Synthetic glycolipid-based TLR4 antagonists negatively regulate TRIF-dependent TLR4 signalling in human macrophages

TLRs, including TLR4, play a crucial role in inflammatory-based diseases, and TLR4 has been identified as a therapeutic target for pharmacological intervention. In previous studies, we investigated the potential of FP7, a novel synthetic glycolipid active as a TLR4 antagonist, to inhibit haematopoietic and non-haematopoietic MyD88-dependent TLR4 pro-inflammatory signalling. The main aim of this study was to investigate the action of FP7 and its derivative FP12 on MyD88-independent TLR4 signalling in THP-1 derived macrophages. Western blotting, Ab array and ELISA approaches were used to explore the effect of FP7 and FP12 on TRIF-dependent TLR4 functional activity in response to LPS and other endogenous TLR4 ligands in THP-1 macrophages. A different kinetic in the inhibition of endotoxin-driven TBK1, IRF3 and STAT1 phosphorylation was observed using different LPS chemotypes. Following activation of TLR4 by LPS, data revealed that FP7 and FP12 inhibited TBK1, IRF3 and STAT1 phosphorylation which was associated with down-regulation IFN-β and IP-10. Specific blockage of the IFN type one receptor showed that these novel molecules inhibited TRIF-dependent TLR4 signalling via IFN-β pathways. These results add novel information on the mechanism of action of monosaccharide FP derivatives. The inhibition of the TRIF-dependent pathway in human macrophages suggests potential therapeutic uses for these novel TLR4 antagonists in pharmacological interventions on inflammatory diseases.     

Dual character of Toll-like receptor signaling: Pro-tumorigenic effects and anti-tumor functions

As a major class of pattern-recognition receptors, Toll-like receptors (TLRs) play a critical role in defense against invading pathogens. Increasing evidence demonstrates that, in addition to infection, TLRs are involved in other important pathological processes, such as tumorigenesis. Activation of TLRs results in opposing outcomes, pro-tumorigenic effects and anti-tumor functions. TLR signaling can inhibit apoptosis and promote chronic inflammation-induced tumorigenesis. TLR activation in tumor cells and immune cells can induce production of cytokines, increase tumor cell proliferation and apoptosis resistance, promote invasion and metastasis, and inhibit immune cell activity resulting in tumor immune escape. In contrast, the engagement of other TLRs directly induces growth inhibition and apoptosis of tumor cells and triggers activation of immune cells enhancing anti-tumor immune responses. Thus, the interpretation of the precise function of each TLR in tumors is very important for targeting TLRs and using TLR agonists in tumor therapy. We review the role of TLR signaling in tumors and discuss the factors that affect outcomes of TLR activation.     

MMP-12-mediated by SARM-TRIF signaling pathway contributes to IFN-γ-independent airway inflammation and AHR post RSV infection in nude mice

Background

Respiratory syncytial virus (RSV) is one of the most frequently observed pathogens during infancy and childhood. However, the corresponding pathogenesis has not been determined to date. We previously demonstrated that IFN-γ plays an important role in RSV pathogenesis, and SARM-TRIF-signaling pathway could regulate the production of IFN-γ. This study is to investigate whether T cells or innate immune cells are the predominant producers of IFN-γ, and further to explore other culprits in addition to IFN-γ in the condition of RSV infection.

Methods

Normal BALB/c mice and nude mice deficient in T cells were infected intranasally with RSV. Leukocytes in bronchoalveolar lavage fluid were counted, lung histopathology was examined, and airway hyperresponsiveness (AHR) was measured by whole-body plethysmography. IFN-γ and MMP-12 were detected by ELISA. MMP408, a selective MMP-12 inhibitor, was given intragastrically. Resveratrol, IFN-γ neutralizing antibody and recombinant murine IFN-γ were administered intraperitoneally. SARM and TRIF protein were semi-quantified by Western blot. siRNA was used to knock-down SARM expression.

Results

RSV induced significant airway inflammation and AHR in both mice; IFN-γ was significantly increased in BALB/c mice but not in nude mice. MMP-12 was dramatically increased in both mice but earlier in nude mice. When MMP-12 was inhibited by MMP408, RSV-induced respiratory symptoms were alleviated. SARM was significantly suppressed while TRIF was significantly enhanced in both mice strains. Following resveratrol administration in nude mice, 1) SARM inhibition was prevented, 2) TRIF and MMP-12 were correspondingly down-regulated and 3) airway disorders were subsequently alleviated. Moreover, when SARM was efficiently knocked down using siRNA, TRIF and MMP-12 were markedly enhanced, and the anti-RSV effects of resveratrol were remarkably abrogated. MMP-12 was significantly increased in the IFN-γ neutralizing antibody-treated BALB/c mice but reduced in the recombinant murine IFN-γ-treated nude mice.

Conclusions

MMP-12 can result in at least part of the airway inflammation and AHR independent of IFN-γ. And SARM-TRIF- signaling pathway is involved in regulating the overproduction of MMP-12. To the best of our knowledge, this study is the first that has examined the effects of SARM on MMP-12 and further highlights the potential to target SARM-TRIF-MMP-12 cascades to treat RSV infection.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0176-8) contains supplementary material, which is available to authorized users.     

