TIR Domain-containing Adaptor Inducing IFN-beta Research Articles

MYELOID-DERIVED TLR4-TRIF SIGNALING PATHWAY MEDIATES OXIDATIVE STRESS IN LPS/D-GALN-INDUCED ACUTE LIVER FAILURE.

Background: Acute liver failure (ALF) is a severe clinical syndrome characterized by massive hepatocyte death in a short time due to viruses, drugs, alcohol, or other factors. Oxidative stress is an important pathogenic mechanism of ALF. LPS-induced internalization of toll-like receptor 4 (TLR4) and the subsequent activation of the toll/IL-1R domain-containing adaptor-inducing IFN-beta (TRIF) signaling pathway widely mediate inflammatory responses in a series of diseases. However, whether the TLR4-TRIF signaling pathway contributes to ALF by mediating oxidative stress processes remains unclear. Methods: An ALF mouse model was induced by lipopolysaccharide (LPS)/D-galactosamine (D-GalN). TLR4-TRIF systemic knockout mice and TLR4 conditional knockout mice were used to determine the role of the TLR4-TRIF signaling pathway in ALF. The effects of TLR4 or TRIF deficiency on oxidative stress were investigated. In addition, we examined the protective role of the clodronate liposomes (macrophage scavengers) and the antioxidant N-acetylcysteine (NAC) in ALF. Results: TLR4 or TRIF deficiency significantly alleviated LPS/D-GalN-induced lethality, hepatic dysfunction, and hepatic pathologic injury, which was dependent on myeloid-derived TLR4. Hence, macrophage clearance exhibits a similar protective effect. Mechanically, TLR4 or TRIF deficiency was observed to inhibit oxidative stress by increasing glutathione, while decreasing malondialdehyde, 8-hydroxy-2-deoxyguanosine, and γ-H2AX. Therefore, the pharmacologic antioxidant NAC exhibited significant hepato-protective effects. Conclusions: Targeting myeloid-derived TLR4-TRIF signaling pathway or antioxidant therapy may be a potential therapeutic direction to treat ALF.

MyD88 and TRIF synergistic interaction is required for TH1‐cell polarization with a synthetic TLR4 agonist adjuvant

Glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) is a synthetic adjuvant TLR4 agonist that promotes potent poly-functional T(H)1 responses. Different TLR4 agonists may preferentially signal via MyD88 or TIR-domain-containing adapter inducing IFN-beta (TRIF) to exert adjuvant effects; however, the contribution of MyD88 and TRIF signaling to the induction of polyclonal T(H)1 responses by TLR4 agonist adjuvants has not been studied in vivo. To determine whether GLA-SE preferentially signals through MyD88 or TRIF, we evaluated the immune response against a candidate tuberculosis (TB) vaccine Ag following immunization of mice lacking either signaling adapter compared with that of wild-type mice. We find that both MyD88 and TRIF are necessary for GLA-SE to induce a poly-functional T(H)1 immune response characterized by CD4(+) T cells producing IFN-γ, TNF, and IL-2, as well as IgG2c class switching, when paired with the TB vaccine Ag ID93. Accordingly, the protective efficacy of ID93/GLA-SE immunization against aerosolized Mycobacterium tuberculosis was lost when either signaling molecule was ablated. We demonstrate that MyD88 and TRIF must be expressed in the same cell for the in vivo T(H)1-skewing adjuvant activity, indicating that these two signaling pathways cooperate on an intracellular level. Thus engagement of both the MyD88 and TRIF signaling pathways are essential for the effective adjuvant activity of this TLR4 agonist.

