Inducible microRNA-155 feedback promotes type I IFN signaling in antiviral innate immunity by targeting suppressor of cytokine signaling 1

Effective recognition of viral infection and subsequent triggering of antiviral innate immune responses are essential for the host antiviral defense, which is tightly regulated by multiple regulators, including microRNAs. Our previous study showed that a panel of microRNAs, including miR-155, was markedly upregulated in macrophages upon vesicular stomatitis virus infection; however, the biological function of miR-155 during viral infection remains unknown. In this paper, we show that RNA virus infection induces miR-155 expression in macrophages via TLR/MyD88-independent but retinoic acid-inducible gene I/JNK/NF-κB-dependent pathway. And the inducible miR-155 feedback promotes type I IFN signaling, thus suppressing viral replication. Furthermore, suppressor of cytokine signaling 1 (SOCS1), a canonical negative regulator of type I IFN signaling, is targeted by miR-155 in macrophages, and SOCS1 knockdown mediates the enhancing effect of miR-155 on type I IFN-mediated antiviral response. Therefore, we demonstrate that inducible miR-155 feedback positively regulates host antiviral innate immune response by promoting type I IFN signaling via targeting SOCS1.     

Myxoma virus lacking the pyrin-like protein M013 is sensed in human myeloid cells by both NLRP3 and multiple Toll-like receptors, which independently activate the inflammasome and NF-κB innate response pathways

The myxoma virus (MYXV)-encoded pyrin domain-containing protein M013 coregulates inflammatory responses mediated by both the inflammasome and the NF-κB pathways. Infection of human THP-1 monocytic cells with a MYXV construct deleted for the M013 gene (vMyxM013-KO), but not the parental MYXV, activates both the inflammasome and NF-κB pathways and induces a spectrum of proinflammatory cytokines and chemokines, like interleukin-1β (IL-1β), tumor necrosis factor (TNF), IL-6, and monocyte chemoattractant protein 1. Here, we report that vMyxM013-KO virus-mediated activation of inflammasomes and secretion of IL-1β are dependent on the adaptor protein ASC, caspase-1, and NLRP3 receptor. However, vMyxM013-KO virus-mediated activation of NF-κB signaling, which induces TNF secretion, was independent of ASC, caspase-1, and either the NLRP3 or AIM2 inflammasome receptors. We also report that early synthesis of pro-IL-1β in response to vMyxM013-KO infection is dependent upon the components of the inflammasome complex. Activation of the NLRP3 inflammasome and secretion of IL-1β was also dependent on the release of cathepsin B and production of reactive oxygen species (ROS). By using small interfering RNA screening, we further demonstrated that, among the RIG-I-like receptors (RLRs) and Toll-like receptors (TLRs), only TLR2, TLR6, TLR7, and TLR9 contribute to the NF-κB-dependent secretion of TNF and the inflammasome-dependent secretion of IL-1β in response to vMyxM013-KO virus infection. Additionally, we demonstrate that early triggering of the mitogen-activated protein kinase pathway by vMyxM013-KO virus infection of THP-1 cells plays a critical common upstream role in the coordinate induction of both NF-κB and inflammasome pathways. We conclude that an additional cellular sensor(s)/receptor(s) in addition to the known RLRs/TLRs plays a role in the M013 knockout virus-induced activation of NF-κB pathway signaling, but the activation of inflammasomes entirely depends on sensing by the NLRP3 receptor in response to vMyxM013-KO infection of human myeloid cells.     

Dectin-1 interaction with Mycobacterium tuberculosis leads to enhanced IL-12p40 production by splenic dendritic cells

Dectin-1 is a fungal pattern recognition receptor that binds to beta-glucans and triggers cytokine production by facilitating interaction with TLR2 or by directly activating spleen tyrosine kinase (Syk). To assess the possible role of Dectin-1 in the innate response to mycobacteria, we used an in vitro system in which IL-12p40 production is measured in splenic dendritic cells (SpDC) following exposure to live Mycobacterium tuberculosis bacilli. Treatment of SpDC with laminarin or glucan phosphate, two molecules known to block Dectin-1-dependent activity, led to a reduction in M. tuberculosis-induced IL-12p40 as well as IL-12p70 production. Moreover, SpDC from Dectin-1-/- chimeric mice displayed reduced IL-12p40 production in response to mycobacteria when compared with Dectin-sufficient DC. Laminarin treatment also inhibited mycobacterial-induced IL-12p40 production in DC from TLR2-/- mice, arguing that Dectin-1 functions independently of TLR2 signaling in this system. Importantly, a Dectin-1 fusion protein was found to directly bind to live mycobacteria in a laminarin-inhibitable manner indicating the presence of ligands for the receptor in the bacterium and laminarin pretreatment resulted in reduced association of mycobacteria to SpDC. In additional experiments, mycobacterial stimulation was shown to be associated with increased phosphorylation of Syk and this response was inhibited by laminarin. Furthermore, pharmacologic inhibition of Syk reduced the M. tuberculosis-induced IL-12p40 response. Together, these findings support a role for Dectin-1 in promoting M. tuberculosis-induced IL-12p40 production by DC in which the receptor augments bacterial-host cell interaction and enhances the subsequent cytokine response through an unknown mechanism involving Syk signaling.     

