other hand, consumption of β-caryophyllene significantly reversed the infarcted size, ECG and BP alterations, ameliorated the ISO elevation in cardiac indicators; it also notably diminished HSP-60, and subsequently TLR2, TLR4, MYD88, and TRIF expression, with a substantial reduction in inflammatory mediator levels. This study revealed the cardioprotective effect of β-caryophyllene against MI through inhibiting HSP-60/TLR/MyD88/NFκB signaling pathways.

Molecular cloning and functional characterization of TRIF in large yellow croaker Larimichthys crocea

As an adaptor in Toll-like receptor (TLR) signaling pathway, Toll/interleukin-1 receptor (TIR) domain containing adaptor inducing interferon-β (TRIF) mediates downstream signaling cascades and plays important roles in host innate immune responses. In the present study, a TRIF ortholog named Lc-TRIF was identified in large yellow croaker (Larimichthys crocea). Sequence comparison analysis revealed that Lc-TRIF has a conserved TIR domain but without TRAF6 binding motif. The genome structure of Lc-TRIF is conserved, with two exons and one intron. Syntenic comparison showed that the loci of fish TRIF was different from that in mammals or birds, and TRAM was absent in the genomes of fish, amphibians, and birds, but present in mammals and reptiles. Expression analysis revealed that Lc-TRIF was broadly expressed in examined organs/tissues, with the highest expression level in gill and weakest in brain, and could be up-regulated under poly I:C, LPS, PGN, and Pseudomonas plecoglossicida stimulation. Fluorescence microscopy results showed that Lc-TRIF exhibited a global localization throughout the entire cell including the nucleus in HEK 293T cells. Additionally, luciferase assays demonstrated that Lc-TRIF expression could significantly induce NF-κB, type I IFN, IRF3 as well as IRF7 promoter activation. These results collectively indicated that Lc-TRIF was function in host antiviral and antibacterial responses via NF-κB and IRF3/7 related signaling pathway.

Bacteriophage trigger antiviral immunity and prevent clearance of bacterial infection.

Bacteriophage are abundant at sites of bacterial infection, but their effects on mammalian hosts are unclear. We have identified pathogenic roles for filamentous Pf bacteriophage produced by Pseudomonas aeruginosa (Pa) in suppression of immunity against bacterial infection. Pf promote Pa wound infection in mice and are associated with chronic human Pa wound infections. Murine and human leukocytes endocytose Pf, and internalization of this single-stranded DNA virus results in phage RNA production. This triggers Toll-like receptor 3 (TLR3)- and TIR domain-containing adapter-inducing interferon-β (TRIF)-dependent type I interferon production, inhibition of tumor necrosis factor (TNF), and the suppression of phagocytosis. Conversely, immunization of mice against Pf prevents Pa wound infection. Thus, Pf triggers maladaptive innate viral pattern-recognition responses, which impair bacterial clearance. Vaccination against phage virions represents a potential strategy to prevent bacterial infection.

Targeting Negative Regulators of TRIF-dependent TLR Signaling Pathway as a Novel Therapeutic Strategy

Background: Toll-Like Receptors (TLRs) are a subclass of pathogen-associated molecular patterns (PAMPs). There is a growing interest in the use of TLR agonists for various pathological dysfunctions, including cancer, microbial infections, and inflammatory diseases. TLR3/4 agonists that can induce TIR-domain-containing adapter-inducing interferon-β (TRIF)- dependent pathway have shown fewer toxic immunostimulatory responses in comparison to other small molecules. Furthermore, TLR3 agonists indicate promising anti-tumor potential in cancer immunotherapy either as vaccine adjuvant or monotherapy. </P><P> Objective: It is logical to assume that the induction of the genes that are involved in TRIF pathway to augment their pleiotropic effects on different cells via TLR agonists, could enhance the treatment process of disease while minimizing the toxicity related to using other small molecules. Methods: An extensive literature search to identify the negative regulators of TRIF-dependent signaling pathway and their biological functions was performed from two databases PubMed and Scopus. Results: Negative regulators of TRIF signaling pathways were identified. In addition, structure and function of sterile α- and armadillo-motif containing protein (SARM), the only TIR domaincontaining adaptor protein that inhibits TRIF-dependent activation, were briefly reviewed. Conclusion: We proposed that the manipulation of TRIF signaling pathway via targeting its negative regulators could be used as an approach to modulate the functions of this pathway without undesired toxic proinflammatory responses.