Interaction of Hepatitis C virus proteins with pattern recognition receptors

Hepatitis C virus (HCV) is an important human pathogen that causes acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma worldwide. This positive stranded RNA virus is extremely efficient in establishing persistent infection by escaping immune detection or hindering the host immune responses. Recent studies have discovered two important signaling pathways that activate the host innate immunity against viral infection. One of these pathways utilizes members of Toll-like receptor (TLR) family and the other uses the RNA helicase retinoic acid inducible gene I (RIG-I) as the receptors for intracellular viral double stranded RNA (dsRNA), and activation of transcription factors. In this review article, we summarize the interaction of HCV proteins with various host receptors/sensors through one of these two pathways or both, and how they exploit these interactions to escape from host defense mechanisms. For this purpose, we searched data from Pubmed and Google Scholar. We found that three HCV proteins; Core (C), non structural 3/4 A (NS3/4A) and non structural 5A (NS5A) have direct interactions with these two pathways. Core protein only in the monomeric form stimulates TLR2 pathway assisting the virus to evade from the innate immune system. NS3/4A disrupts TLR3 and RIG-1 signaling pathways by cleaving Toll/IL-1 receptor domain-containing adapter inducing IFN-beta (TRIF) and Cardif, the two important adapter proteins of these signaling cascades respectively, thus halting the defense against HCV. NS5A downmodulates the expressions of NKG2D on natural killer cells (NK cells) via TLR4 pathway and impairs the functional ability of these cells. TLRs and RIG-1 pathways have a central role in innate immunity and despite their opposing natures to HCV proteins, when exploited together, HCV as an ever developing virus against host immunity is able to accumulate these mechanisms for near unbeatable survival.

Levocetrizine has anti-inflammatory effects against Toll-like receptor (TLR)3 through the inhibition of Toll-IL-1 receptor (TIR)-domain-containing adapter inducing IFN-beta (TRIF) and receptor-interacting protein (RIP)

To investigate the anti-inflammatory effect of levocetrizine (LCEZ) on the intracellular adhesion molecule-1 (ICAM-1) in human nasal epithelial cells stimulated by TLR3 and further analyze the anti-inflammatory mechanism of LCEZ in the MyD88-independent pathway before NF-κB is activated. A primary culture of human nasal epithelial cells (HNECs) was generated from nasal polyps. After stimulation of epithelial cells with LTA, double-stranded RNA (dsRNA), and LPS, reverse transcription-polymerase chain reaction (RT-PCR) was performed at 1, 6, and 24 h to clarify the optimal stimulation of ICAM-1 in HNECs. To investigate the anti-inflammatory effects of LCEZ, HNECs were pretreated with three different concentrations of LCEZ (500, 50, and 5 nM) for 2 h. HNECs were washed and then stimulated with dsRNA. At 1, 6, and 24 h after stimulation, the level of ICAM-1 was measured by RT-PCR and ELISA. Western blots for TRIF and RIP were performed. The level of ICAM-1 was significantly elevated by dsRNA. Pretreatment with LCEZ decreased the secretion of ICAM-1, which was observed in RT-PCR and Western blots but not in ELISA analyses. The expression of TRIF and RIP, measured by Western blot, was decreased by pretreatment with LCEZ. The activation of HNECs by TLRs (especially TLR3) could trigger an inflammatory process, which might be inhibited by LCEZ through the suppression of TRIF and RIP proteins.

MYD88‐dependent and ‐independent activation of TREM‐1 via specific TLR ligands

Triggering receptor expressed on myeloid cells (TREM)-1 plays an important role in myeloid cell-activated inflammatory responses. Although TLR ligands such as LPS and lipoteichoic acid have been shown to upregulate TREM-1 expression in macrophage and neutrophils, the role of specific TLR in inducing the expression of TREM-1 remains unclear. In this study, we investigated whether the presence of TLR is necessary for the expression of TREM-1. We show that BM-derived macrophages from TLR4 and TLR2 KO mice failed to induce expression of TREM-1 message and protein in response to their specific ligands. Interestingly, the expression of TREM-1 in response to LPS is not altered in myeloid differentiation factor 88 (MyD88) KO macrophages, suggesting that downstream of TLR a MyD88-independent pathway induces the expression of TREM-1. Inhibiting toll/IL-1R domain-containing adaptor-inducing IFN-beta (TRIF) expression by siRNA decreased TREM-1 expression in response to LPS, suggesting that the expression of TREM-1 in response to LPS was mediated by the TRIF signaling pathway. On the other hand, the expression of TREM-1 in response to lipoteichoic acid is dependent on MyD88 expression. These data indicate that the expression of TREM-1 in response to TLR ligands occurs secondary to downstream signaling events and that the presence of TLR is necessary for the expression of TREM-1 in response to their specific ligands. However, the downstream signaling required for the expression of TREM-1 is dependent on the stimulus and the surface receptor through which the signaling is initiated.

