The TLR4-TRIF pathway protects against H5N1 influenza virus infection

Prestimulation of the TLR4 pathway with lipopolysaccharide (LPS) protects mice from lethal infection with H5N1 influenza virus. Here, we reveal that the TLR4-TRIF pathway is required for this protective effect by using mice whose TLR4-related molecules were knocked out. Microarray analysis of primary mouse lung culture cells that were LPS pretreated and infected with an H5N1 virus indicated that TLR3 mRNA was upregulated. Primary lung culture cells of TLR3 knockout mice showed no response to LPS pretreatment against H5N1 virus infection, suggesting that TLR3 is also involved in the preventive effect of LPS. Our data suggest that the TLR4-TRIF axis has an important role in stimulating protective innate immunity against H5N1 influenza A virus infection and that TLR3 signaling is involved in this pathway.     

The contribution of the Toll-like/IL-1 receptor superfamily to innate and adaptive immunity to fungal pathogens in vivo

In vitro studies have indicated the importance of Toll-like receptor (TLR) signaling in response to the fungal pathogens Candida albicans and Aspergillus fumigatus. However, the functional consequences of the complex interplay between fungal morphogenesis and TLR signaling in vivo remain largely undefined. In this study we evaluate the impact of the IL-1R/TLR/myeloid differentiation primary response gene 88 (MyD88)-dependent signaling pathway on the innate and adaptive Th immunities to C. albicans and A. fumigatus in vivo. It was found that 1) the MyD88-dependent pathway is required for resistance to both fungi; 2) the involvement of the MyD88 adapter may occur through signaling by distinct members of the IL-1R/TLR superfamily, including IL-1R, TLR2, TLR4, and TLR9, with the proportional role of the individual receptors varying depending on fungal species, fungal morphotypes, and route of infection; 3) individual TLRs and IL-1R activate specialized antifungal effector functions on neutrophils, which correlates with susceptibility to infection; and 4) MyD88-dependent signaling on dendritic cells is crucial for priming antifungal Th1 responses. Thus, the finding that the innate and adaptive immunities to C. albicans and A. fumigatus require the coordinated action of distinct members of the IL-1R/TLR superfamily acting through MyD88 makes TLR manipulation amenable to the induction of host resistance to fungi.     

Intracellular TLR signaling: a structural perspective on human disease

TLRs are crucial sensors of microbial infection. Maintaining structural integrity of TLR signaling components is essential for subsequent immunological protection. Alterations to the structure of these signaling molecules are often associated with profound clinical outcomes and susceptibility to various infectious diseases. These changes in structure are sometimes the result of a single nucleotide polymorphism (SNP). Numerous SNPs have been found in components of the TLR signaling pathway. Recently, the medical consequences and effects on TLR signaling of several of these SNPs have been elucidated. In addition, there have been numerous structures solved that are important to our understanding of the TLR signaling pathway at the molecular level. The scope of this review is to tie together current structural, biochemical, and genetic information of TLR signaling.     

Cutting edge: TLR2-deficient and MyD88-deficient mice are highly susceptible to Staphylococcus aureus infection

Toll-like receptor (TLR) family acts as pattern recognition receptors for pathogen-specific molecular patterns. We previously showed that TLR2 recognizes Gram-positive bacterial components whereas TLR4 recognizes LPS, a component of Gram-negative bacteria. MyD88 is shown to be an adaptor molecule essential for TLR family signaling. To investigate the role of TLR family in host defense against Gram-positive bacteria, we infected TLR2- and MyD88-deficient mice with Staphylococcus aureus. Both TLR2- and MyD88-deficient mice were highly susceptible to S. aureus infection, with more enhanced susceptibility in MyD88-deficient mice. Peritoneal macrophages from MyD88-deficient mice did not produce any detectable levels of cytokines in response to S. aureus. In contrast, TLR2-deficient macrophages produced reduced, but significant, levels of the cytokines, and TLR4-deficient macrophages produced the same amounts as wild-type cells, indicating that S. aureus is recognized not only by TLR2, but also by other TLR family members except for TLR4.     