Toll-Like Receptor 4 Signaling Licenses the Cytosolic Transport of Lipopolysaccharide From Bacterial Outer Membrane Vesicles

Outer membrane vesicles (OMVs), released by variety of bacteria, are membrane-enclosed entities enriched in microbial components, toxins, and virulent factors. OMVs could deliver lipopolysaccharide (LPS) into the cytosol of host cells and subsequently activate caspase-11, which critically orchestrates immune responses and mediates septic shock. Although it is known that caspase-11 is activated by intracellular LPS, how OMVs deliver LPS into the cytosol remains largely unknown. Here we show that the activation of toll-like receptor 4 (TLR4), a LPS receptor on the cytoplasmic membrane, licenses macrophages to transport LPS from OMVs into the cytosol through TIR domain-containing adaptor-inducing interferon-β (TRIF). TRIF-mediated cytosolic delivery of LPS from OMVs depends on the production of type 1 interferon and the expression of guanylate-binding proteins (GBPs). Deletion of TRIF or GBPs prevents pyroptosis and lethality induced by OMVs or OMVs-releasing Escherichia coli. Together, these findings provide novel insight into how host coordinates extracellular and intracellular LPS sensing to orchestrate immune responses during gram-negative bacterial infection.

Prestimulation of Microglia Through TLR4 Pathway Promotes Interferon Beta Expression in a Rat Model of Alzheimer's Disease.

Soluble amyloid beta (Aβ) oligomers are the most common forms of Aβ in the early stage of Alzheimer's disease (AD). They are highly toxic to the neurons but their capability to activate microglia remains controversial. Microglia develop two distinct phenotypes, classic (M1) and alternative (M2). Tuning of microglia to the alternative (anti-inflammatory) state is of major interest in treatment of neuroinflammatory disease. This study aimed to assess tuning the microglia to produce interferon beta (IFN-β) as an anti-inflammatory cytokine through TLR4 pathway in a rat model of AD. Microglial BV-2 cells were treated with 1μg/ml lipopolysaccharides (LPS), Monophosphoryl lipid A (MPL), or vehicles for 24h, and then incubated with Aβ oligomer. After 24h, cell pellets were harvested and TIR-domain-containing adapter-inducing interferon-β (TRIF), interferon regulatory factor 3 (IRF3), and IFN-β levels were measured. The ligands/vehicle were microinjected into the right ventricle of male Wistar rats every 3days. Two weeks later, an osmotic pump filled with oligomeric Aβ/vehicle was implanted in the left ventricle. After 2weeks, TRIF, IRF3, and IFN-β levels were measured in the hippocampal tissue. TNF-α and IFN-β levels were assessed in the hippocampus using immunohistochemistry. The oligomeric Aβ did not change TRIF, IRF3, and IFN-β levels in both cell culture and hippocampal tissue. However, pretreatment with LPS or MPL increased the level of these proteins. BV-2 cells morphologically express M1 state in presence of higher dose of Aβ oligomer (10μM). Pretreatment with LPS or MPL decreased the TNF-α and increased the number of IFN-β positive cells in the hippocampus of Aβ-treated rats. In conclusion, pretreatment with low dose TLR4 agonists could induce microglia to produce neuroprotective cytokines including IFN-β which may be considered as a potential strategy to combat neuronal degeneration in AD.