Toll-like receptor 3 and TICAM genes in catfish: species-specific expression profiles following infection with Edwardsiella ictaluri

Toll-like receptors (TLRs) are a family of transmembrane proteins that recognize specific pathogen-associated molecular patterns and use conserved signaling pathways to activate proinflammatory cytokines and type-1 interferons to fight infection. TLR3 in mammals is best known for its recognition of dsRNA as ligand and its MyD88-independent signaling. TLR3, upon recognition of dsRNA, recruits and binds its adaptor protein TIR domain-containing adapter molecule (TICAM) 1. Here we report the genomic sequences and structures of TLR3 and a TICAM adaptor from channel catfish (Ictalurus punctatus). Whereas a partial TLR3 cDNA sequence has been reported from channel catfish, and complete TLR3 genes are known from other teleost fish species, a complete TICAM sequence has not been previously reported from a nonmammalian species. Analysis of catfish TLR3 and TICAM expression after infection with Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish (ESC), suggested a conserved TLR3-TICAM receptor–adaptor relation in catfish. Comparison of TLR3 and TICAM expression profiles in channel catfish with those from the closely related blue catfish species (Ictalurus furcatus), which exhibits strong resistance to ESC, revealed a striking pattern of species-specific expression. A dramatic downregulation of TLR3 and TICAM gene expression was observed in blue catfish head kidney and spleen, which we speculate may be the result of maturation and migration of different cell types to and from the lymphoid tissues following infection.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.Puttharat Baoprasertkul and Eric Peatman contributed equally to this work.     

TRIM56 Is an Essential Component of the TLR3 Antiviral Signaling Pathway

Members of the tripartite motif (TRIM) proteins are being recognized as important regulators of host innate immunity. However, specific TRIMs that contribute to TLR3-mediated antiviral defense have not been identified. We show here that TRIM56 is a positive regulator of TLR3 signaling. Overexpression of TRIM56 substantially potentiated extracellular dsRNA-induced expression of interferon (IFN)-β and interferon-stimulated genes (ISGs), while knockdown of TRIM56 greatly impaired activation of IRF3, induction of IFN-β and ISGs, and establishment of an antiviral state by TLR3 ligand and severely compromised TLR3-mediated chemokine induction following infection by hepatitis C virus. The ability to promote TLR3 signaling was independent of the E3 ubiquitin ligase activity of TRIM56. Rather, it correlated with a physical interaction between TRIM56 and TRIF. Deletion of the C-terminal portion of TRIM56 abrogated the TRIM56-TRIF interaction as well as the augmentation of TLR3-mediated IFN response. Together, our data demonstrate TRIM56 is an essential component of the TLR3 antiviral signaling pathway and reveal a novel role for TRIM56 in innate antiviral immunity.     

Delayed administration of pituitary adenylate cyclase-activating polypeptide 38 ameliorates renal ischemia/reperfusion injury in mice by modulating Toll-like receptors

We investigated whether pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) ameliorates kidney injury after ischemia/reperfusion (IR) by modulating Toll-like receptor (TLR)-associated signaling pathways. Male C57BL/6 mice were subjected to bilateral renal ischemia for 45 min. PACAP38, 20 μg in 100 μl of saline, was administered i.p. at 24 and 48 h after IR, and mice were euthanized at 72 h. In IR mice, PACAP38 maintained serum creatinine near control levels (0.81 ± 0.08 vs. 0.69 ± 0.17 mg/dl in controls, p = NS, vs. 1.8 ± 0.03 in saline-treated IR mice, p < 0.01) and significantly reduced the expression of kidney injury biomarkers. PACAP38 significantly reduced the levels of apoptosis and neutrophil infiltration, and protected against tubular damage. With PCR arrays, 59 of 83 TLR-related genes significantly changed their expression after IR. TLR2 increased 162 fold, followed by Fas-associated death domain (37 fold) and TLR6 (24 fold), while ubiquitin-conjugating enzyme E2 variant 1 (UBE2V1) decreased 55 fold. PACAP38 given 24 and 48 h after IR injury significantly reversed these changes in 56 genes, including TLR2, TLR3, TLR4, TLR6, and genes in the NF-κB pathways. The alterations in TLR2, TLR3, TLR6, and UBE2V1 were confirmed by RT-PCR. After IR, PACAP38 also suppressed protein levels of TLR-associated cytokines. PACAP38 reversed the changes in IR-activated TLR-associated NF-κB signaling pathways even when treatment was delayed 24 h. Therefore, PACAP38 could be an effective therapeutic for unexpected IR-mediated renal injury. The prominently IR-induced TLR-related genes identified in this study could be novel drug targets.     