Suppressors of cytokine signaling (SOCS)-1 and SOCS-3 are induced by CpG-DNA and modulate cytokine responses in APCs

During infection, the functional status of the innate immune system is tightly regulated. Although signals resulting in activation have been well characterized, counterregulative mechanisms are poorly understood. Suppressor of cytokine signaling (SOCS) proteins have been characterized as cytokine-inducible negative regulators of Janus kinase/STAT signaling in cells of hemopoietic origin. To analyze whether SOCS proteins could also be induced by pathogen-derived stimuli, we investigated the induction of SOCS-1 and SOCS-3 after triggering of macrophage cell lines, bone marrow-derived dendritic cells, and peritoneal macrophages with CpG-DNA. In this study, we show that CpG-DNA, but not GpC-DNA, induces expression of mRNA for SOCS-1 and SOCS-3 in vitro and in vivo. SOCS mRNA expression could be blocked by chloroquine and was independent of protein synthesis. Inhibitors of the mitogen-activated protein kinase pathway triggered by CpG-DNA were able to impede induction of SOCS mRNA. CpG-DNA triggered synthesis of SOCS proteins that could be detected by Western blotting. SOCS proteins were functional because they inhibited IFN-gamma as well as IL-6- and GM-CSF-induced phosphorylation of STAT proteins. Furthermore, IFN-gamma-induced up-regulation of MHC class II molecules was also prevented. The same effects could be achieved by overexpression of SOCS-1. Hence, the results indicate a substantial cross-talk between signal pathways within cells. They provide evidence for regulative mechanisms of Janus kinase/STAT signaling after triggering Toll-like receptor signal pathways.     

Gas6 negatively regulates the Staphylococcus aureus-induced inflammatory response via TLR signaling in the mouse mammary gland

Staphylococcus aureus (S. aureus)-induced mastitis is the most frequent, pathogenic, and prevalent infection of the mammary gland. The ligand growth arrest-specific 6 (Gas6) is a secretory protein that binds to and activates Tyro3, Axl, and MerTK receptors. This study explored the role of Gas6 in S. aureus-induced mastitis. Our results revealed that TLR receptors initiate the innate immune response in mammary gland tissues and epithelial cells and that introducing S. aureus activates TLR2 and TLR6 to drive multiple intracellular mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) pathways. Moreover, S. aureus also induces Gas6, which then activates the TAM receptor kinase pathway, which is related to the inhibition of TLR2- and TLR6-mediated inflammatory pathways through SOCS1 and SOCS3 induction. Gas6 absence alone was found to be involved in the downregulation of TAM receptor-mediated anti-inflammatory effects by inducing significantly prominent expression of TRAF6 and low protein and messenger RNA expression of SOCS1 and SOCS3. S. aureus-induced MAPK and NF-ĸB p65 phosphorylation were also dependent on Gas6, which negatively regulated the production of Pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) in S. aureus-treated mammary tissues and mammary epithelial cells. Our in vivo and in vitro study uncovered the Gas6-mediated negative feedback mechanism, which inhibits TLR2- and TLR6-mediated MAPK and NF-ĸB signaling by activating TAM receptor kinase (MerTK, Axl, and Tyro3) through the induction of SOCS1/SOCS3 proteins.     

