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Host pathogen-recognition receptors detect nucleic acid from invading viruses and initiate a series of signaling pathways that lead to the production of type I interferons (IFNs) and proinflammatory cytokines. Here, we found that a viral infection-induced deubiquitinase (DUB), ubiquitin-specific protease 25 (USP25) was required for host defense against RNA and DNA viruses. The activation of transcription factors IRF3 and NF-κB was impaired and the production of type I IFNs and proinflammatory cytokines was inhibited inUsp25−/− cells compared with the wild-type counterparts after RNA or DNA viruses infection. Consistently, USP25 deficient mice were more susceptible to H5N1 or HSV-1 infection compared with the wild-type mice. USP25 was associated with TRAF3 and TRAF6 after infection by RNA or DNA viruses and protected virus-induced proteasome-dependent or independent degradation of TRAF3 and TRAF6, respectively. Moreover, reconstitution of TRAF3 and TRAF6 intoUsp25−/− MEFs restored virus-triggered production of type I IFNs and proinflammatory cytokines. Our findings thus reveal a previously uncovered positive feedback regulation of innate immune responses against RNA and DNA viruses by USP25.

How IL-17 signaling is regulated remains poorly understood. Dong and colleagues identify the ubiquitin-specific protease USP25 as a negative regulator of IL-17-mediated signaling and inflammation. Interleukin 17 (IL-17) is important in infection and autoimmunity; how it signals remains poorly understood. In this study, we identified the ubiquitin-specific protease USP25 as a negative regulator of IL-17-mediated signaling and inflammation. Overexpression of USP25 inhibited IL-17-triggered signaling, whereas USP25 deficiency resulted in more phosphorylation of the inhibitor IκBα and kinase Jnk and higher expression of chemokines and cytokines, as well as a prolonged half-life for chemokine CXCL1–encoding mRNA after treatment with IL-17. Consistent with that, Usp25 −/− mice showed greater sensitivity to IL-17-dependent inflammation and autoimmunity in vivo . Mechanistically, stimulation with IL-17 induced the association of USP25 with the adaptors TRAF5 and TRAF6, and USP25 induced removal of Lys63-linked ubiquitination in TRAF5 and TRAF6 mediated by the adaptor Act1. Thus, our results demonstrate that USP25 is a deubiquitinating enzyme (DUB) that negatively regulates IL-17-triggered signaling.

Many microbes create and maintain pathogen-containing vacuoles (PVs) as an intracellular niche permissive for microbial growth and survival. The destruction of PVs by IFNγ-inducible guanylate binding protein (GBP) and immunity-related GTPase (IRG) host proteins is central to a successful immune response directed against numerous PV-resident pathogens. However, the mechanism bywhich IRGs and GBPs cooperatively detect and destroy PVs is unclear. We find that host cell priming with IFNγ prompts IRG-dependent association ofToxoplasma-andChlamydia-containing vacuoles with ubiquitin through regulated translocation of the E3 ubiquitin ligase tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6). This initial ubiquitin labeling elicits p62-mediated escort and deposition of GBPs to PVs, thereby conferring cell-autonomous immunity. Hypervirulent strains ofToxoplasma gondiievade this process via specific rhoptry protein kinases that inhibit IRG function, resulting in blockage of downstream PV ubiquitination and GBP delivery. Our results define a ubiquitin-centered mechanism by which host cells deliver GBPs to PVs and explain how hypervirulent parasites evade GBP-mediated immunity.

Sensors of the NLR family generally activate innate immunity. Ting et al ., however, demonstrate that the little-known NLRC3 negatively regulates Toll-like receptor signaling by altering ubiquitination of the signaling adaptor TRAF6. Several members of the NLR family of sensors activate innate immunity. In contrast, we found here that NLRC3 inhibited Toll-like receptor (TLR)-dependent activation of the transcription factor NF-κB by interacting with the TLR signaling adaptor TRAF6 to attenuate Lys63 (K63)-linked ubiquitination of TRAF6 and activation of NF-κB. We used bioinformatics to predict interactions between NLR and TRAF proteins, including interactions of TRAF with NLRC3. In vivo , macrophage expression of Nlrc3 mRNA was diminished by the administration of lipopolysaccharide (LPS) but was restored when cellular activation subsided. To assess biologic relevance, we generated Nlrc3 −/− mice. LPS-treated Nlrc3 −/− macrophages had more K63-ubiquitinated TRAF6, nuclear NF-κB and proinflammatory cytokines. Finally, LPS-treated Nlrc3 −/− mice had more signs of inflammation. Thus, signaling via NLRC3 and TLR constitutes a negative feedback loop. Furthermore, prevalent NLR-TRAF interactions suggest the formation of a 'TRAFasome' complex.