Upregulation of the TLR3 pathway by Kaposi's sarcoma-associated herpesvirus during primary infection

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with several different human malignancies, including Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. KSHV establishes lifelong latency in the host and modulates the host immune response. Innate immunity is critical for controlling de novo viral infection. Toll-like receptors (TLRs) are key components of the innate immune system, and they serve as pathogen recognition receptors that stimulate the host antiviral response. In particular, TLR3 has been implicated in RNA virus recognition. Currently, there is no information regarding how KSHV infection modulates any TLR pathway. We report the first evidence that KSHV upregulates TLR3 expression in human monocytes during primary infection. This is also the first demonstration of a human DNA tumor virus upregulating TLR3, a TLR that thus far has been associated with the recognition of RNA viruses. We found that KSHV upregulates the TLR3 pathway and induces TLR3-specific cytokines and chemokines, including beta 1 interferon (IFN-beta1) and CXCL10 (IP-10). Small interfering RNAs directed against TLR3 greatly reduced the ability of KSHV to upregulate IFN-beta1 and CXCL10 upon infection.     

Direct TLR2 Signaling Is Critical for NK Cell Activation and Function in Response to Vaccinia Viral Infection

Natural killer (NK) cells play an essential role in innate immune control of poxviral infections in vivo. However, the mechanism(s) underlying NK cell activation and function in response to poxviruses remains poorly understood. In a mouse model of infection with vaccinia virus (VV), the most studied member of the poxvirus family, we identified that the Toll-like receptor (TLR) 2-myeloid differentiating factor 88 (MyD88) pathway was critical for the activation of NK cells and the control of VV infection in vivo. We further showed that TLR2 signaling on NK cells, but not on accessory cells such as dendritic cells (DCs), was necessary for NK cell activation and that this intrinsic TLR2-MyD88 signaling pathway was required for NK cell activation and played a critical role in the control of VV infection in vivo. In addition, we showed that the activating receptor NKG2D was also important for efficient NK activation and function, as well as recognition of VV-infected targets. We further demonstrated that VV could directly activate NK cells via TLR2 in the presence of cytokines in vitro and TLR2-MyD88-dependent activation of NK cells by VV was mediated through the phosphatidylinositol 3-kinase (PI3K)-extracellular signal-regulated kinase (ERK) pathway. Taken together, these results represent the first evidence that intrinsic TLR signaling is critical for NK cell activation and function in the control of a viral infection in vivo, indicate that multiple pathways are required for efficient NK cell activation and function in response to VV infection, and may provide important insights into the design of effective strategies to combat poxviral infections.     

Toll-like receptors that sense viral infection

Anti-viral host defense harbors a variety of strategies to coup with viral infection. Recent findings suggested that Toll-like receptors (TLRs) and their signaling pathways involve type I IFN induction in response to virus-specific molecular patterns. TLR 3 and TLR 4 in myeloid dendritic cells (mDCs) recognize viral dsRNA and putative viral products, respectively, to induce IFN-beta via IRF-3 activation. On the other hand, TLR 7 and TLR 9 in plasmacytoid DCs (pDCs) induce IFN-alpha in response to their ligands, U/G-rich ssRNA and non-methylated CpG DNA. We identified TICAM-1 which is recruited to the cytoplasmic domain (designated TIR) of TLR 3 and allows to select the pathway to activation of IRF-3. We also identified TICAM-2 which binds TLR 4 and together with TICAM-1 activates IRF-3. TICAM-1 knockdown by RNAi supported the key role of TICAM-1 in IFN-beta induction. Hence, the IFN-beta induction in mDCs appears in part due to the function of TICAM-1. Viruses are known to activate kinases that directly activate IRF-3 inside the cells, and this pathway may merge with the TLR 3-TICAM-1 pathway. Here we review the relationship between the TLR 3-TICAM-1 pathway and viral infection.     

Orange-spotted grouper (Epinephelus coioides) TLR2, MyD88 and IL-1β involved in anti-Cryptocaryon irritans response

Cryptocaryon irritans is one of the most important ectoparasites of marine fish, and can have a devastating effect on aquacultured fish populations. The role of TLR signaling pathways in anti-parasitic immune responses is poorly understood in fish. In this paper, we first cloned Epinephelus coioides MyD88 full-length cDNA (EcMyD88) and its respective gene. The open reading frame (ORF) of cDNA is 873 bp encoding 291 amino acid residues. Similar to other species, the EcMyD88 gene contains of five conserved exons and four diverse introns. The constitutive expression of EcMyD88 was detected in the gill, trunk kidney, head kidney, spleen, and heart in high concentrations, while the skin, brain, liver, and muscles contained much lower titers, indicating that EcMyD88 may play a crucial role in host innate immunologic surveillance. To identify the potential role of TLR pathways in fish anti-C. irritans immune responses, we chose three important molecules involved in anti-parasite responses, TLR2, MyD88 and IL-1β to indicate TLR pathway’s signal-in, signal transduction, and signal-out functions, respectively. The expression profile of these three genes was detected in grouper infected by C. irritans. Results showed these molecules each experience significant changes within the skin, gill (two infected mucosal sites), head kidney and spleen (two systematic immune organs) after C. irritans infection. These findings indicate the TLR signaling pathway may play an important role in host defense against C. irritans.     

