Hepatitis C virus fails to activate NF-κB signaling in plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs) respond to viral infection by production of alpha interferon (IFN-α), proinflammatory cytokines, and cell differentiation. The elimination of hepatitis C virus (HCV) in more than 50% of chronically infected patients by treatment with IFN-α suggests that pDCs can play an important role in the control of HCV infection. pDCs exposed to HCV-infected hepatoma cells, in contrast to cell-free HCV virions, produce large amounts of IFN-α. To further investigate the molecular mechanism of HCV sensing, we studied whether exposure of pDCs to HCV-infected hepatoma cells activates, in parallel to interferon regulatory factor 7 (IRF7)-mediated production of IFN-α, nuclear factor kappa B (NF-κB)-dependent pDC responses, such as expression of the differentiation markers CD40, CCR7, CD86, and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and secretion of the proinflammatory cytokines TNF-α and interleukin 6 (IL-6). We demonstrate that exposure of pDCs to HCV-infected hepatoma cells surprisingly did not induce phosphorylation of NF-κB or cell surface expression of CD40, CCR7, CD86, or TRAIL or secretion of TNF-α and IL-6. In contrast, CpG-A and CpG-B induced production of TNF-α and IL-6 in pDCs exposed to the HCV-infected hepatoma cells, showing that cell-associated virus did not actively inhibit Toll-like receptor (TLR)-mediated NF-κB phosphorylation. Our results suggest that cell-associated HCV signals in pDCs via an endocytosis-dependent mechanism and IRF7 but not via the NF-κB pathway. In spite of IFN-α induction, cell-associated HCV does not induce a full functional response of pDCs. These findings contribute to the understanding of evasion of immune responses by HCV.     

CMRF-35-like molecule 3 preferentially promotes TLR9-triggered proinflammatory cytokine production in macrophages by enhancing TNF receptor-associated factor 6 ubiquitination

TLRs are critical innate immune sensors in the induction of proinflammatory cytokines to eliminate invading pathogens. However, the mechanisms for the full activation of TLR-triggered innate immune response need to be fully understood. The murine CMRF-35-like molecule (CLM)-3 is a representative of CLM family belonging to the Ig superfamily. Considering that CLM-3 is selectively expressed in macrophages and the roles of CLM members in innate immune response remain unclear, in this study we investigated the role of CLM-3 in the regulation of TLR-triggered innate response. We found that CLM-3 was an endosome/lysosome-localized molecule, and was downregulated in macrophages by stimulation with TLR9 ligand, but not TLR4 and TLR3 ligands. Interestingly, CLM-3 selectively promoted production of TNF-α and IL-6 in macrophages triggered by TLR9, but not TLR4 or TLR3. CLM-3 enhanced activation of MAPKs and NF-κB pathways in TLR9-triggered macrophages. Furthermore, CLM-3-transgenic mice were generated, and CLM-3 expression was confirmed by mAb against CLM-3 that we prepared. Accordingly, the macrophages derived from CLM-3-transgenic mice were more sensitive to TLR9 ligand stimulation, with more pronounced production of TNF-α, IL-6, and increased activation of MAPKs and NF-κB pathways. Moreover, ubiquitination of TNFR-associated factor 6, a crucial signaling transducer of TLR-triggered MAPKs and NF-κB activation, was found to be significantly promoted by CLM-3 in macrophages. Collectively, the endosome/lysosome-localized CLM-3 can promote full activation of TLR9-triggered innate responses by enhancing TNFR-associated factor 6 ubiquitination and subsequently activating MAPKs and NF-κB.     

Essential role for IKKβ in production of type 1 interferons by plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs) are characterized by their ability to produce high levels of type 1 interferons in response to ligands that activate TLR7 and TLR9, but the signaling pathways required for IFN production are incompletely understood. Here we exploit the human pDC cell line Gen2.2 and improved pharmacological inhibitors of protein kinases to address this issue. We demonstrate that ligands that activate TLR7 and TLR9 require the TAK1-IKKβ signaling pathway to induce the production of IFNβ via a pathway that is independent of the degradation of IκBα. We also show that IKKβ activity, as well as the subsequent IFNβ-stimulated activation of the JAK-STAT1/2 signaling pathway, are essential for the production of IFNα by TLR9 ligands. We further show that TLR7 ligands CL097 and R848 fail to produce significant amounts of IFNα because the activation of IKKβ is not sustained for a sufficient length of time. The TLR7/9-stimulated production of type 1 IFNs is inhibited by much lower concentrations of IKKβ inhibitors than those needed to suppress the production of NFκB-dependent proinflammatory cytokines, such as IL-6, suggesting that drugs that inhibit IKKβ may have a potential for the treatment of forms of lupus that are driven by self-RNA and self-DNA-induced activation of TLR7 and TLR9, respectively.     