固有免疫信号因子TRIF在肝纤维化中的表达

目的初步探讨TOLL样受体下游重要信号因子TRIF与肝纤维化发生发展的病理机制关系。方法以四氯化碳皮下注射+低蛋白高脂饮食+酒精饮料的方法复制大鼠肝纤维化模型。将SD大鼠随机分成正常对照组和模型组,在完成制备模型实验后进行取材。部分实验鼠进行心脏生理盐水和多聚甲醛灌注后,取肝脏组织制备石蜡切片,进行常规HE染色和免疫组化实验;另一部分实验鼠脱颈安乐死后,取新鲜肝组织进行电镜标本的制备和Western Blot实验检测。结果 HE染色结果显示与而正常对照组大鼠相比,模型组大鼠肝小叶结构明显破坏,肝细胞数量明显减少和肝纤维化程度等特点明显;电镜结果也显示,肝纤维化组可见大量胶原纤维沉积现象,胞质可见明显的溶解现象,在狄氏腔内,肝星状细胞的细胞核溶解,血窦内皮细胞胞质、胞核皆溶解;免疫组化模型组大鼠肝组织均显示内皮细胞、星形细胞等TRIF都有强烈高表达,并且以胞核表达为主,亦见胞质表达,而正常组呈现弱阳性表达;与正常对照组相比,模型组大鼠肝组织TRIF蛋白表达都明显升高,呈显著性差异(P<0.01),与形态学的表达特点相一致。结论 TRIF在肝纤维化中表达显著增强,说明在肝纤维化过程中,TOLL样受体明显激活,并且通过下游信号转导途径,在机体内产生一系列的免疫应答反应。通过该现象的观察,我们初步证实了TOLL样受体固有免疫信号因子TRIF在纤维化形成中起着重要的作用。     

Mass Spectrometry-based Structural Analysis and Systems Immunoproteomics Strategies for Deciphering the Host Response to Endotoxin

One cause of sepsis is systemic maladaptive immune response of the host to bacteria and specifically, to Gram-negative bacterial outer-membrane glycolipid lipopolysaccharide (LPS). On the host myeloid cell surface, proinflammatory LPS activates the innate immune system via Toll-like receptor-4/myeloid differentiation factor-2 complex. Intracellularly, LPS is also sensed by the noncanonical inflammasome through caspase-11 in mice and 4/5 in humans. The minimal functional determinant for innate immune activation is the membrane anchor of LPS called lipid A. Even subtle modifications to the lipid A scaffold can enable, diminish, or abolish immune activation. Bacteria are known to modify their LPS structure during environmental stress and infection of hosts to alter cellular immune phenotypes. In this review, we describe how mass spectrometry-based structural analysis of endotoxin helped uncover major determinations of molecular pathogenesis. Through characterization of LPS modifications, we now better understand resistance to antibiotics and cationic antimicrobial peptides, as well as how the environment impacts overall endotoxin structure. In addition, mass spectrometry-based systems immunoproteomics approaches can assist in elucidating the immune response against LPS. Many regulatory proteins have been characterized through proteomics and global/targeted analysis of protein modifications, enabling the discovery and characterization of novel endotoxin-mediated protein translational modifications.     

Elevated expression of EBV and TLRs in the brain is associated with Rasmussen’s encephalitis

Rasmussen's encephalitis(RE) is a rare pediatric neurological disorder, the etiology of which remains unclear. It has been speculated that the immunopathogenesis of RE involves damage to neurons, which eventually leads to the occurrence of RE. Viral infection may be a critical factor in triggering RE immunopathogenesis. In this study, we analyzed the expression of Epstein-Barr virus(EBV) antigens as well as of Toll-like receptor 3(TLR3), TLR9, and downstream adapter TIRdomain-containing adapter-inducing interferon-β(TRIF) in the brain tissues of 26 patients with RE and 16 control individuals using immunohistochemistry(IHC). In the RE group, EBV antigens were detected in 53% of individuals at various expression levels. In contrast, there was no detectable EBV antigen expression in control brain tissues. Moreover, we found marked increases in the expression of TLR3, TLR9, and TRIF in the brain tissues of RE patients compared with levels in the control group. Furthermore, among RE cases, EBV expression and high TLR3 expression were associated with more severe brain atrophy. Our results suggest that the elevated expression of EBV and TLRs may be involved in RE occurrence through the activation of downstream molecules.     

The role of tumor necrosis factor-α for interleukin-10 production by murine dendritic cells

In the present study, we examined the role of tumor necrosis factor (TNF) in interleukin (IL)-10 production by dendritic cells (DCs) using bone-marrow derived DCs from wild type (WT) and TNF-α knockout (TNF-α^−/−) mice. Toll-like receptor (TLR) stimulation induced substantial level of IL-10 production by WT DCs, but significantly low level of IL-10 production by TNF-α^−/− DCs. In contrast, no significant difference was detected in IL-12 p40 production between WT and TNF-α^−/− DCs. Addition of TNF-α during TLR stimulation recovered the impaired ability of TNF-α^−/− DCs for IL-10 production. This recovery appeared to be associated with an activation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and phosphatidylinositol 3-kinase/Akt following the TNF-α addition. Blocking these kinases significantly inhibited IL-10 production by TNF-α^−/− DCs stimulated with TLR ligands plus TNF-α. Thus, TNF-α may be a key molecule to regulate the balance between anti-inflammatory versus inflammatory cytokine production in DCs.