TLR2 signaling contributes to rapid inflammasome activation during F. novicida infection

BACKGROUND: Early detection of microorganisms by the innate immune system is provided by surface-expressed and endosomal pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs). Detection of microbial components by TLRs initiates a signaling cascade leading to the expression of proinflammatory cytokines including IL-6 and IL-1β. Some intracellular bacteria subvert the TLR response by rapidly escaping the phagosome and entering the cytosol. However, these bacteria may be recognized by the inflammasome, a multi-protein complex comprised of a sensor protein, ASC and the cysteine protease caspase-1. Inflammasome activation leads to release of the proinflammatory cytokines IL-1β and IL-18 and death of the infected cell, an important host defense that eliminates the pathogen's replicative niche. While TLRs and inflammasomes are critical for controlling bacterial infections, it is unknown whether these distinct host pathways cooperate to activate defenses against intracellular bacteria. METHODOLOGY/SIGNIFICANT FINDINGS: Using the intracellular bacterium Francisella novicida as a model, we show that TLR2(-/-) macrophages exhibited delayed inflammasome activation compared to wild-type macrophages as measured by inflammasome assembly, caspase-1 activation, cell death and IL-18 release. TLR2 also contributed to inflammasome activation in response to infection by the cytosolic bacterium Listeria monocytogenes. Components of the TLR2 signaling pathway, MyD88 and NF-κB, were required for rapid inflammasome activation. Furthermore, TLR2(-/-) mice exhibited lower levels of cell death, caspase-1 activation, and IL-18 production than wild-type mice upon F. novicida infection. CONCLUSIONS/SIGNIFICANCE: These results show that TLR2 is required for rapid inflammasome activation in response to infection by cytosolic bacterial pathogens. In addition to further characterizing the role of TLR2 in host defense, these findings broaden our understanding of how the host integrates signals from spatiotemporally separated PRRs to coordinate an innate response against intracellular bacteria.     

The transforming growth factor-beta-Smad3/4 signaling pathway acts as a positive regulator for TLR2 induction by bacteria via a dual mechanism involving functional cooperation with NF-kappaB and MAPK phosphatase 1-dependent negative cross-talk with p38 MAPK

The transforming growth factor beta (TGF-beta) pathway represents an important signaling pathway involved in the regulation of diverse biological processes, including cell proliferation, differentiation, and apoptosis. Despite the known role of TGF-betaR-mediated signaling in suppressing immune response, its role in regulating human Toll-like receptors (TLRs), key host defense receptors that recognize invading bacterial pathogens, however, remains unknown. Here, we show for the first time that TGF-betaR-Smad3/4 signaling pathway acts as a positive regulator for TLR2 induction by bacterium nontypeable Hemophilus influenzae (NTHi) in vitro and in vivo. The positive regulation of TLR2 induction by TGF-betaR is mediated via a dual mechanism involving distinct signaling pathways. One mechanism involves functional cooperation between the TGF-betaR-Smad3/4 pathway and NF-kappaB pathway. Another involves MAP kinase phosphatase 1 (MKP-1)-dependent inhibition of p38 MAPK, a known negative regulator for TLR2 induction. Moreover, we showed that TbetaR-mediated signaling is probably activated by NTHi-derived TGF-beta mimicry molecule via an autocrine-independent mechanism. Thus, our study provides new insights into the role of TGF-beta signaling in positively regulating host defense response by tightly controlling the expression level of TLR2 during bacterial infections and may lead to new therapeutic strategies for modulating host defense and inflammatory response.     