Phospholipase Cγ-2 and intracellular calcium are required for lipopolysaccharide-induced Toll-like receptor 4 (TLR4) endocytosis and interferon regulatory factor 3 (IRF3) activation

Toll-like receptor 4 (TLR4) is unique among the TLRs in its use of multiple adaptor proteins leading to activation of both the interferon regulatory factor 3 (IRF3) and nuclear factor κB (NF-κB) pathways. Previous work has demonstrated that TLR4 initiates NF-κB activation from the plasma membrane, but that subsequent TLR4 translocation to the endosomes is required for IRF3 activation. Here we have characterized several components of the signaling pathway that governs TLR4 translocation and subsequent IRF3 activation. We find that phospholipase C γ2 (PLCγ2) accounts for LPS-induced inositol 1,4,5-trisphosphate (IP(3)) production and subsequent calcium (Ca(2+)) release. Blockage of PLCγ2 function by inhibitors or knockdown of PLCγ2 expression by siRNAs in RAW 264.7 macrophages lead to reduced IRF3, but enhanced NF-κB activation. In addition, bone marrow-derived macrophages from PLCγ2-deficient mice showed impaired IRF3 phosphorylation and expression of IRF3-regulated genes after LPS stimulation. Using cell fractionation, we show that PLCγ2-IP(3)-Ca(2+) signaling cascade is required for TLR4 endocytosis following LPS stimulation. In conclusion, our results describe a novel role of the PLCγ2-IP(3)-Ca(2+) cascade in the LPS-induced innate immune response pathway where release of intracellular Ca(2+) mediates TLR4 trafficking and subsequent activation of IRF3.     

Pellino-1 Positively Regulates Toll-like Receptor (TLR) 2 and TLR4 Signaling and Is Suppressed upon Induction of Endotoxin Tolerance

Endotoxin tolerance reprograms Toll-like receptor (TLR) 4-mediated macrophage responses by attenuating induction of proinflammatory cytokines while retaining expression of anti-inflammatory and antimicrobial mediators. We previously demonstrated deficient TLR4-induced activation of IL-1 receptor-associated kinase (IRAK) 4, IRAK1, and TANK-binding kinase (TBK) 1 as critical hallmarks of endotoxin tolerance, but mechanisms remain unclear. In this study, we examined the role of the E3 ubiquitin ligase Pellino-1 in endotoxin tolerance and TLR signaling. LPS stimulation increased Pellino-1 mRNA and protein expression in macrophages from mice injected with saline and in medium-pretreated human monocytes, THP-1, and MonoMac-6 cells, whereas endotoxin tolerization abrogated LPS inducibility of Pellino-1. Overexpression of Pellino-1 in 293/TLR2 and 293/TLR4/MD2 cells enhanced TLR2- and TLR4-induced nuclear factor κB (NF-κB) and expression of IL-8 mRNA, whereas Pellino-1 knockdown reduced these responses. Pellino-1 ablation in THP-1 cells impaired induction of myeloid differentiation primary response protein (MyD88), and Toll-IL-1R domain-containing adapter inducing IFN-β (TRIF)-dependent cytokine genes in response to TLR4 and TLR2 agonists and heat-killed Escherichia coli and Staphylococcus aureus, whereas only weakly affecting phagocytosis of heat-killed bacteria. Co-expressed Pellino-1 potentiated NF-κB activation driven by transfected MyD88, TRIF, IRAK1, TBK1, TGF-β-activated kinase (TAK) 1, and TNFR-associated factor 6, whereas not affecting p65-induced responses. Mechanistically, Pellino-1 increased LPS-driven K63-linked polyubiquitination of IRAK1, TBK1, TAK1, and phosphorylation of TBK1 and IFN regulatory factor 3. These results reveal a novel mechanism by which endotoxin tolerance re-programs TLR4 signaling via suppression of Pellino-1, a positive regulator of MyD88- and TRIF-dependent signaling that promotes K63-linked polyubiquitination of IRAK1, TBK1, and TAK1.     

microRNA-210 negatively regulates LPS-induced production of proinflammatory cytokines by targeting NF-κB1 in murine macrophages

Ligation of TLR4 with LPS in macrophages leads to the production of proinflammatory cytokines, which are central to eliminate viral and bacterial infection. However, uncontrolled TLR4 activation may contribute to pathogenesis of inflammatory diseases such as septic shock. In this study, we found microRNA-210 was induced in murine macrophages by LPS. Transfection of miR-210 mimics significantly inhibited LPS-induced production of inflammatory cytokines. In contrast, transfection of anti-miR-210 inhibitors increased LPS-induced expression of proinflammatory cytokines. Furthermore, we demonstrated that miR-210 targets NF-κB1. Therefore, our data identify miR-210 as a very important feedback negative regulator for LPS-induced production of proinflammatory cytokines.     

