Toll-interleukin-1 Receptor Domain-containing Adapter Inducing Interferon-beta Research Articles

Activation of Toll-like receptor 3 reduces actin polymerization and adhesion molecule expression in endometrial cells, a potential mechanism for viral-induced implantation failure.

Does activation of endometrial Toll-like receptor 3 (TLR 3) affect cell receptivity to trophoblast adhesion? TLR 3 activation in vitro reduces the attachment of trophoblast cells to endometrial cells by altering the cell cytoskeleton and reducing the expression of adhesion molecules in human endometrial cells. It is well documented that the presence of an infection at the time of implantation can lead to implantation failure. The female reproductive tract recognizes invading micro-organisms through the innate pathogen recognition receptors such as the TLRs. Poly I:C was used as a TLR 3-specific ligand and endometrial cells were either treated or not with Poly I:C (treated versus control) in vitro. The experiments were performed in three replicates on three separate days. An in vitro assay was developed using RL95-2 (a human endometrial cell line) and JAr (a human trophoblast cell line) cells. Initially, the percentage of attached JAr spheroids to RL95-2 was measured in response to TLR 3 activation. Next, actin polymerization in RL95-2 cells was assessed in response to TLR 2/6, 3 and 5 activation. Phalloidin was used to assess the mean fluorescence intensity of F-actin by flow cytometry or confocal microscopy. Secondly, the influence of TLR 2/6, 3 and 5 activation on the expression of cluster of differentiation 98 (CD98) and β3 integrin was determined. To further understand through which pathways the TLR 3-induced alterations occur, inhibitors were applied for Toll/interleukin-1 receptor domain-containing adaptor inducing interferon-beta (TRIF), myeloid differentiation primary response 88 (MYD88), mitogen-activated protein kinases (MAPK) and nuclear factor pathways. We observed that stimulation of TLR 3 in endometrial cells with different concentrations of Poly I:C led to a reduction in the percentage of trophoblasts attached to the endometrial cells in a dose-dependent manner (P < 0.05). This decrease was consistent in the Poly I:C treated group regardless of the co-incubation time (P < 0.05). In addition, our results demonstrated that actin polymerization and CD98 expression significantly decreased only in response to TLR 3 activation (P < 0.05). Activation of endometrial cells with TLR 2/6, 3 and 5 significantly reduced β3 integrin expression (P < 0.05). These alterations were shown to work via MYD88-MAPK pathways (P < 0.05). This study has been performed in vitro. Future in vivo studies will be required in order to confirm our data. This is a novel discovery which extends our current knowledge concerning diagnosis and treatment of viral-induced infertility cases. This research was supported by the COST Action FA1201 (GEMINI) by granting a Short Term Scientific Mission and the Instituto de Salud Carlos III by granting Grant PI11/01645. The authors have no conflict of interest to declare.

TRIF Is Required for TLR4 Mediated Adjuvant Effects on T Cell Clonal Expansion

Toll like receptor 4 (TLR4) is an important pattern recognition receptor with the ability to drive potent innate immune responses and also to modulate adaptive immune responses needed for long term protection. Activation of TLR4 by its ligands is mediated by engagement of the adapter proteins MyD88 (myeloid differentiation factor 88) and TRIF (Toll-interleukin 1 receptor domain-containing adapter inducing interferon-beta). Previously, we showed that TRIF, but not MyD88, plays an important role in allowing TLR4 agonists to adjuvant early T cell responses. In this study, we investigated the T cell priming events that are regulated specifically by the TRIF signaling branch of TLR4. We found that TRIF deficiency prevented the TLR4 agonist lipid A from enhancing T cell proliferation and survival in an adoptive transfer model of T cell priming. TRIF deficient DC showed defective maturation as evidenced by their failure to upregulate co-stimulatory molecules in response to lipid A stimulation. Importantly, TRIF alone caused CD86 and CD40 upregulation on splenic DC, but both TRIF and MyD88 were required for CD80 upregulation. The impairment of T cell adjuvant effects and defective DC maturation in TRIF lps/lps mice after TLR4 stimulation was mainly due to loss of type I IFN production, indicating that type I interferons are central to TLR4's adjuvant effects. These results are useful for the continued development of TLR4 based vaccine adjuvants that avoid inflammatory risks while retaining beneficial immune response.

TBK1-targeted suppression of TRIF-dependent signaling pathway of toll-like receptor 3 by auranofin

Toll-like receptors (TLRs) play an important role in induction of innate immune responses. The stimulation of TLRs by microbial components triggers two branches of downstream signaling pathways: myeloid differential factor 88 (MyD88)- and toll-interleukin-1 receptor domain-containing adapter inducing interferon-beta (TRIF)-dependent signaling pathways. Auranofin, a sulfur-containing gold compound (Au[I]), has been widely used for the treatment of rheumatoid arthritis. Since dysregulation of TLRs can lead to severe systemic inflammatory and joint destructive process in rheumatoid arthritis, auranofin-mediated modulation of TLR activation may have therapeutic potential against such diseases. Previously, we demonstrated that auranofin suppressed TLR4 signaling pathway by inhibiting TLR4 dimerization induced by LPS. Here, we examined the effect of auranofin on signal transduction via the TRIF-dependent pathway induced by a TLR3 agonist. Auranofin inhibited nuclear factor-kappaB and interferon (IFN) regulatory factor 3 (IRF3) activation induced by polyinosinic-polycytidylic acid (poly[I:C]). Auranofin inhibited poly[I:C]-induced phosphorylation of IRF3 as well as IFN-inducible genes such as IFN inducible protein-10. Furthermore, auranofin inhibited TBK1 kinase activity in vitro. All the results suggest that auranofin suppress TLR signaling at multiple steps.

