Wei Zhao

E3 Ubiquitin Ligase Tripartite Motif 38 Negatively Regulates TLR-Mediated Immune Responses by Proteasomal Degradation of TNF Receptor-Associated Factor 6 in Macrophages

Activation of TLR signaling in the innate immune cells is critical for the elimination of invading microorganisms. However, uncontrolled activation may lead to autoimmune and inflammatory diseases. In this article, we report the identification of tripartite motif (TRIM) 38 as a negative feedback regulator in TLR signaling by targeting TNFR-associated factor 6 (TRAF6). TRIM38 was induced by TLR stimulation in an NF-κB–dependent manner in macrophages. Knockdown of TRIM38 expression by small interfering RNA resulted in augmented activation of NF-κB and MAPKs, and enhanced expression of proinflammatory cytokines, whereas overexpression of TRIM38 has an opposite effect. As an E3 ligase, TRIM38 bound to TRAF6 and promoted K48-linked polyubiquitination, which led to the proteasomal degradation of TRAF6. Consistently, knockdown of TRIM38 expression resulted in higher protein level of TRAF6 in primary macrophages. Our findings defined a novel function for TRIM38 to prevent excessive TLR-induced inflammatory responses through proteasomal degradation of TRAF6.

TRAF-interacting protein (TRIP) negatively regulates IFN-β production and antiviral response by promoting proteasomal degradation of TANK-binding kinase 1.

TRAF-interacting protein (TRIP) negatively regulates TLR3/4- and RIG-I–induced IFN-β signaling by promoting K48-linked ubiquitination and proteasomal degradation of TBK1.

Negative regulation of TBK1‐mediated antiviral immunity

TANK‐binding kinase 1 (TBK1) plays pivotal roles in antiviral innate immunity. TBK1 mediates the activation of interferon regulatory factor (IRF) 3, leading to the induction of type I IFNs (IFN‐α/β) following viral infections. TBK1 must be tightly regulated to effectively control viral infections and maintain immune homeostasis. TBK1 activity can be regulated in a variety of ways, such as phosphorylation, ubiquitination, kinase activity modulation and prevention of functional TBK1‐containing complexes formation. Furthermore, multiple viruses have evolved elaborate strategies to circumvent IFN responses by targeting TBK1. Here we provide an overview of TBK1 in antiviral immunity and recent developments on the regulation of TBK1 activity.

USP4 positively regulates RIG-I-mediated antiviral response through deubiquitination and stabilization of RIG-I

ABSTRACT Protein ubiquitination plays an essential role in the regulation of retinoic acid-inducible gene I (RIG-I) activation and the antiviral immune response. However, the function of the opposite process of deubiquitination in RIG-I activation remains elusive. In this study, we have identified the deubiquitinating enzyme ubiquitin-specific protease 4 (USP4) as a new regulator for RIG-I activation through deubiquitination and stabilization of RIG-I. USP4 expression was attenuated after virus-induced RIG-I activation. Overexpression of USP4 significantly enhanced RIG-I protein expression and RIG-I-triggered beta interferon (IFN-β) signaling and, at the same time, inhibited vesicular stomatitis virus (VSV) replication. Small interfering RNA (siRNA) knockdown of USP4 expression had an opposite effect. Furthermore, USP4 was found to interact with RIG-I and remove K48-linked polyubiquitination chains from RIG-I. Therefore, we identified USP4 as a new positive regulator for RIG-I that acts through deubiquitinating K48-linked ubiquitin chains and stabilizing RIG-I.

Peroxisome Proliferator-activated Receptor γ Negatively Regulates IFN-β Production in Toll-like Receptor (TLR) 3- and TLR4-stimulated Macrophages by Preventing Interferon Regulatory Factor 3 Binding to the IFN-β Promoter