NLRC5 negatively regulates LTA-induced inflammation via TLR2/NF-κB and participates in TLR2-mediated allergic airway inflammation

NLRC5, the largest member of the Nod-like receptor (NLR) family, has been reported to play a pivotal role in regulating inflammatory responses. Recent evidence suggests that NLRC5 participates in Toll-like receptor (TLR) signaling pathways and negatively modulates nuclear factor-κB (NF-κB) activation. In this study, we investigated the interaction between NLRC5 and TLR2 in the NF-κB inflammatory signaling pathway and the involvement of NLRC5 in TLR2-mediated allergic airway inflammation. We knocked down TLR2 and NLRC5, respectively in the RAW264.7 macrophage cell line by small interfering RNA (siRNA) and then stimulated the knockdown cells with lipoteichoic acid (LTA). In comparison with the negative siRNA group, the level of NLRC5 expression was lower in the TLR2 siRNA group, with a reduction in the NF-κB-related inflammatory response. Conversely, in the NLRC5 knockdown cells, after LTA-treated the level of TLR2 expression did not change but the expression levels of both NF-κB pp65 and NLRP3 increased remarkably. Thus, we hypothesize that NLRC5 participates in the LTA-induced inflammatory signaling pathway and regulates the inflammation via TLR2/NF-κB. Similarly, in subsequent in vivo experiments, we demonstrated that the expression level of NLRC5 was significantly increased in the ovalbumin-induced allergic airway inflammation. However, this effect disappeared in TLR2-deficient (TLR2 ^−/−) mice and was accompanied by reduced levels of NF-κB expression and airway inflammation. In conclusion, NLRC5 negatively regulates LTA-induced inflammatory response via a TLR2/NF-κB pathway in macrophages and also participates in TLR2-mediated allergic airway inflammation.     

Heterotrimeric Gi/Go proteins modulate endothelial TLR signaling independent of the MyD88-dependent pathway

The innate immune recognition of bacterial lipopolysaccharide (LPS) is mediated by Toll-like receptor 4 (TLR4) and results in activation of proinflammatory signaling including NF-κB and MAPK pathways. Heterotrimeric G proteins have been previously implicated in LPS signaling in macrophages and monocytes. In the present study, we show that pertussis toxin sensitive heterotrimeric G proteins (Gα(i/o)) are involved in the activation of MAPK and Akt downstream of TLR2, TLR3, and TLR4 in endothelial cells. Gα(i/o) are also required for full activation of interferon signaling downstream of TLR3 and TLR4 but are not required for the activation of NF-κB. We find that Gα(i/o)-mediated activation of the MAPK is independent of the canonical MyD88, interleukin-1 receptor-associated kinase, and tumor necrosis factor receptor-associated factor 6 signaling cascade in LPS-stimulated cells. Taken together, the data presented here suggest that heterotrimeric G proteins are widely involved in TLR pathways along a signaling cascade that is distinct from MyD88-TRAF6.     

Abnormal trafficking and degradation of TLR4 underlie the elevated inflammatory response in cystic fibrosis

Morbidity and mortality in cystic fibrosis (CF) are due not only to abnormal epithelial cell function, but also to an abnormal immune response. We have shown previously that macrophages lacking CF transmembrane conductance regulator (CFTR), the gene mutated in CF, contribute significantly to the hyperinflammatory response observed in CF. In this study, we show that lack of functional CFTR in murine macrophages causes abnormal TLR4 subcellular localization. Upon LPS stimulation, CFTR macrophages have prolonged TLR4 retention in the early endosome and reduced translocation into the lysosomal compartment. This abnormal TLR4 trafficking leads to increased LPS-induced activation of the NF-κB, MAPK, and IFN regulatory factor-3 pathways and decreased TLR4 degradation, which affects downregulation of the proinflammatory state. In addition to primary murine cells, mononuclear cells isolated from CF patients demonstrate similar defects in response to LPS. Moreover, specific inhibition of CFTR function induces abnormal TLR4 trafficking and enhances the inflammatory response of wild-type murine cells to LPS. Thus, functional CFTR in macrophages influences TLR4 spatial and temporal localization and perturbs LPS-mediated signaling in both murine CF models and patients with CF.     