The Toll-like receptor adaptor molecule TRIF enhances DNA vaccination against classical swine fever

To evaluate the effects of the Toll/interleukin-1 receptor domain-containing adaptor-inducing interferon-beta (TRIF) on immune responses induced by DNA vaccines, mice were immunized with the eukaryotic expression plasmid pcDNA/E2 encoding classical swine fever virus (CSFV) E2 alone or in combination with the TRIF genetic adjuvant. Immune responses were examined in immunized mice. Our data demonstrates that co-delivery of the DNA vaccine pcDNA/E2 with the TRIF adjuvant augmented specific humoral and cellular immune responses in a mouse model. Vaccination of pigs confirmed that the pcDNA/E2 in combination with TRIF conferred total protection against lethal challenge with highly virulent CSFV. We conclude that TRIF enhances the effects of the DNA vaccine against CSFV infection and could be used as a potential genetic adjuvant for DNA vaccines in large animal species.

Isoliquiritigenin Suppresses the Toll−Interleukin-1 Receptor Domain-Containing Adapter Inducing Interferon-β (TRIF)-Dependent Signaling Pathway of Toll-Like Receptors by Targeting TBK1

Toll-like receptors (TLRs) play an important role in induction of innate immune responses. TLRs can trigger the activation of myeloid differential factor 88 (MyD88)- and Toll-interleukin-1 receptor domain-containing adapter inducing interferon-beta (TRIF)-dependent downstream signaling pathways. Expression of more than 70% of lipopolysaccharide (LPS)-induced target genes is mediated through a TRIF-dependent signaling pathway. To evaluate the therapeutic potential of isoliquiritigenin (ILG), we examined its effect on signal transduction via the TRIF-dependent pathway of TLRs. ILG inhibited interferon regulatory factor 3 activation induced by LPS or polyinosinic-polycytidylic acid, as well as interferon-inducible genes, such as interferon-inducible protein-10. ILG attenuated ligand-independent activation of IRF3 induced by TRIF or TBK1. Furthermore, ILG inhibited TBK1 kinase activity in vitro. Together, these results demonstrate that TBK1 is the molecular target of ILG, resulting in the downregulation of the TRIF-dependent signaling pathways of TLRs.

Viral RNA Induces Type I Interferon-Dependent Cytokine Release and Cell Death in Mesangial Cells via Melanoma-Differentiation-Associated Gene-5: Implications for Viral Infection-Associated Glomerulonephritis

Viral RNA can trigger interferon signaling in dendritic cells via the innate recognition receptors melanoma-differentiation-associated gene (MDA)-5 and retinod-inducible gene (RIG)-I in the cytosol or via Toll-like receptors (TLRs) in intracellular endosomes. We hypothesized that viral RNA would also activate glomerular mesangial cells to produce type I interferon (IFN) via TLR-dependent and TLR-independent pathways. To test this hypothesis, we examined Toll/Interleukin-1 receptor domain-containing adaptor-inducing interferon-beta (TRIF)-deficient mice, which lack a key adaptor for TLR3 signaling. In primary mesangial cells, poly I:C RNA-mediated IFN-beta induction was partially TRIF dependent; however, when poly I:C RNA was complexed with cationic lipids to enhance cytosolic uptake, mesangial cells produced large amounts of IFN-alpha and IFN-beta independent of TRIF. Mesangial cells expressed RIG-I and MDA-5 and their mitochondrial adaptor IFN-beta promoter stimulator-1 as well, and small interfering RNA studies revealed that MDA5 but not RIG-I was required for cytosolic poly I:C RNA signaling. In addition, mesangial cells produced Il-6 on stimulation with IFN-alpha and IFN-beta, suggesting an autocrine proinflammatory effect. Indeed, blockade of IFN-alphabeta or lack of the IFNA receptor reduced viral RNA-induced Il-6 production and apoptotic cell death in mesangial cells. Furthermore, viral RNA/cationic lipid complexes increased focal necrosis in murine nephrotoxic serum nephritis in association with increased renal mRNA expression of IFN-related genes. Thus, TLR-independent recognition of viral RNA is a potent inducer of type I interferon in mesangial cells, which can be an important mediator of virally induced glomerulonephritis.