Thalidomide inhibits lipopolysaccharide-induced nitric oxide production and prevents lipopolysaccharide-mediated lethality in mice

The effect of thalidomide on lipopolysaccharide-induced nitric oxide (NO) production was studied using RAW 264.7 macrophage-like cells. Thalidomide significantly inhibited lipopolysaccharide-induced NO production via reduced expression of an inducible NO synthase. Thalidomide reduced the phosphorylation of the p65 nuclear factor-kappaB subunit, inhibitory kappaB (IkappaB) and IkappaB kinase in lipopolysaccharide-stimulated cells. However, thalidomide did not affect the expression of interferon-beta (IFN-beta) and interferon regulatory factor-1 in response to lipopolysaccharide. Further, thalidomide inhibited the MyD88 augmentation in lipopolysaccharide-stimulated cells, whereas it did not alter the expression of TIR domain-containing adaptor-inducing IFN-beta in the MyD88-independent pathway. Thalidomide significantly inhibited the NO production in response to Pam(3)Cys, CpG DNA and imiquimod as MyD88-dependent Toll-like receptor (TLR) ligands, but not polyI:C as a MyD88-independent TLR ligand. Therefore, thalidomide was suggested to inhibit lipopolysaccharide-induced NO production via downregulation of the MyD88-dependent signal pathway. The anti-inflammatory action of thalidomide might be involved in the prevention of lipopolysaccharide-mediated lethality in mice.

Balancing inflammation and tolerance in vivo through dendritic cells by the commensal Candida albicans

We analyzed the contribution of intracellular signaling to the functional plasticity of dendritic cells (DCs) presenting Candida albicans, a human commensal associated with severe diseases. Distinct intracellular pathways were activated by recognition of different fungal morphotypes in distinct DC subsets and in Peyer's patches DCs. Inflammatory DCs initiated Th17/Th2 responses to yeasts through the adaptor myeloid differentiation factor-88 (MyD88), whereas tolerogenic DCs activate Th1/T regulatory cell (Treg) differentiation programs to hyphae involving Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF) as an intermediary of signaling. In addition, signal transducer and activator of transcription 3 (STAT3), affecting the balance between canonical and non-canonical activation of nuclear factor-kappaB (NF-κB) and 2,3 indoleamine dioxygenase (IDO), pivotally contributed to DC plasticity and functional specialization. As Candida-induced tolerogenic DCs ameliorated experimental colitis, our data qualify Candida as a commensal with immunoregulatory activity, resulting from the orchestrated usage of multiple, yet functionally distinct, receptor–signaling pathways in DCs. Ultimately, affecting the local Th17/Treg balance might likely be exploited by the fungus for either commensalism or pathogenicity.

Mechanisms of Toll-Like Receptor 4 (TLR4)-Mediated Inflammation After Cold Ischemia/Reperfusion in the Heart