Herpes simplex virus 1 ICP6 impedes TNF receptor 1–induced necrosome assembly during compartmentalization to detergent-resistant membrane vesicles

Receptor-interacting protein (RIP) kinase 3 (RIPK3)-dependent necroptosis directs inflammation and tissue injury, as well as anti-viral host defense. In human cells, herpes simplex virus 1 (HSV1) UL39-encoded ICP6 blocks RIP homotypic interacting motif (RHIM) signal transduction, preventing this leakage form of cell death and sustaining viral infection. TNF receptor 1 (TNFR1)-induced necroptosis is known to require the formation of a RIPK1-RIPK3-mixed lineage kinase domain-like pseudokinase (MLKL) signaling complex (necrosome) that we find compartmentalizes exclusively to caveolin-1-associated detergent-resistant membrane (DRM) vesicles in HT-29 cells. Translocation proceeds in the presence of RIPK3 kinase inhibitor GSK'840 or MLKL inhibitor necrosulfonomide but requires the kinase activity, as well as RHIM signaling of RIPK1. ICP6 impedes the translocation of RIPK1, RIPK3, and MLKL to caveolin-1-containing DRM vesicles without fully blocking the activation of RIPK3 or phosphorylation of MLKL. Consistent with the important contribution of RIPK1 RHIM-dependent recruitment of RIPK3, overexpression of RHIM-deficient RIPK3 results in phosphorylation of MLKL, but this does not lead to either translocation or necroptosis. Combined, these data reveal a critical role of RHIM signaling in the recruitment of the MLKL-containing necrosome to membrane vesicle-associated sites of aggregation. A similar mechanism is predicted for other RHIM-containing signaling adaptors, Z-nucleic acid-binding protein 1 (ZBP1) (also called DAI and DLM1), and TIR domain-containing adapter-inducing interferon-β (TRIF).

Cluster of differentiation 14 and toll-like receptor 4 are involved in the anti-inflammatory effects of tyrosol

We investigated effects of tyrosol on upstream pathway regulating lipopolysaccharide (LPS)-induced inflammatory response, including cluster of differentiation 14 (CD14)-mediated LPS–macrophage binding, toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD2) expression and subsequent myeloid differentiation primary response gene 88 (MyD88) and TIR-domain-containing adapter-inducing interferon-β (TRIF) recruitments. Flow cytometry revealed that, compared with LPS-treated RAW264.7 cells (LPS group), those treated with LPS and tyrosol (1.2 mM) [LPS+T(1.2) group] demonstrated lower LPS–macrophage binding and membrane-bound CD14 (mCD14) and TLR4/MD2 expression (all p < 0.05). Immunoprecipitation/immunoblotting assay revealed lower MyD88 and TRIF concentrations in the LPS+T(1.2) group than in the LPS group (both p < 0.05). Soluble CD14 concentration was higher in the LPS+T(1.2) group than in the LPS group (p = 0.013); protease inhibition counteracted this effect. Thus, tyrosol inhibits LPS–macrophage binding, mCD14 and TLR4/MD2 expression, and MyD88 and TRIF recruitment, all possibly by enhancing protease-mediated mCD14 cleavage.

TRIF deficiency protects non-obese diabetic mice from type 1 diabetes by modulating the gut microbiota and dendritic cells

The incidence of type 1 diabetes (T1D) is determined by both genetic and environmental factors. In recent years, the gut microbiota have been identified to be an important environmental factor that could modify diabetes susceptibility. We have previously shown that Myeloid differentiation primary response gene 88 (MyD88), a major adaptor protein downstream of most innate immune Toll-like receptor (TLR) signaling, is important for mediating diabetes susceptibility in the non-obese diabetic (NOD) mouse model of human T1D. Here we report the role of TIR-domain-containing adapter-inducing interferon-β (TRIF) in T1D development, as TRIF is an important adaptor protein downstream of TLR3 and TLR4 signaling. We found that TRIF-deficient (TRIF−/-) NOD mice were protected from development of diabetes, but only when housed with TRIF-deficient (TRIF−/-) NOD mice. When housed with TRIF-sufficient wild type (WT, i.e., TRIF+/+) NOD mice, the mice developed diabetes. We further investigated the gut microbiota as a potential cause for the altered diabetes development. Interestingly, TRIF−/−NOD mice had a different microbiota composition compared to WT NOD mice, only if they were housed with TRIF−/−NOD mice. However, the composition of gut microbiota in the TRIF−/−NOD mice was indistinguishable from WT NOD mice, if they were housed with WT NOD mice. The difference in the gut microbiota in TRIF−/−NOD mice, due to cohousing, accorded with the diabetes development in TRIF−/−NOD mice. Comparing the gut microbiota in TRIF−/- and WT NOD mice, we identified changes in percentage of Sutterella, Rikenella and Turicibacter species. Moreover, bacteria from WT NOD mice induced significantly stronger inflammatory immune responses in vitro compared to those from TRIF−/−NOD mice. Further immunological analysis revealed impaired function of dendritic cells and reduced T cell activation and proliferation in TRIF−/−NOD mice. Our data show that TRIF-deficiency protects NOD mice from diabetes development through alteration of the gut microbiota and reduced immune cell activation; however, that protection is over-ridden upon exposure to WT NOD bacteria. Therefore exposure to different microbiota can modify disease susceptibility determined by genetic factors related to innate immunity.