Toll-like receptors (TLRs) in innate immune cells are the prime cellular sensors for microbial components. TLR activation leads to the production of proinflammatory mediators and thus TLR signaling must be properly regulated by various mechanisms to maintain homeostasis. TLR4-ligand lipopolysaccharide (LPS)-induced tolerance or cross-tolerance is one such mechanism, and it plays an important role in innate immunity. Tolerance is established and sustained by the activity of the microRNA miR-146a, which is known to target key elements of the myeloid differentiation factor 88 (MyD88) signaling pathway, including IL-1 receptor-associated kinase (IRAK1), IRAK2 and tumor-necrosis factor (TNF) receptor-associated factor 6 (TRAF6). In this review, we comprehensively examine the TLR signaling involved in innate immunity, with special focus on LPS-induced tolerance. The function of TLR ligand-induced microRNAs, including miR-146a, miR-155 and miR-132, in regulating inflammatory mediators, and their impact on the immune system and human diseases, are discussed. Modulation of these microRNAs may affect TLR pathway activation and help to develop therapeutics against inflammatory diseases.

A critical role of the Toll-like receptor(TLR) and its downstream molecules, including IL-1 receptor-associated kinase 1(IRAK1) and tumor necrosis factor receptor- associated factor 6(TRAF6), in the pathogenesis of liver ischemia/reperfusion (I/R) injury has been documented. Recently a microRNA, miR-146a, was identified as a potent negative regulator of the TLR signaling pathway. In this study, we investigated the role of miR-146a to attenuate TLR signaling and liver I/R injury in vivo and in vitro. miR-146a was decreased in mice Kupffer cells following hepatic I/R, whereas IRAK1 and TRAF6 increased. Overexpression of miR-146a directly decreased IRAK1 and TRAF6 expression and attenuated the release of proinflammatory cytokines through the inactivation of NF-κB P65 in hypoxia/reoxygenation (H/R)-induced macrophages, RAW264.7 cells. Knockdown experiments demonstrated that IRAK1 and TRAF6 are two potential targets for reducing the release of proinflammatory cytokines. Moreover, co-culture assays indicated that miR-146a decreases the apoptosis of hepatocytes after H/R. In vivo administration of Ago-miR-146a, a stable version of miR-146a in vivo, protected against liver injury in mice after I/R via inactivation of the TLR signaling pathway. We conclude that miR-146a ameliorates liver ischemia/reperfusion injury in vivo and hypoxia/reoxygenation injury in vitro by directly suppressing IRAK1 and TRAF6.

Activation of the Toll‐like receptor (TLR) family of innate immune sensors stimulates multiple signal transduction pathways. Previous studies have suggested that TLR2, TLR4 and TLR9 induce serine phosphorylation of Signal Transducers and Activators of Transcription‐1 (STAT1) at residue 727 (S727), although its role in TLR signaling was unclear. We report here that STAT1 rapidly undergoes phosphorylation following TLR4 challenge with lipopolysaccharide (LPS) in a model of LPS hypersensitivity in vivo. Importantly, genetic ablation of STAT1 protected against LPS‐induced lethality suggesting that STAT1 may have a key role in TLR‐induced inflammation. We have found that multiple TLRs induce Ser727 phosphorylation of STAT1, which is dependent on MyD88 and TRIF signaling, but independent of interferon (IFN) regulatory factor (IRF)‐3, IRF7 and the IFN receptor complex, suggesting that activation is a direct TLR response rather than autocrine activation via IFN. Importantly, we found that STAT1 interacts with tumor necrosis factor (TNF) receptor‐associated factor‐6 (TRAF6), a key mediator of TLR signaling after TLR challenge and that following activation, STAT1 translocates to the nucleus. Critically, macrophages generated from mice in which the S727 residue was replaced with alanine (STAT1 S727A mice) display significantly reduced TNFα protein production, but not reduced interleukin‐6 or RANTES protein in response to multiple TLR challenges, as compared with wild‐type macrophages. These results clearly demonstrate cross‐talk between the TLR and JAK/STAT signaling pathways with direct recruitment of STAT1 by TRAF6 and that the direct activation of STAT1 by TLR signaling suggests a crucial role for STAT1 in TLR‐induced inflammation.