Toll-Like Receptor 4 (TLR4) signaling mediates early inflammation after cold ischemia-reperfusion (I/R). We hypothesized that the TLR4 coreceptor CD14, the intracellular adaptor proteins myeloid differentiation factor 88 (MyD88) and TIR domain-containing-adaptor inducing IFNbeta (TRIF) would be required for cold I/R induced inflammation. High mobility group box 1 (HMGB1) is a putative endogenous activator of TLR4. Therefore, we also assessed the contribution of HMGB1 in cold I/R induced inflammation. Syngeneic heart transplants were performed in mice deficient in CD14, MyD88, TRIF, or wild-type mice. In other experiments, anti-HMGB1 neutralizing antibody or control IgG was administered at reperfusion. Donor hearts were subjected to 2 hr of cold ischemia and retrieved after 3 hr of reperfusion. After cold I/R, grafts revealed striking translocation of HMGB1 out of the nucleus in cardiac myocytes. Administration of an anti-HMGB1 neutralizing antibody resulted in reduced systemic interleukin (IL)-6, tumor necrosis factor alpha (TNFalpha), and intercellular adhesion molecule-1 (ICAM-1) messenger RNA (mRNA) levels (P< or =0.05). Compared with controls, CD14 knock-out (KO) mice exhibited significantly lower (P< or =0.05) systemic IL-6 and JE/monocyte chemotractant protein-1 levels after cold I/R. Intragraft TNFalpha and IL-1beta mRNA levels were also significantly lower (P< or =0.05) in CD14 KO grafts. MyD88 KO mice exhibited significantly lower (P< or=0.05) systemic IL-6 levels compared with control mice after cold I/R. Intragraft TNFalpha, IL-6, and ICAM-1 mRNA levels were also significantly lower (P< or =0.05) in MyD88 KO grafts. Significantly lower levels (P< or =0.05) of serum IL-6, monocyte chemotractant protein-1 as well as intragraft TNFalpha, IL-6, IL-1beta, and ICAM-1 were observed after cold I/R in TRIF deficient animals compared with controls. CD14, MyD88, TRIF, and HMGB1 contribute to the inflammatory response that occurs after cold I/R. These results provide insight into the mechanisms of TLR4-mediated inflammation after cold I/R.

Contribution of TIR domain-containing adapter inducing IFN-β-mediated IL-18 release to LPS-induced liver injury in mice

After treatment with heat-killed Propionibacterium acnes mice show dense hepatic granuloma formation. Such mice develop liver injury in an interleukin (IL)-18-dependent manner after challenge with a sublethal dose LPS. As previously shown, LPS-stimulated Kupffer cells secrete IL-18 depending on caspase-1 and Toll-like receptor (TLR)-4 but independently of its signal adaptor myeloid differentiation factor 88 (MyD88), suggesting importance of another signal adaptor TIR domain-containing adapter inducing IFN-beta (TRIF). Nalp3 inflammasome reportedly controls caspase-1 activation. Here we investigated the roles of MyD88 and TRIF in P. acnes-induced hepatic granuloma formation and LPS-induced caspase-1 activation for IL-18 release. Mice were sequentially treated with P. acnes and LPS, and their serum IL-18 levels and liver injuries were determined by ELISA and ALT/AST measurement, respectively. Active caspase-1 in LPS-stimulated Kupffer cells was determined by Western blotting. Macrophage-ablated mice lacked P. acnes-induced hepatic granuloma formation and LPS-induced serum IL-18 elevation and liver injury. Myd88(-/-) Kupffer cells, but not Trif(-/-) cells, exhibited normal caspase-1 activation upon TLR4 engagement in vitro. Myd88(-/-) mice failed to develop hepatic granulomas after P. acnes treatment and liver injury induced by LPS challenge. In contrast, Trif(-/-) mice normally formed the hepatic granulomas, but could not release IL-18 or develop the liver injury. Nalp3(-/-) mice showed the same phenotypes of Trif(-/-) mice. Propionibacterium acnes treatment MyD88-dependently induced hepatic granuloma formation. Subsequent LPS TRIF-dependently activated caspase-1 via Nalp3 inflammasome and induced IL-18 release, eventually leading to the liver injury.

Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1β production

Systems for protein degradation are essential for tight control of the inflammatory immune response. Autophagy, a bulk degradation system that delivers cytoplasmic constituents into autolysosomes, controls degradation of long-lived proteins, insoluble protein aggregates and invading microbes, and is suggested to be involved in the regulation of inflammation. However, the mechanism underlying the regulation of inflammatory response by autophagy is poorly understood. Here we show that Atg16L1 (autophagy-related 16-like 1), which is implicated in Crohn's disease, regulates endotoxin-induced inflammasome activation in mice. Atg16L1-deficiency disrupts the recruitment of the Atg12-Atg5 conjugate to the isolation membrane, resulting in a loss of microtubule-associated protein 1 light chain 3 (LC3) conjugation to phosphatidylethanolamine. Consequently, both autophagosome formation and degradation of long-lived proteins are severely impaired in Atg16L1-deficient cells. Following stimulation with lipopolysaccharide, a ligand for Toll-like receptor 4 (refs 8, 9), Atg16L1-deficient macrophages produce high amounts of the inflammatory cytokines IL-1beta and IL-18. In lipopolysaccharide-stimulated macrophages, Atg16L1-deficiency causes Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF)-dependent activation of caspase-1, leading to increased production of IL-1beta. Mice lacking Atg16L1 in haematopoietic cells are highly susceptible to dextran sulphate sodium-induced acute colitis, which is alleviated by injection of anti-IL-1beta and IL-18 antibodies, indicating the importance of Atg16L1 in the suppression of intestinal inflammation. These results demonstrate that Atg16L1 is an essential component of the autophagic machinery responsible for control of the endotoxin-induced inflammatory immune response.

TLR4-mediated activation of dendritic cells by the heat shock protein DnaK fromFrancisella tularensis

Francisella tularensis is the causative agent of tularemia, a severe, debilitating disease of humans and other mammals. As this microorganism is also classified as a "category-A pathogen" and a potential biowarfare agent, there is a need for an effective vaccine. Several antigens of F. tularensis, including the heat shock protein DnaK, have been proposed for use in a potential subunit vaccine. In this study, we characterized the innate immune response of murine bone marrow-derived dendritic cells (DC) to F. tularensis DnaK. Recombinant DnaK was produced using a bacterial expression system and purified using affinity, ion-exchange, and size-exclusion chromatography. DnaK induced the activation of MAPKs and NF-kappaB in DC and the production of the proinflammatory cytokines IL-6, TNF-alpha, and IL-12 p40, as well as low levels of IL-10. DnaK induced phenotypic maturation of DC, as demonstrated by an up-regulation of costimulatory molecules CD40, CD80, and CD86. DnaK stimulated DC through TLR4 and the adapters MyD88 and Toll/IL-1R domain-containing adaptor-inducing IFN-beta (TRIF) that mediated differential responses. DnaK induced activation of MAPKs and NF-kappaB in a MyD88- or TRIF-dependent manner. However, the presence of MyD88- and TRIF-dependent signaling pathways was essential for an optimal, DnaK-induced cytokine response in DC. In contrast, DnaK induced DC maturation in a TRIF-dependent, MyD88-independent manner. These results provide insight about the molecular interactions between an immunodominant antigen of F. tularensis and host immune cells, which is crucial for the rational design and development of a safe and efficacious vaccine against tularemia.

Porcine reproductive and respiratory syndrome virus (PRRSV) suppresses interferon-β production by interfering with the RIG-I signaling pathway

Porcine reproductive and respiratory syndrome virus (PRRSV) is the cause of an economically important swine disease that has been devastating the swine industry since the late 1980s. Accumulating evidences have revealed that PRRSV infection fails to induce type I interferon (IFN-α/β), which are normally induced rapidly during virus replication in virus-infected cells. However, the potential mechanisms remain largely unclear. In this study, we showed that PRRSV infection activated the signal transduction components of NF-κB and AP-1, but not of interferon regulatory factor 3 (IRF3), an essential IFN-β transcription factor. Furthermore, PRRSV infection significantly blocked synthetic dsRNA-induced IFN-β production and IRF3 nuclear translocation. To better understand the upstream signaling events that suppress IRF3 activation, we further investigated the roles of individual components of the retinoic acid-inducible gene I (RIG-I)- and Toll-like receptor 3 (TLR3)-mediated signaling pathway for IFN-β production during PRRSV infection. We observed that PRRSV infection significantly inhibited dsRNA-induced IRF3 activation and IFN-β generation by inactivating IFN-β promoter stimulator 1 (IPS-1), an adaptor molecule of RIG-I. In contrast, PRRSV infection only partially reduced the activation of TIR domain-containing adaptor inducing IFN-β (TRIF), an adaptor molecule of TLR3. Our results suggest that PRRSV infection suppresses production of IFN-β primarily by interfering with the IPS-1 activation in the RIG-I signaling pathway.