Regulator of calcineurin 1 differentially regulates TLR-dependent MyD88 and TRIF signaling pathways.

Toll-like receptors (TLRs) recognize the conserved molecular patterns in microorganisms and trigger myeloid differentiation primary response 88 (MyD88) and/or TIR-domain-containing adapter-inducing interferon-β (TRIF) pathways that are critical for host defense against microbial infection. However, the molecular mechanisms that govern TLR signaling remain incompletely understood. Regulator of calcineurin-1 (RCAN1), a small evolutionarily conserved protein that inhibits calcineurin phosphatase activity, suppresses inflammation during Pseudomonas aeruginosa infection. Here, we define the roles for RCAN1 in P. aeruginosa lipopolysaccharide (LPS)-activated TLR4 signaling. We compared the effects of P. aeruginosa LPS challenge on bone marrow-derived macrophages from both wild-type and RCAN1-deficient mice and found that RCAN1 deficiency increased the MyD88-NF-κB-mediated cytokine production (IL-6, TNF and MIP-2), whereas TRIF-interferon-stimulated response elements (ISRE)-mediated cytokine production (IFNβ, RANTES and IP-10) was suppressed. RCAN1 deficiency caused increased IκBα phosphorylation and NF-κB activity in the MyD88-dependent pathway, but impaired ISRE activation and reduced IRF7 expression in the TRIF-dependent pathway. Complementary studies of a mouse model of P. aeruginosa LPS-induced acute pneumonia confirmed that RCAN1-deficient mice displayed greatly enhanced NF-κB activity and MyD88-NF-κB-mediated cytokine production, which correlated with enhanced pulmonary infiltration of neutrophils. By contrast, RCAN1 deficiency had little effect on the TRIF pathway in vivo. These findings demonstrate a novel regulatory role of RCAN1 in TLR signaling, which differentially regulates MyD88 and TRIF pathways.

Gender difference in the effect of progesterone on neonatal hypoxic/ischemic brain injury in mouse

This study investigated the effects of progesterone (PROG) on neonatal hypoxic/ischemic (NHI) brain injury, the differences in effects between genders, and the underlying mechanisms. NHI brain injury was established in both male and female neonatal mice induced by occlusion of the left common carotid artery followed by hypoxia. The mice were treated with PROG or vehicle. Fluoro-Jade B staining (F-JB), long term behavior testing, and brain magnetic resonance image (MRI) were applied to evaluate neuronal death, neurological function, and brain damage. The underlying molecular mechanisms were also investigated by Western blots. The results showed that, in the male mice, administration of PROG significantly reduced neuronal death, improved the learning and memory function impaired by cerebral HI, decreased infarct size, and maintained the thickness of the cortex after cerebral HI. PROG treatment, however, did not show significant neuroprotective effects on female mice subjected to HI. In addition, the data demonstrated a gender difference in the expression of tumor necrosis factor receptor 1 (TNFR1), TNF receptor associated factor 6 (TRAF6), Fas associated protein with death domain (FADD), and TIR-domain-containing adapter-inducing interferon-β (TRIF) between males and females. Our results indicated that treatment with PROG had beneficial effects on NHI injured brain in acute stage and improved the long term cognitive function impaired by cerebral HI in male mice. In addition, the activation of TNF and TRIF mediated signaling in response to cerebral HI and the treatment of PROG varied between genders, which highly suggested that gender differences should be emphasized in evaluating neonatal HI brain injury and PROG effects, as well as the underlying mechanisms.