The role of intermediary domain of MyD88 in cell activation and therapeutic inhibition of TLRs

Adaptor MyD88 has a pivotal role in TLR and IL-1R signaling and is involved in mediating excessive inflammation. MyD88 is composed of a death domain and a Toll/IL-1R domain connected by an intermediary domain (INT). The alternatively spliced form of MyD88 lacking the INT prevents signaling through MyD88-dependent TLRs. We designed a peptide from the INT and showed that it inhibits TLR4 activation by LPS when linked to a cell-penetrating peptide. As a new approach for the delivery of signaling-inhibitory peptides, INT peptide acylation also provided efficient cell translocation and inhibition of activation. We determined that INT peptide targets IL-1R-associated kinase 4. Furthermore, MyD88 mutant and molecular modeling refines the MyD88- IL-1R-associated kinase 4 interaction model based on the Myddosome structure. In addition to TLR4, INT peptide also inhibited TLR5, TLR2, TLR9, and IL-1R signaling but not TLR3, which uses Toll/IL-1R domain-containing adapter inducing IFN-β signaling adaptor. Inhibition of signaling in murine and human cells was observed by decreased NF-κB activation, cytokine mRNA synthesis, and phosphorylation of downstream kinases. In the endotoxemic mouse model, INT peptide suppressed production of inflammatory cytokines and improved survival, supporting therapeutic application of INT peptides for the suppression of inflammatory conditions mediated by MyD88.     

TLR2 hypersensitivity of astrocytes as functional consequence of previous inflammatory episodes

Precedent inflammatory episodes may drastically modify the function and reactivity of cells. We investigated whether priming of astrocytes by microglia-derived cytokines alters their subsequent reaction to pathogen-associated danger signals not recognized in the quiescent state. Resting primary murine astrocytes expressed little TLR2, and neither the TLR2/6 ligand fibroblast-stimulating lipopeptide-1 (FSL1) nor the TLR1/2 ligand Pam(3)CysSK(4) (P3C) triggered NF-κB translocation or IL-6 release. We made use of single-cell detection of NF-κB translocation as easily detectable and sharply regulated upstream indicator of an inflammatory response or of c-Jun phosphorylation to measure restimulation events in astrocytes under varying conditions. Cells prestimulated with IL-1β, with a TLR3 ligand, with a complete cytokine mix consisting of TNF-α, IL-1β, and IFN-γ, or with media conditioned by activated microglia responded strongly to FSL1 or P3C stimulation, whereas the sensitivity of the NF-κB response to other pattern recognition receptors was unchanged. This sensitization to TLR2 ligands was associated with an initial upregulation of TLR2, displayed a "memory" window of several days, and was largely independent of the length of prestimulation. The altered signaling led to altered function, as FSL1 or P3C triggered the release of IL-6, CCL-20, and CXCL-2 in primed cells, but not in resting astrocytes. These data confirmed the hypothesis that astrocytes exposed to activated microglia assume a different functional phenotype involving longer term TLR2 responsiveness, even after the initial stimulation by inflammatory mediators has ended.     

Discovery of substituted 4-aminoquinazolines as selective Toll-like receptor 4 ligands

The Toll-like receptors (TLRs) are critical components of the innate immune system that regulate immune recognition in part through NF-κB activation. A human cell-based high throughput screen (HTS) revealed substituted 4-aminoquinazolines to be small molecular weight activators of NF-κB. The most potent hit compound predominantly stimulated through the human TLR4/MD2 complex, and had less activity with the mouse TLR4/MD2. There was no activity with other TLRs and the TLR4 activation was MD-2 dependent and CD14 independent. Synthetic modifications of the quinazoline scaffold at the 2 and 4 positions revealed trends in structure–activity relationships with respect to TLR dependent production of the NF-κB associated cytokine IL-8 in human peripheral blood mononuclear cells, as well as IL-6 in mouse antigen presenting cells. Furthermore, the hit compound in this series also activated the interferon signaling pathway resulting in type I interferon production. Substitution at the O-phenyl moiety with groups such as bromine, chlorine and methyl resulted in enhanced immunological activity. Computational studies indicated that the 4-aminoquinazoline compounds bind primarily to human MD-2 in the TLR4/MD-2 complex. These small molecules, which preferentially stimulate human rather than mouse innate immune cells, may be useful as adjuvants or immunotherapeutic agents.     

CD300F blocks both MyD88 and TRIF-mediated TLR signaling through activation of Src homology region 2 domain-containing phosphatase 1

CD300F is known to exhibit inhibitory activity in myeloid cells through its intracellular ITIM. To investigate the effect of CD300F stimulation on TLR signaling, the human acute monocytic leukemia cell line THP-1 was treated with CD300F-specific mAbs or two synthetic peptides that represented the ITIM-like domains of CD300F. Treatment with these agents blocked TLR2-, 3-, 4-, and 9-mediated expression of proinflammatory mediators such as IL-8 and matrix metalloproteinase-9. The luciferase reporter assay in 293T cells and Western blot analysis of THP-1 cells revealed that these inhibitory actions were effective in pathways involving MyD88 and/or TRIF of TLR signaling and associated with marked suppression of IκB kinase activation, phosphorylation/degradation of IκB, and subsequent activation of NF-κB. Use of specific inhibitors and immunoprecipitation analysis further indicated that the inhibitory effects were mediated by Src homology 2 domain-containing phosphatase-1, a protein tyrosine phosphatase with inhibitory activity in hematopoietic cells. These data indicate that CD300F is an active regulator of TLR-mediated macrophage activation through its association with Src homology 2 domain-containing phosphatase-1 and that the synthetic peptides can be applied for the regulation of immune responses that are induced by TLRs.     