Polyinosinic Acid Is a Ligand for Toll-like Receptor 3

Innate immune responses are critical in controlling viral infections. Viral proteins and nucleic acids have been shown to be recognized by pattern recognition receptors of the Toll-like receptor (TLR) family, triggering downstream signaling cascades that lead to cellular activation and cytokine production. Viral DNA is sensed by TLR9, and TLRs 3, 7, and 8 have been implicated in innate responses to RNA viruses by virtue of their ability to sense double-stranded (ds) RNA (TLR3) or single-stranded RNA (murine TLR7 and human TLR8). Viral and synthetic dsRNAs have also been shown to be a potent adjuvant, promoting enhanced adaptive immune responses, and this property is also dependent on their recognition by TLR3. It has recently been shown that mRNA that is largely single-stranded is a ligand for TLR3. Here we have investigated the ability of single-stranded homopolymeric nucleic acids to induce innate responses by murine immune cells. We show for the first time that polyinosinic acid (poly(I)) activates B lymphocytes, dendritic cells, and macrophages and that these responses are dependent on the expression of both TLR3 and the adaptor molecule, Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF). We therefore conclude that TLR3 is able to sense both single-stranded RNA and dsRNA.

Interferon-beta is activated by hepatitis C virus NS5B and inhibited by NS4A, NS4B, and NS5A

Innate immunity is part of the antiviral response. Interferon (IFN)-beta plays a leading role in this system. To investigate the influence of hepatitis C virus (HCV) on innate immunity, we examined the effect of viral proteins on IFN-beta induction. HepG2 cells were co-transfected with plasmids for seven HCV proteins (core protein, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) and the IFN-beta promoter luciferase. Toll-like receptor (TLR) 3 and Toll/IL-1 receptor domain-containing adapter inducing IFN-beta (TRIF) play key roles in dsRNA-mediated activation of interferon regulatory factor (IRF)-3 and IFN-beta; therefore, the participation of TLR3/TRIF in NS5B-mediated IFN induction was examined. Among seven HCV proteins, only NS5B, a viral RNA-dependent RNA polymerase (RdRp), activated the IFN-beta promoter. However, mutant NS5B without RdRp activity or template/primer association did not activate the IFN-beta promoter. Activation of the IFN-beta promoter by NS5B required the positive regulatory domain III, a binding sequence for IRF-3. Moreover, IRF-3 was phosphorylated by NS5B. Both inhibition of TLR3 expression by small interfering RNA and expression of the dominant negative form of TRIF significantly reduced NS5B-induced activation of IFN-beta. Of the six other HCV proteins, NS4A, NS4B, and NS5A efficiently inhibited this activation. HCV NS5B is a potent activator of the host innate immune system, possibly through TLR3/TRIF and synthesis of dsRNA. Meanwhile, NS4A, NS4B, and NS5A block IFN-beta induction by NS5B, which may contribute toward the persistence of this virus.