Toll-like receptors 3 and 4 utilize adaptor TRIF to activate interferon regulatory factor 3 (IRF3), resulting in IFN-β production to mediate anti-viral and bacterial infection. Peroxisome proliferator-activated receptor (PPAR)-γ is a ligand-activated transcription factor expressed in various immune cells and acts as a transcriptional repressor to inhibit the transcription of many proinflammatory cytokines. But, the function of PPAR-γ in TLR3- and -4-mediated IFN-β production is not well elucidated. Here, we have analyzed the effect of the PPAR-γ agonists on IFN-β production in peritoneal primary macrophages in response to LPS and poly(I:C). PPAR-γ agonists inhibited LPS and poly(I:C)-induced IFN-β transcription and secretion. siRNA knockdown of PPAR-γ expression and transfection of PPAR-γ expression plasmid demonstrated that PPAR-γ agonist inhibits IFN-β production in a PPAR-γ-dependent manner. The ability of the PPAR-γ agonist to inhibit IFN-β production was confirmed in vivo as mice treated with troglitazone exhibited decreased levels of IFN-β upon LPS and poly(I:C) challenge. Chromatin immunoprecipitation (CHIP) assay and electrophoretic mobility shift assay (EMSA) demonstrated that troglitazone treatment impaired IRF3 binding to the IFN-β promoter. Furthermore, troglitazone could inhibit LPS and poly(I:C)-induced STAT1 phosphorylation and subsequent ISRE activation. These results demonstrate that PPAR-γ negatively regulates IFN-β production in TLR3- and 4-stimulated macrophages by preventing IRF3 binding to the IFN-β promoter.

Aryl hydrocarbon receptor negatively regulates NLRP3 inflammasome activity by inhibiting NLRP3 transcription

NLRP3 inflammasome is a multi-protein complex, which plays crucial roles in host defense against pathogens. The NLRP3 protein level is considered rate limiting for the activation of the inflammasome, thus its expression must be tightly controlled to maintain immune homeostasis. However, the molecular mechanisms that modulate NLRP3 expression, especially at the transcriptional level, remain largely unknown. In the present study, we show that aryl hydrocarbon receptor (AhR) activation inhibits NLRP3 expression, caspase-1 activation and subsequent IL-1β secretion in peritoneal macrophages, whereas siRNA knockdown of AhR has opposite effects. AhR could bind to the xenobiotic response element (XRE) in the NLRP3 promoter and inhibit NLRP3 transcription. Furthermore, AhR activation suppresses Alum-induced peritonitis in vivo. Therefore, we identified AhR as a negative regulator of NLRP3 inflammasome activity by inhibiting the transcription of NLRP3 and suggested AhR as a potential target for the intervention of diseases with uncontrolled inflammasome activation.

Tripartite Motif-Containing Protein 38 Negatively Regulates TLR3/4- and RIG-I–Mediated IFN-β Production and Antiviral Response by Targeting NAP1

Recognition of RNA virus through TLR and RIG-I–like receptor results in rapid expression of type I IFNs, which play an essential role in host antiviral responses. However, the mechanisms to terminate the production of type I IFNs are not well defined. In the current study, we identified a member of the tripartite motif (TRIM) family, TRIM38, as a negative regulator in TLR3/4- and RIG-I–mediated IFN-β signaling. Knockdown of TRIM38 expression by small interfering RNA resulted in augmented activation of IFN regulatory factor 3 and enhanced expression of IFN-β, whereas overexpression of TRIM38 had opposite effects. Coimmunoprecipitation and colocalization experiments demonstrated that TRIM38 interacted with NF-κB–activating kinase-associated protein 1 (NAP1), which is required for TLR-induced IFN regulatory factor 3 activation and IFN-β production. As an E3 ligase, TRIM38 promoted K48-linked polyubiquitination and proteasomal degradation of NAP1. Thus, knockdown of TRIM38 expression resulted in higher protein level of NAP1 in primary macrophages. Consistent with the inhibitory roles in TLR3/4- and RIG-I–mediated IFN-β signaling, knockdown of TRIM38 significantly inhibited the replication of vesicular stomatitis virus. Overexpression of TRIM38 resulted in enhanced replication of vesicular stomatitis virus. Therefore, our results demonstrate that TRIM38 is a negative regulator for TLR and RIG-I–mediated IFN-β production by targeting NAP1 for ubiquitination and subsequent proteasome-mediated degradation.