Mechanical Ventilation Induces a Toll/Interleukin-1 Receptor Domain-containing Adapter-inducing Interferon β–dependent Inflammatory Response in Healthy Mice

Mechanical ventilation (MV) can induce lung injury. Proinflammatory cytokines have been shown to play an important role in the development of ventilator-induced lung injury. Previously, the authors have shown a role for Toll-like receptor 4 signaling. The current study aims to investigate the role of Toll/interleukin-1 receptor domain-containing adapter-inducing interferon-beta (TRIF), a protein downstream of Toll-like receptors, in the development of the inflammatory response after MV in healthy mice. Wild-type C57BL6 and TRIF mutant mice were mechanically ventilated for 4 h. Lung tissue and plasma was used to investigate changes in cytokine profile, leukocyte influx, and nuclear factor-kappaB activity. In addition, experiments were performed to assess the role of TRIF in changes in cardiopulmonary physiology after MV. MV significantly increased messenger RNA expression of interleukin (IL)-1beta in wild-type mice, but not in TRIF mutant mice. In lung homogenates, MV increased levels of IL-1alpha, IL-1beta, and keratinocyte-derived chemokine in wild-type mice. In contrast, in TRIF mutant mice, only a minor increase in IL-1beta and keratinocyte-derived chemokine was found after MV. Nuclear factor-kappaB activity after MV was significantly lower in TRIF mutant mice compared with wild-type mice. In plasma, MV increased levels of IL-6 and keratinocyte-derived chemokine. In TRIF mutant mice, no increase of IL-6 was found after MV, and the increase in keratinocyte-derived chemokine appeared less pronounced. TRIF deletion did not affect cardiopulmonary physiology after MV. The current study supports a prominent role for TRIF in the development of the pulmonary and systemic inflammatory response after MV.

Suppression of the TRIF-dependent signaling pathway of toll-like receptors by isoliquiritigenin in RAW264.7 macrophages.

Toll-like receptors (TLRs) play an important role in host defense by sensing invading microbial pathogens and initiating innate immune responses. The stimulation of TLRs by microbial components triggers the activation of myeloid differential factor 88 (MyD88)- and toll-interleukin-1 receptor domain-containing adapter inducing interferon-beta (TRIF)-dependent downstream signaling pathways. Isoliquiritigen in (ILG), an active ingredient of Licorice, has been used for centuries to treat many chronic diseases. ILG inhibits the MyD88-dependent pathway by inhibiting the activity of inhibitor-kappaB kinase. However, it is not known whether ILG inhibits the TRIF-dependent pathway. To evaluate the therapeutic potential of ILG, we examined its effect on signal transduction via the TRIF-dependent pathway of TLRs induced by several agonists. ILG inhibited nuclear factor-kappaB and interferon regulatory factor 3 activation induced by lipopolysaccharide or polyinosinic-polycytidylic acid. ILG inhibited the lipopolysaccharide-induced phosphorylation of interferon regulatory factor 3 as well as interferon-inducible genes such as interferon inducible protein-10, and regulated activation of normal T-cell expressed and secreted (RANTES). These results suggest that ILG can modulate TRIF-dependent signaling pathways of TLRs, leading to decreased inflammatory gene expression.

Toll-like Receptor 4 Is a Sensor for Autophagy Associated with Innate Immunity

Autophagy has recently been shown to be an important component of the innate immune response. The signaling pathways leading to activation of autophagy in innate immunity are not known. Here we showed that Toll-like receptor 4 (TLR4) served as a previously unrecognized environmental sensor for autophagy. Autophagy was induced by lipopolysaccharide (LPS) in primary human macrophages and in the murine macrophage RAW264.7 cell line. We defined a new molecular pathway in which LPS-induced autophagy was regulated through a Toll-interleukin-1 receptor domain-containing adaptor-inducing interferon-beta (TRIF)-dependent, myeloid differentiation factor 88 (MyD88)-independent TLR4 signaling pathway. Receptor-interacting protein (RIP1) and p38 mitogen-activated protein kinase were downstream components of this pathway. This signaling pathway did not affect cell viability, indicating that it is distinct from the autophagic death signaling pathway. We further showed that LPS-induced autophagy could enhance mycobacterial colocalization with the autophagosomes. This study links two ancient processes, autophagy and innate immunity, together through a shared signaling pathway.

P.217 High rate of spontaneous viral clearance in patients with symptomatic acute hepatitis C

To evaluate the effects of the Toll/interleukin-1 receptor domain-containing adaptor-inducing interferon-beta (TRIF) on immune responses induced by DNA vaccines, mice were immunized with the eukaryotic expression plasmid pcDNA/E2 encoding classical swine fever virus (CSFV) E2 alone or in combination with the TRIF genetic adjuvant. Immune responses were examined in immunized mice. Our data demonstrates that co-delivery of the DNA vaccine pcDNA/E2 with the TRIF adjuvant augmented specific humoral and cellular immune responses in a mouse model. Vaccination of pigs confirmed that the pcDNA/E2 in combination with TRIF conferred total protection against lethal challenge with highly virulent CSFV. We conclude that TRIF enhances the effects of the DNA vaccine against CSFV infection and could be used as a potential genetic adjuvant for DNA vaccines in large animal species.