TLR3 signaling in a hepatoma cell line is skewed towards apoptosis

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPS) leading to the activation of the innate immune response and subsequently to the shaping of the adaptive immune response. Of the known human TLRs, TLR3, 7, 8, and 9 were shown to recognize nucleic acid ligands. TLR3 signaling is induced by double-stranded (ds)RNA, a molecular signature of viruses, and is mediated by the TRIF (TIR domain-containing adaptor-inducing IFNbeta) adaptor molecule. Thus, TLR3 plays an important role in the host response to viral infections. The liver is constantly exposed to a large variety of foreign substances, including pathogens such as HBV (hepatitis B virus) and HCV (hepatitis C virus), which frequently establish persistent liver infections. In this work, we investigated the expression and signaling pathway of TLR3 in different hepatoma cell lines. We show that hepatocyte lineage cells express relatively low levels of TLR3 mRNA. TLR3 signaling in HEK293 cells (human embryonic kidney cells) activated NF-kappaB and IRF3 (interferon regulatory factor 3) and induced IFNbeta (interferon beta) promoter expression, which are known to lead to pro-inflammatory cytokine secretion. In Huh7 cells, there was only a short-term IRF3 activation, and a very low level of IFNbeta expression. In HepG2 cells on the other hand, while no induction of pro-inflammatory factors was observed, signaling by TLR3 was skewed towards the induction of apoptosis. These results indicate preferential induction of the apoptotic pathway over the cytokine induction pathway by TLR3 signaling in hepatocellular carcinoma cells with potential implications for therapeutic strategies.

Pathological role of Toll-like receptor signaling in cerebral malaria

Toll-like receptors (TLRs) recognize malaria parasites or their metabolites; however, their physiological roles in malaria infection in vivo are not fully understood. Here, we show that myeloid differentiation primary response gene 88 (MyD88)-dependent TLR signaling mediates brain pathogenesis of severe malaria infection, namely cerebral malaria (CM). A significant number of MyD88-, but not TIR domain containing adaptor-inducing IFN-beta (TRIF)-deficient or wild-type (WT) mice survived CM caused by Plasmodium berghei ANKA (PbA) infection. Although systemic parasitemia was comparable, sequestration of parasite and hemozoin load in the brain blood vessels was significantly lower in MyD88-deficient mice compared with those in TRIF-deficient or WT mice. Moreover, brain-specific pathological changes were associated with MyD88-dependent infiltration of CD8+, CCR5+ T cells and CD11c+ dendritic cells, including CD11c+, NK1.1+ and B220+ cells, and up-regulation of genes such as Granzyme B, Lipocalin 2, Ccl3 and Ccr5. Further studies using mice lacking various TLRs suggest that TLR2 and TLR9, but not TLR4, 5 and 7, were involved in CM. These results strongly suggest that TLR2- and/or TLR9-mediated, MyD88-dependent brain pathogenesis may play a critical role in CM, the lethal complication during PbA infection.

TRIF–GEFH1–RhoB pathway is involved in MHCII expression on dendritic cells that is critical for CD4 T-cell activation

Dendritic cells (DC) play a central role in immune responses by presenting antigenic peptides to CD4+ T cells through MHCII molecules. Here, we demonstrate a TRIF-GEFH1-RhoB pathway is involved in MHCII surface expression on DC. We show the TRIF (TIR domain-containing adapter inducing IFNbeta)- but not the myeloid differentiation factor 88 (MyD88)-dependent pathway of lipopolysaccharide (LPS)-signaling in DC is crucial for the MHCII surface expression, followed by CD4+ T-cell activation. LPS increased the activity of RhoB, but not of RhoA, Cdc42, or Rac1/2 in a manor dependent on LPS-TRIF- but not LPS-Myd88-signaling. RhoB colocalized with MHCII+ lysosomes in DC. A dominant-negative (DN) form of RhoB (DN-RhoB) or RhoB's RNAi in DC inhibited the LPS-induced MHCII surface expression. Moreover, we found GEFH1 associated with RhoB, and DN-GEFH1 or GEFH1's RNAi suppressed the LPS-mediated RhoB activation and MHCII surface expression. DN-RhoB attenuated the DC's CD4+ T-cell stimulatory activity. Thus, our results provide a molecular mechanism relating how the MHCII surface expression is regulated during the maturation stage of DC. The activation of GEFH1-RhoB through the TRIF-dependent pathway of LPS in DC might be a critical target for controlling the activation of CD4+ T cells.