Differential Expression of Intracellular and Secreted Osteopontin Isoforms by Murine Macrophages in Response to Toll-like Receptor Agonists

Osteopontin (OPN), expressed by various immune cells, modulates both innate and adaptive immune responses. Different immune cells have shown differential expression of the two isoforms of OPN: secreted form of OPN (sOPN) and intracellular form of OPN (iOPN). However, the molecular mechanisms that control opn gene expression and the OPN isoforms produced by immune cells remain largely unknown. In this study, we demonstrate that OPN mRNA and protein expression are significantly up-regulated upon stimulation with TLR agonists in macrophages. Interestingly, we find that macrophages constitutively express the secreted form of OPN (sOPN), while the intracellular form of OPN (iOPN) is induced following the stimulation with TLR agonists. Phosphoinositide 3-kinase (PI3K), extracellular signal-regulated kinase (ERK), and c-Jun NH2-terminal kinase (JNK) that are activated by LPS stimulation were shown to upregulate OPN expression. In addition, chromatin immunoprecipitation (CHIP) assays showed that AP-1 binds to the proximal AP-1 site in the OPN promoter from LPS-stimulated macrophages. Mutation of the AP-1 site in OPN promoter completely ablates LPS-induced OPN promoter activation. Knockdown of c-Jun and c-Fos expression by small interfering RNA (siRNA) significantly decreases LPS-induced OPN expression. Stable cell lines with iOPN overexpression and knockdown showed that TLR-induced iOPN expression is a negative regulator for interferon-β (IFN-β) production. Our findings provide new insight into the transcriptional regulation of opn gene and further clarify the isoforms and functions of OPN produced by macrophages.

NF-κB– and AP-1–Mediated DNA Looping Regulates Osteopontin Transcription in Endotoxin-Stimulated Murine Macrophages

Osteopontin (OPN) is expressed by various immune cells and modulates both innate and adaptive immune responses. However, the molecular mechanisms that control opn gene expression, especially at the chromatin level, remain largely unknown. We have previously demonstrated many specific cis- and trans-regulatory elements that determine the extent of endotoxin (LPS)-mediated induction of OPN synthesis in murine macrophages. In the present study, we confirm that NF-κB also plays an important role in the setting of LPS-stimulated OPN expression through binding to a distal regulatory element. Importantly, we demonstrate that LPS stimulates chromosomal loops in the OPN promoter between NF-κB binding site and AP-1 binding site using chromosome conformation capture technology. The crucial role of NF-κB and AP-1 in LPS-stimulated DNA looping was confirmed, as small interfering RNA knock-down of NF-κB p65 and AP-1 c-Jun exhibited decreased levels of DNA looping. Furthermore, we demonstrate that p300 can form a complex with NF-κB and AP-1 and is involved in DNA looping and LPS-induced OPN expression. Therefore, we have identified an essential mechanism to remodel the local chromatin structures and spatial conformations to regulate LPS-induced OPN expression.

The E3 ubiquitin ligase TRIM31 attenuates NLRP3 inflammasome activation by promoting proteasomal degradation of NLRP3

The NLRP3 inflammasome has a fundamental role in host defence against microbial pathogens and its deregulation may cause diverse inflammatory diseases. NLRP3 protein expression is a rate-limiting step for inflammasome activation, thus its expression must be tightly controlled to maintain immune homeostasis and avoid detrimental effects. However, how NLRP3 expression is regulated remains largely unknown. In this study, we identify E3 ubiquitin ligase TRIM31 as a feedback suppressor of NLRP3 inflammasome. TRIM31 directly binds to NLRP3, promotes K48-linked polyubiquitination and proteasomal degradation of NLRP3. Consequently, TRIM31 deficiency enhances NLRP3 inflammasome activation and aggravates alum-induced peritonitis in vivo. Furthermore, TRIM31 deficiency attenuates the severity of dextran sodium sulfate (DSS)-induced colitis, an inflammatory bowel diseases model in which NLRP3 possesses protective roles. Thus, our research describes a mechanism by which TRIM31 limits NLRP3 inflammasome activity under physiological conditions and suggests TRIM31 as a potential therapeutic target for the intervention of NLRP3 inflammasome related diseases.