Damage-associated molecular patterns in resuscitated hemorrhagic shock are mitigated by peritoneal fluid administration.

Conventional resuscitation (CR) of hemorrhagic shock (HS), a significant cause of trauma mortality, is intravenous blood and fluids. CR restores central hemodynamics, but vital organ flow can drop, causing hypoperfusion, hypoxia, damage-associated molecular patterns (DAMPs), and remote organ dysfunction (i.e., lung). CR plus direct peritoneal resuscitation (DPR) prevents intestinal and hepatic hypoperfusion. We hypothesized that DPR prevents lung injury in HS/CR by altering DAMPs. Anesthetized male Sprague-Dawley rats were randomized to groups ( n = 8/group) in one of two sets: 1) sham (no HS, CR, or DPR), 2) HS/CR (HS = 40% mean arterial pressure (MAP) for 60 min, CR = shed blood + 2 volumes normal saline), or 3) HS/CR + DPR. The first set underwent whole lung blood flow by colorimetric microspheres. The second set underwent tissue collection for Luminex, ELISAs, and histopathology. Lipopolysaccharide (LPS) and DAMPs were measured in serum and/or lung, including cytokines, hyaluronic acid (HA), high-mobility group box 1 (HMGB1), Toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 protein (MYD88), and TIR-domain-containing adapter-inducing interferon-β (TRIF). Statistics were by ANOVA and Tukey-Kramer test with a priori P < 0.05. HS/CR increased serum LPS, HA, HMGB1, and some cytokines [interleukin (IL)-1α, IL-1β, IL-6, and interferon-γ]. Lung TLR4 and MYD88 were increased but not TRIF compared with Shams. HS/CR + DPR decreased LPS, HA, cytokines, HMGB1, TLR4, and MYD88 levels but did not alter TRIF compared with HS/CR. The data suggest that gut-derived DAMPs can be modulated by adjunctive DPR to prevent activation of lung TLR-4-mediated processes. Also, DPR improved lung blood flow and reduced lung tissue injury. Adjunctive DPR in HS/CR potentially improves morbidity and mortality by downregulating the systemic DAMP response.

Increased A20-E3 ubiquitin ligase interactions in bid-deficient glia attenuate TLR3- and TLR4-induced inflammation

BackgroundChronic pro-inflammatory signaling propagates damage to neural tissue and affects the rate of disease progression. Increased activation of Toll-like receptors (TLRs), master regulators of the innate immune response, is implicated in the etiology of several neuropathologies including amyotrophic lateral sclerosis, Alzheimer’s disease, and Parkinson’s disease. Previously, we identified that the Bcl-2 family protein BH3-interacting domain death agonist (Bid) potentiates the TLR4-NF-κB pro-inflammatory response in glia, and specifically characterized an interaction between Bid and TNF receptor associated factor 6 (TRAF6) in microglia in response to TLR4 activation.MethodsWe assessed the activation of mitogen-activated protein kinase (MAPK) and interferon regulatory factor 3 (IRF3) inflammatory pathways in response to TLR3 and TLR4 agonists in wild-type (wt) and bid-deficient microglia and macrophages, using Western blot and qPCR, focusing on the response of the E3 ubiquitin ligases Pellino 1 (Peli1) and TRAF3 in the absence of microglial and astrocytic Bid. Additionally, by Western blot, we investigated the Bid-dependent turnover of Peli1 and TRAF3 in wt and bid−/− microglia using the proteasome inhibitor Bortezomib. Interactions between the de-ubiquitinating Smad6-A20 and the E3 ubiquitin ligases, TRAF3 and TRAF6, were determined by FLAG pull-down in TRAF6-FLAG or Smad6-FLAG overexpressing wt and bid-deficient mixed glia.ResultsWe elucidated a positive role of Bid in both TIR-domain-containing adapter-inducing interferon-β (TRIF)- and myeloid differentiation primary response 88 (MyD88)-dependent pathways downstream of TLR4, concurrently implicating TLR3-induced inflammation. We identified that Peli1 mRNA levels were significantly reduced in PolyI:C- and lipopolysaccharide (LPS)-stimulated bid-deficient microglia, suggesting disturbed IRF3 activation. Differential regulation of TRAF3 and Peli1, both essential E3 ubiquitin ligases facilitating TRIF-dependent signaling, was observed between wt and bid−/− microglia and astrocytes. bid deficiency resulted in increased A20-E3 ubiquitin ligase protein interactions in glia, specifically A20-TRAF6 and A20-TRAF3, implicating enhanced de-ubiquitination as the mechanism of action by which E3 ligase activity is perturbed. Furthermore, Smad6-facilitated recruitment of the de-ubiquitinase A20 to E3-ligases occurred in a bid-dependent manner.ConclusionsThis study demonstrates that Bid promotes E3 ubiquitin ligase-mediated signaling downstream of TLR3 and TLR4 and provides further evidence for the potential of Bid inhibition as a therapeutic for the attenuation of the robust pro-inflammatory response culminating in TLR activation.