Immunobiology of Toll‐like receptors: Emerging trends

Toll-like receptors (TLR), a family of evolutionarily conserved pathogen recognition receptors, play pivotal role as primary sensors of invading pathogens. TLR identify molecular motifs of infectious agents (pathogen associated molecular patterns) and elicit an effective defensive response against them. Mammalian TLR derive their name from the Drosophila Toll protein, with which they share sequence similarity. So far, expression of 10 TLR is known in humans. The adaptor proteins, myeloid differentiation factor 88 and Toll IL-1 receptor (TIR) domain containing adaptor inducing IFN-beta (TRIF) are the key players in the TLR signalling cascade leading to the activation of nuclear factor (NF)-kappaB and interferon regulatory factor-3, respectively. Targeted manipulation of the TLR signalling pathway has immense therapeutic potential and may eventually prove to be a boon in the development of innovative treatments for diverse disease conditions. There is accumulating evidence that TLR agonists have tremendous potential as novel therapeutic targets. In this review, we have discussed the immunobiology of TLR and emphasize significant advances made within the ever-expanding field of TLR that provide intriguing insights efficacious in unravelling the complexities associated with TLR.

Mechanism of pathogen‐specific TLR4 activation in the mucosa: Fimbriae, recognition receptors and adaptor protein selection

The mucosal host defence discriminates pathogens from commensals, and prevents infection while allowing the normal flora to persist. Paradoxically, Toll-like receptors (TLR) control the mucosal defence against pathogens, even though the TLR recognise conserved molecules like LPS, which are shared between pathogens and commensals. This study proposes a mechanism of pathogen-specific mucosal TLR4 activation, involving adhesive ligands and their host cell receptors. TLR4 signalling was activated in CD14-negative, LPS-unresponsive epithelial cells by P fimbriated, uropathogenic Escherichia coli but not by a mutant lacking fimbriae. Epithelial TLR4 signalling in vivo involved the glycosphingolipid receptors for P fimbriae and the adaptor proteins Toll/IL-1R (TIR) domain-containing adaptor inducing IFN-beta (TRIF)/TRIF-related adaptor molecule (TRAM), but myeloid differentiation protein 88 (MyD88)/TIR domain-containing adaptor protein were not required for the epithelial response. Substituting the P fimbriae with type 1 fimbriae changed TLR4 signalling from the TRIF to the MyD88 adaptor pathway. In addition, the adaptor proteins and the fimbrial type were found to influence bacterial clearance. Trif(-/-) and Tram(-/-) mice remained infected with P fimbriated E. coli but cleared the type 1 fimbriated strain, while Myd88(-/-) mice became carriers of both the P and the type 1 fimbriated bacteria. Thus, TLR4 may be engaged specifically by pathogens, when the proper cell surface receptors are engaged by virulence ligands.

Specificity in Toll-like receptor signalling through distinct effector functions of TRAF3 and TRAF6

Toll-like receptors (TLRs) are activated by pathogen-associated molecular patterns to induce innate immune responses and production of pro-inflammatory cytokines, interferons and anti-inflammatory cytokines. TLRs activate downstream effectors through adaptors that contain Toll/interleukin-1 receptor (TIR) domains, but the mechanisms accounting for diversification of TLR effector functions are unclear. To dissect biochemically TLR signalling, we established a system for isolating signalling complexes assembled by dimerized adaptors. Using MyD88 as a prototypical adaptor, we identified TNF receptor-associated factor 3 (TRAF3) as a new component of TIR signalling complexes that is recruited along with TRAF6. Using myeloid cells from TRAF3- and TRAF6-deficient mice, we show that TRAF3 is essential for the induction of type I interferons (IFN) and the anti-inflammatory cytokine interleukin-10 (IL-10), but is dispensable for expression of pro-inflammatory cytokines. In fact, TRAF3-deficient cells overproduce pro-inflammatory cytokines owing to defective IL-10 production. Despite their structural similarity, the functions of TRAF3 and TRAF6 are largely distinct. TRAF3 is also recruited to the adaptor TRIF (Toll/IL-1 receptor domain-containing adaptor-inducing IFN-beta) and is required for marshalling the protein kinase TBK1 (also called NAK) into TIR signalling complexes, thereby explaining its unique role in activation of the IFN response.