Interleukin-10 activates Toll-like receptor 4 and requires MyD88 for cardiomyocyte survival

Toll-like receptors (TLRs) are important in a variety of inflammatory diseases including acute cardiac disorders. TLR4 innate signaling regulates the synthesis of anti-inflammatory cytokine, interleukin-10 (IL-10) upon TLR4 agonists’ re-stimulation. Anti-apoptotic action of IL-10 in cardiac dysfunction is generally accepted but its protective mechanism through TLR4 is not yet understood. We studied the effect of IL-10 in the activation of TLR4 downstream signals leading to cardiomyocytes survival. IL-10 caused a significant increase in the expression of CD14, MyD88 and TLR4. TLR4 activation led to the translocation of the interferon regulatory factor 3 (IRF3) into the nucleus. Phosphorylation of IRF3 enhanced mRNA synthesis for IL-1β but not TNF-α and was elevated even after removal of IL-10 stimulation. Furthermore, degradation of inhibitory kappa B (IκB) kinase (Ikk) suggested that IκBβ was the main activating kinase for IRF3-regulated NF-κB activation and phosphorylation of p65. Phosphorylated NF-κB p65 was translocated into the nucleus. Concomitantly, an increase in Bcl-xL activity inhibited Bax and the proteolytic activity of caspase 3 as well as a decrease in PARP cleavage. An inhibition of MyD88, modulated the above listed responses to IL-10 as there was a decrease in TLR4 and IRF3 and an increase in TNF-α mRNA. This was associated with a decrease in NF-κB p65, Bcl-xL mRNA and protein levels as well as there was an activation of Bax and PARP cleavage independent of caspase 3 activation. These data in cardiomyocytes suggest that IL-10 induced anti-apoptotic signaling involves upregulation of TLR4 through MyD88 activation.     

Bacterial lipoprotein-induced tolerance is reversed by overexpression of IRAK-1

Tolerance to bacterial cell wall components including bacterial lipoprotein (BLP) represents an essential regulatory mechanism during bacterial infection. Reduced Toll-like receptor 2 (TLR2) and IL-1 receptor-associated kinase 1 (IRAK-1) expression is a characteristic of the downregulated TLR signaling pathway observed in BLP-tolerised cells. In this study, we attempted to clarify whether TLR2 and/or IRAK-1 are the key molecules responsible for BLP-induced tolerance. Transfection of HEK293 cells and THP-1 cells with the plasmid encoding TLR2 affected neither BLP tolerisation-induced NF-κB deactivation nor BLP tolerisation-attenuated pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α) production, indicating that BLP tolerance develops despite overexpression of TLR2 in these cells. In contrast, overexpression of IRAK-1 reversed BLP-induced tolerance, as transfection of IRAK-1 expressing vector resulted in a dose-dependent NF-κB activation and TNF-α release in BLP-tolerised cells. Furthermore, BLP-tolerised cells exhibited markedly repressed NF-κB p65 phosphorylation and impaired binding of p65 to several pro-inflammatory cytokine gene promoters including TNF-α and interleukin-6 (IL-6). Overexpression of IRAK-1 restored the nuclear transactivation of p65 at both TNF-α and IL-6 promoters. These results indicate a crucial role for IRAK-1 in BLP-induced tolerance, and suggest IRAK-1 as a potential target for manipulation of the TLR-mediated inflammatory response during microbial sepsis.     

The molecular mechanisms of the effect of Dexamethasone and Cyclosporin A on TLR4 /NF-κB signaling pathway activation in oral lichen planus