1161 TIR-domain-containing adapter-inducing interferon-β (TRIF) protects mice from UVB induced suppression of cell-mediated immune responses

1161 TIR-domain-containing adapter-inducing interferon-β (TRIF) protects mice from UVB induced suppression of cell-mediated immune responses

LincRNA-Cox2 regulates NLRP3 inflammasome and autophagy mediated neuroinflammation

Inflammasome activation plays key roles in host defense, but also contributes to the pathogenesis of auto-inflammatory, and neurodegenerative diseases. As autophagy is connected with both the innate and adaptive immune systems, autophagic dysfunction is also closely related to inflammation, infection, and neurodegeneration. Here we identify that lincRNA-Cox2, previously known as a mediator of both the activation and repression of immune genes expression in innate immune cells, could bind NF-κB p65 and promote its nuclear translocation and transcription, modulating the expression of inflammasome sensor NLRP3 and adaptor ASC. Knockdown of lincRNA-Cox2 inhibited the inflammasome activation and prevented the lincRNA-Cox2-triggered caspase-1 activation, leading to decreased IL-1β secretion and weakened TIR-domain-containing adapter-inducing interferon-β (TRIF) cleavage, thereby enhancing TRIF-mediated autophagy. Elucidation of the link between lincRNA-Cox2 and the inflammasome-autophagy crosstalk in macrophage and microglia reveals a role for lncRNAs in activation of NLRP3 inflammasome and autophagy, and provides new opportunities for therapeutic intervention in neuroinflammation-dependent diseases.

Innate immune adaptor TRIF deficiency accelerates disease progression of ALS mice with accumulation of aberrantly activated astrocytes

There is compelling evidence that glial-immune interactions contribute to the progression of neurodegenerative diseases. The adaptive immune response has been implicated in disease processes of amyotrophic lateral sclerosis (ALS), but it remains unknown if innate immune signaling also contributes to ALS progression. Here we report that deficiency of the innate immune adaptor TIR domain-containing adaptor inducing interferon-β (TRIF), which is essential for certain Toll-like receptor (TLR) signaling cascades, significantly shortens survival time and accelerates disease progression of ALS mice. While myeloid differentiation factor 88 (MyD88) is also a crucial adaptor for most TLR signaling pathways, MyD88 deficiency had only a marginal impact on disease course. Moreover, TRIF deficiency reduced the number of natural killer (NK), NK-T-lymphocytes, and CD8-T cells infiltrating into the spinal cord of ALS mice, but experimental modulation of these populations did not substantially influence survival time. Instead, we found that aberrantly activated astrocytes expressing Mac2, p62, and apoptotic markers were accumulated in the lesions of TRIF-deficient ALS mice, and that the number of aberrantly activated astrocytes was negatively correlated with survival time. These findings suggest that TRIF pathway plays an important role in protecting a microenvironment surrounding motor neurons by eliminating aberrantly activated astrocytes.