Secretory leukocyte proteinase inhibitor-competent DNA deposits are potent stimulators of plasmacytoid dendritic cells: implication for psoriasis

Secretory leukocyte proteinase inhibitor (SLPI) is a well-established inhibitor of serine proteases such as human neutrophil elastase (HNE) and a NF-κB regulatory agent in immune cells. In this paper, we report that SLPI plays a previously uncharacterized role in regulating activation of plasmacytoid dendritic cells (pDCs). As the main source of IFN type I (IFNI), pDCs are crucial contributors to inflammatory and likely wound-healing responses associated with psoriasis. The mechanisms responsible for activation of pDCs in psoriatic skin are therefore of substantial interest. We demonstrate that in lesional skin of psoriasis patients, SLPI together with its enzymatic target HNE and DNA, is a component of neutrophil extracellular traps (NETs). Whereas SLPI(+) neutrophils and NETs were found to colocalize with pDCs in psoriatic skin, a mixture of SLPI with neutrophil DNA and HNE induced a marked production of IFNI by pDCs. IFNI synthesis by stimulated pDCs was dependent on intracellular DNA receptor TLR9. Thus, SLPI may contribute to psoriasis by enabling pDCs to sense extracellular DNA and produce IFNI.     

EBI2 Is a Negative Regulator of Type I Interferons in Plasmacytoid and Myeloid Dendritic Cells

Epstein-Barr virus induced receptor 2 (EBI2), a Gα_i-coupled G protein-coupled receptor, is a chemotactic receptor for B, T and dendritic cells (DC). Genetic studies have also implicated EBI2 as a regulator of an interferon regulatory factor 7 (IRF7)-driven inflammatory network (IDIN) associated with autoimmune diseases, although the corollary in primary type I IFN-producing cells has not been reported. Here we demonstrate that EBI2 negatively regulates type I IFN responses in plasmacytoid DC (pDCs) and CD11b⁺ myeloid cells. Activation of EBI2^−/− pDCs and CD11b⁺ cells with various TLR ligands induced elevated type I IFN production compared to wild-type cells. Moreover, in vivo challenge with endosomal TLR agonists or infection with lymphocytic choriomeningitis virus elicited more type I IFNs and proinflammatory cytokines in EBI2^−/− mice compared to normal mice. Elevated systemic cytokines occurred despite impaired ability of EBI2-deficient pDCs and CD11b⁺ cells to migrate from the blood to the spleen and peritoneal cavity under homeostatic conditions. As reported for other immune cells, pDC migration was dependent on the ligand for EBI2, 7α,25-dihydroxycholesterol. Consistent with a cell intrinsic role for EBI2, type I IFN-producing cells from EBI2-deficient mice expressed higher levels of IRF7 and IDIN genes. Together these data suggest a negative regulatory role for EBI2 in balancing TLR-mediated responses to foreign and to self nucleic acids that may precipitate autoimmunity.     

Vasoactive intestinal peptide downregulates proinflammatory TLRs while upregulating anti-inflammatory TLRs in the infected cornea

TLRs recognize microbial pathogens and trigger an immune response, but their regulation by neuropeptides, such as vasoactive intestinal peptide (VIP), during Pseudomonas aeruginosa corneal infection remains unexplored. Therefore, C57BL/6 (B6) mice were injected i.p. with VIP, and mRNA, protein, and immunostaining assays were performed. After VIP treatment, PCR array and real-time RT-PCR demonstrated that proinflammatory TLRs (conserved helix-loop-helix ubiquitous kinase, IRAK1, TLR1, TLR4, TLR6, TLR8, TLR9, and TNFR-associated factor 6) were downregulated, whereas anti-inflammatory TLRs (single Ig IL-1-related receptor [SIGIRR] and ST2) were upregulated. ELISA showed that VIP modestly downregulated phosphorylated inhibitor of NF-κB kinase subunit α but upregulated ST2 ~2-fold. SIGIRR was also upregulated, whereas TLR4 immunostaining was reduced in cornea; all confirmed the mRNA data. To determine whether VIP effects were cAMP dependent, mice were injected with small interfering RNA for type 7 adenylate cyclase (AC7), with or without VIP treatment. After silencing AC7, changes in mRNA levels of TLR1, TNFR-associated factor 6, and ST2 were seen and unchanged with addition of VIP, indicating that their regulation was cAMP dependent. In contrast, changes were seen in mRNA levels of conserved helix-loop-helix ubiquitous kinase, IRAK1, 2, TLR4, 9 and SIGIRR following AC7 silencing alone; these were modified by VIP addition, indicating their cAMP independence. In vitro studies assessed the effects of VIP on TLR regulation in macrophages and Langerhans cells. VIP downregulated mRNA expression of proinflammatory TLRs while upregulating anti-inflammatory TLRs in both cell types. Collectively, the data provide evidence that VIP downregulates proinflammatory TLRs and upregulates anti-inflammatory TLRs and that this regulation is both cAMP dependent and independent and involves immune cell types found in the infected cornea.