Toll-like receptors (TLRs) and the nuclear factor-kappa B (NF-κB) signaling transduction pathway play important roles in the pathogenesis of several chronic inflammatory diseases, but its function in oral lichen planus (OLP) remains unclear. In this study, we examined the expression of TLR4 and NF-κB-p65 and inflammatory cytokines TNF-α and IL-1β by immunohistochemistry in OLP tissues, and found that TLR4 and NF-κB-p65 were significantly upregulated in OLP compared to normal oral mucosa (P<0.05). We used keratinocytes HaCaT stimulated with lipopolysaccharide (LPS) to simulate the local OLP immune environment to some extent. RT-PCR and immunoblotting analyses showed significant activation of TLR4 and NF-κB-p65 in the circumstance of LPS-induced inflammatory response. The high expression of TLR4 and NF-κB-p65 are correlated with expression of cytokines TNF-α and IL-1β (P<0.05). We further showed that NF-κB could act as an anti-apoptotic molecule in OLP. We conclude that TLR4 and the NF-κB signaling pathway may interact with the perpetuation of OLP. Steroids and cyclosporine are effective in the treatment of symptomatic OLP. However, there was some weak evidence for the mechanism over Dexamethasone (DeX) and Cyclosporine A (CsA) for the palliation of symptomatic OLP. In the present study, we found that Dexamethasone and Cyclosporine A negatively regulated NF-κB signaling pathway under LPS simulation in HaCaT cells by inhibiting TLR4 expression, on the other hand, Cyclosporine A could inhibit HaCaT cell proliferation by the induction of the apoptosis of HaCaT cells to protect OLP from the destruction of epidermal cells effectively.     

CpG oligonucleotides induce an immune response of odontoblasts through the TLR9, MyD88 and NF-κB pathways

Odontoblasts are the first-line defense cells against invading microorganisms. Toll-like receptors (TLRs) play a crucial role in innate immunity, and TLR9 is involved in the recognition of microbial DNA. This study aimed to investigate whether odontoblasts can respond to CpG DNA and to determine the intracellular signaling pathways triggered by CpG DNA. We found that the mouse odontoblast-like cell line MDPC-23 constitutively expressed TLR9. Exposure to CpG ODN induced a potent proinflammatory response based on an increase of IL-6 and TNF-α expression. Pretreatment with an inhibitory MyD88 peptide or a specific inhibitor for TLR9, NF-κB or IκBα markedly inhibited CpG ODN-induced IL-6 and TNF-α expression. Moreover, the CpG ODN-mediated increase of κB-luciferase activity in MDPC-23 cells was suppressed by the overexpression of dominant negative mutants of TLR9, MyD88 and IκBα, but not by the dominant negative mutant of TLR4. This result suggests a possible role for the CpG DNA-mediated immune response in odontoblasts and indicates that TLR9, MyD88 and NF-κB are involved in this process.     

Neu1 sialidase and matrix metalloproteinase-9 cross-talk regulates nucleic acid-induced endosomal TOLL-like receptor-7 and -9 activation,cellular signaling and pro-inflammatory responses

The precise mechanism(s) by which intracellular TOLL-like receptors (TLRs) become activated by their ligands remains unclear. Here, we report a molecular organizational G-protein coupled receptor (GPCR) signaling platform to potentiate a novel mammalian neuraminidase-1 (Neu1) and matrix metalloproteinase-9 (MMP-9) cross-talk in alliance with neuromedin B GPCR, all of which form a tripartite complex with TLR-7 and -9. siRNA silencing Neu1, MMP-9 and neuromedin-B GPCR in RAW-blue macrophage cells significantly reduced TLR7 imiquimod- and TLR9 ODN1826-induced NF-κB (NF-κB-pSer⁵³⁶) activity. Tamiflu, specific MMP-9 inhibitor, neuromedin B receptor specific antagonist BIM23127, and the selective inhibitor of whole heterotrimeric G-protein complex BIM-46174 significantly block nucleic acid-induced TLR-7 and -9 MyD88 recruitment, NF-κB activation and proinflammatory TNFα and MCP-1 cytokine responses. For the first time, Neu1 clearly plays a central role in mediating nucleic acid-induced intracellular TLR activation, and the interactions involving NMBR–MMP9–Neu1 cross-talk constitute a novel intracellular TLR signaling platform that is essential for NF-κB activation and pro-inflammatory responses.     

Toll-like receptor downstream signaling

The family of Toll-like receptors (TLRs) senses conserved structures found in a broad range of pathogens, causing innate immune responses that include the production of inflammatory cytokines, chemokines and interferons. The signal transduction is initiated from the Toll/interleukin-1 receptor (TIR) domain of TLRs after pathogen recognition. Almost all TLRs use a TIR-containing adapter MyD88 to activate a common signaling pathway that results in the activation of NF-κB to express cytokine genes relevant to inflammation. Recently, three further TIR-containing adapters have been identified and shown to selectively interact with several TLRs. In particular, activation of the TRIF-dependent pathway confers antiviral responses by inducing anti-viral genes including that encoding interferon-β. Taken together, these results indicate that the interaction between individual TLRs and the different combinations of adapters directs appropriate responses against distinct pathogens.