Aspergillus fumigatus induces innate immune responses in alveolar macrophages through the MAPK pathway independently of TLR2 and TLR4

Aspergillus fumigatus causes invasive aspergillosis in immunosuppressed patients. In the immunocompetent host, inhaled conidia are cleared by alveolar macrophages. The signaling pathways of the alveolar macrophage involved in the clearance of A. fumigatus are poorly understood. Therefore, we investigated the role of TLRs in the immune response against A. fumigatus and their contribution to the signaling events triggered in murine alveolar macrophages upon infection with A. fumigatus conidia. Specifically, we examined the MAPKs and NF-kappaB activation and cytokine signaling. Our investigations revealed that immunocompetent TLR2, TLR4, and MyD88 knockout mice were not more susceptible to invasive aspergillosis as compared with wild-type mice and that the in vitro phosphorylation of the MAPKs ERK and p38 was not affected in TLR2, TLR4, or MyD88 knockout mice following stimulation with conidia. In vivo experiments suggest that ERK was an essential MAPK in the defense against A. fumigatus, whereas the activation of NF-kappaB appeared to play only a secondary role. In conclusion, our findings demonstrate that TLR2/4 recognition and MyD88 signaling are dispensable for the clearance of A. fumigatus under immunocompetent situations. Furthermore, our data stress the important role of ERK activation in innate immunity to A. fumigatus.     

Hypoxia attenuates inflammatory mediators production induced by Acanthamoeba via Toll-like receptor 4 signaling in human corneal epithelial cells

Acanthamoeba keratitis (AK) is a vision-threatening corneal infection that is intimately associated with contact lens use which leads to hypoxic conditions on the corneal surface. However, the effect of hypoxia on the Acanthamoeba-induced host inflammatory response of corneal epithelial cells has not been studied. In the present study, we investigated the effect of hypoxia on the Acanthamoeba-induced production of inflammatory mediators interleukin-8 (IL-8) and interferon-β (IFN-β) in human corneal epithelial cells and then evaluated its effects on the Toll-like receptor 4 (TLR4) signaling, including TLR4 and myeloid differentiation primary response gene (88) (MyD88) expression as well as the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and extracellular signal-regulated kinases 1/2 (ERK1/2). We then studied the effect of hypoxia on a TLR4-specific inflammatory response triggered by the TLR4 ligand lipopolysaccharide (LPS). Our data showed that hypoxia significantly decreased the production of IL-8 and IFN-β. Furthermore, hypoxia attenuated Acanthamoeba-triggered TLR4 expression as well as the activation of NF-κB and ERK1/2, indicating that hypoxia abated Acanthamoeba-induced inflammatory responses by affecting TLR4 signaling. Hypoxia also inhibited LPS-induced IL-6 and IL-8 secretion, myeloid differentiation primary response gene (88) MyD88 expression and NF-κB activation, confirming that hypoxia suppressed the LPS-induced inflammatory response by affecting TLR4 signaling. In conclusion, our results demonstrated that hypoxia attenuated the host immune and inflammatory response against Acanthamoeba infection by suppressing TLR4 signaling, indicating that hypoxia might impair the host cell's ability to eliminate the Acanthamoeba invasion and that hypoxia could enhance cell susceptibility to Acanthamoeba infection. These results may explain why contact lens use is one of the most prominent risk factors for AK.     

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.     

Neurotropic EV71 causes encephalitis by engaging intracellular TLR9 to elicit neurotoxic IL12-p40-iNOS signaling

Brainstem encephalitis, a manifestation of severe enterovirus 71 (EV71) infection, is an acute excessive inflammatory response. The mechanisms underlying its development remain poorly understood. Usually neurotropic viruses trigger acute host immune response by engaging cell surface or intracellular receptors. Here, we show that EV71 engagement with intracellular receptor TLR9 elicits IL-12p40-iNOS signaling causing encephalitis in mice. We identified IL-12p40 to be the only prominent cytokine-induced at the early infection stage in the brainstem of mice subjected to a lethal dose of EV71. The upregulated IL-12p40 proteins were expressed in glial cells but not neuronal cells. To better understand the role of IL-12p40 in severe EV71 infection, we treated the EV71-infected mice with an antibody against IL-12p40 and found the mortality rate, brainstem inflammation, and gliosis to be markedly reduced, suggesting that the acute IL-12p40 response plays a critical role in the pathogenesis of brainstem encephalitis. Mechanistically, intracellular TLR9 was found essential to the activation of the IL-12p40 response. Blocking TLR9 signaling with CpG-ODN antagonist ameliorated IL-12p40 response, brainstem inflammation, and limb paralysis in mice with EV71-induced encephalitis. We further found the glial IL-12p40 response might damage neurons by inducing excess production of neurotoxic NO by iNOS. Overall, EV71 engagement with intracellular TLR9 was found to elicit a neurotoxic glial response via IL12p40-iNOS signaling contributing to the neurological manifestation of EV71 infection. This pathway could potentially be targeted for the treatment of brainstem encephalitis.Subject terms: Toll-like receptors, Viral infection