Many Toll-like receptors (TLRs) signal through TNF receptor-associated factor 6 (TRAF6) to activate innate immune responses. Here, we show that somatic nuclear autoantigenic sperm protein (sNASP) binds to TRAF6 to prevent TRAF6 autoubiquitination in unstimulated macrophages. Following LPS stimulation, a complex consisting of sNASP, TRAF6, IRAK4, and casein kinase 2 (CK2) is formed. CK2 phosphorylates sNASP at serine 158, allowing sNASP to dissociate from TRAF6. Free TRAF6 is then autoubiquitinated, followed by activation of downstream signaling pathways. In sNasp S158A knockin (S158A-KI) mice, LPS-treated macrophages could not phosphorylate sNASP, which remained bound to TRAF6. S158A-KI mice were more susceptible to sepsis due to a marked reduction in IL-1β, TNF-α, and IFN-γ production accompanied by an inability to clear bacteria and recruit leukocytes. Furthermore, phosphorylation-regulated release of sNASP from TRAF6 is observed following activation of TLR-1, -2, -4, -5, and -6. Thus, sNASP is a negative regulator of TLR signaling to modulate the innate immune response.

Myeloid-derived suppressor cells (MDSCs) are immature heterogeneous cells derived from the bone marrow and they are the major component of the tumor-induced immunosuppressive environment. Tumor necrosis factor receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase, catalyzes the polyubiquitination of target proteins. TRAF6 plays a critical role in modulating the immune system. However, whether TRAF6 is involved in the regulation of MDSCs has not been thoroughly elucidated to date. In this study, we found that the expression of TRAF6 in MDSCs derived from tumor tissue was significantly upregulated compared with that of MDSCs from spleen of tumor-bearing mice. Knockdown of TRAF6 remarkably attenuated the immunosuppressive effects of MDSCs. Mechanistically, TRAF6 might improve the immunosuppression of MDSCs by mediating K63-linked polyubiquitination and phosphorylation of signal transducer and activator of transcription 3 (STAT3). Additionally, it was discovered that the accumulation of MDSCs was abnormal in peripheral blood of lung cancer patients. TRAF6 and arginase 1 were highly expressed in MDSCs of patients with lung cancer. Taken together, our study demonstrated that TRAF6 participates in promoting the immunosuppressive function of MDSCs and provided a potential target for antitumor immunotherapy.

Retinoid X receptor-alpha (RXRα) is a potent regulator of inflammatory responses; however, its therapeutic potential for inflammatory cancer remains to be explored. We previously discovered that RXRα is abnormally cleaved in tumor cells and tissues, producing a truncated RXRα (tRXRα). Here, we show that transgenic expression of tRXRα in mice accelerates the development of colitis-associated colon cancer (CAC). The tumorigenic effect of tRXRα is primarily dependent on its expression in myeloid cells, which results in interleukin-6 (IL-6) induction and STAT3 activation. Mechanistic studies reveal an extensive interaction between tRXRα and TRAF6 in the cytoplasm of macrophages, leading to TRAF6 ubiquitination and subsequent activation of the NF-κB inflammatory pathway. K-80003, a tRXRα modulator derived from nonsteroidal anti-inflammatory drug (NSAID) sulindac, suppresses the growth of tRXRα-mediated colorectal tumor by inhibiting the NF-κB-IL-6-STAT3 signaling cascade. These results provide new insight into tRXRα action and identify a promising tRXRα ligand for treating CAC. The truncated form of retinoid X receptor-alpha is found in cancer. Here, the authors show that this mutant receptor promotes colorectal tumorigenesis by activation of NF-κB in myeloid cells, leading to subsequent IL-6-induced STAT3 activation in intestinal epithelial cells, which can be suppressed by ligand K-80003.