Dopamine Uses the DRD5-ARRB2-PP2A Signaling Axis to Block the TRAF6-Mediated NF-κB Pathway and Suppress Systemic Inflammation.

The functional relevance and mechanistic basis of the effects of the neurotransmitter dopamine (DA) on inflammation remain unclear. Here we reveal that DA inhibited TLR2-induced NF-κB activation and inflammation via the DRD5 receptor in macrophages. We found that the DRD5 receptor, via the EFD and IYX(X)I/L motifs in its CT and IC3 loop, respectively, can directly recruit TRAF6 and its negative regulator ARRB2 to form a multi-protein complex also containing downstream signaling proteins, such as TAK1, IKKs, and PP2A, that impairs TRAF6-mediated activation of NF-κB and expression of pro-inflammatory genes. Furthermore, the DA-DRD5-ARRB2-PP2A signaling axis can prevent S. aureus-induced inflammation and protect mice against S. aureus-induced sepsis and meningitis after DA treatment. Collectively, these findings provide the first demonstration of DA-DRD5 signaling acting to control inflammation and a detailed delineation of the underlying mechanism and identify the DRD5-ARRB2-PP2A axis as a potential target for future therapy of inflammation-associated diseases such as meningitis and sepsis.

Pellino3 ubiquitinates RIP2 and mediates Nod2-induced signaling and protective effects in colitis.

Mutations that result in loss of function of Nod2, an intracellular receptor for bacterial peptidoglycan, are associated with Crohn's disease. Here we found that the E3 ubiquitin ligase Pellino3 was an important mediator in the Nod2 signaling pathway. Pellino3-deficient mice had less induction of cytokines after engagement of Nod2 and had exacerbated disease in various experimental models of colitis. Furthermore, expression of Pellino3 was lower in the colons of patients with Crohn's disease. Pellino3 directly bound to the kinase RIP2 and catalyzed its ubiquitination. Loss of Pellino3 led to attenuation of Nod2-induced ubiquitination of RIP2 and less activation of the transcription factor NF-κB and mitogen-activated protein kinases (MAPKs). Our findings identify RIP2 as a substrate for Pellino3 and Pellino3 as an important mediator in the Nod2 pathway and regulator of intestinal inflammation.

The Gasdermin D N-terminal fragment acts as a negative feedback system to inhibit inflammasome-mediated activation of Caspase-1/11.

Inflammatory pathways usually utilize negative feedback regulatory systems to prevent tissue damage arising from excessive inflammatory response. Whether such negative feedback mechanisms exist in inflammasome activation remains unknown. Gasdermin D (GSDMD) is the pyroptosis executioner of downstream inflammasome signaling. Here, we found that GSDMD, after its cleavage by caspase-1/11, utilizes its RFWK motif in the N-terminal ß1-ß2 loop to inhibit the activation of caspase-1/11 and downstream inflammation in a negative feedback manner. Furthermore, an RFWK motif-based peptide inhibitor can inhibit caspase-1/11 activation and its downstream substrates GSDMD and interleukin-1ß cleavage, as well as lipopolysaccharide-induced sepsis in mice. Collectively, these findings provide a demonstration of the N-terminal fragment of GSDMD as a negative feedback regulator controlling inflammasome activation and a detailed delineation of the underlying inhibitory mechanism.

Pellino3 targets the IRF7 pathway and facilitates autoregulation of TLR3- and viral-induced expression of type I interferons.

Toll-like receptors (TLRs) sense pathogen-associated molecules and respond by inducing cytokines and type I interferon. Here we show that genetic ablation of the E3 ubiquitin ligase Pellino3 augmented the expression of type I interferon but not of proinflammatory cytokines in response to TLR3 activation. Pellino3-deficient mice had greater resistance against the pathogenic and lethal effects of encephalomyocarditis virus (EMCV). TLR3 signaling induced Pellino3, which in turn interacted with and ubiquitinated TRAF6. This modification suppressed the ability of TRAF6 to interact with and activate IRF7, resulting in downregulation of type I interferon expression. Our findings highlight a new physiological role for Pellino3 and define a new autoregulatory network for controlling type I interferon expression.

Peli1 (rel)ieves autoimmunity.

T cell tolerance is essential to the prevention of autoimmunity. The ubiquitin E3 ligase Peli1 acts as a negative regulator of T cell activation and contributes to the maintenance of self-tolerance.

The E3 ubiquitin ligase Pellino3 protects against obesity-induced inflammation and insulin resistance.

Diet-induced obesity can induce low-level inflammation and insulin resistance. Interleukin-1ß (IL-1ß) is one of the key proinflammatory cytokines that contributes to the generation of insulin resistance and diabetes, but the mechanisms that regulate obesity-driven inflammation are ill defined. Here we found reduced expression of the E3 ubiquitin ligase Pellino3 in human abdominal adipose tissue from obese subjects and in adipose tissue of mice fed a high-fat diet and showing signs of insulin resistance. Pellino3-deficient mice demonstrated exacerbated high-fat-diet-induced inflammation, IL-1ß expression, and insulin resistance. Mechanistically, Pellino3 negatively regulated TNF receptor associated 6 (TRAF6)-mediated ubiquitination and stabilization of hypoxia-inducible factor 1α (HIF1α), resulting in reduced HIF1α-induced expression of IL-1ß. Our studies identify a regulatory mechanism controlling diet-induced insulin resistance by highlighting a critical role for Pellino3 in regulating IL-1ß expression with implications for diseases like type 2 diabetes.

Dendritic Cell-Specific Role for Pellino2 as a Mediator of TLR9 Signaling Pathway.

Ubiquitination regulates immune signaling, and multiple E3 ubiquitin ligases have been studied in the context of their role in immunity. Despite this progress, the physiological roles of the Pellino E3 ubiquitin ligases, especially Pellino2, in immune regulation remain largely unknown. Accordingly, this study aimed to elucidate the role of Pellino2 in murine dendritic cells (DCs). In this study, we reveal a critical role of Pellino2 in regulation of the proinflammatory response following TLR9 stimulation. Pellino2-deficient murine DCs show impaired secretion of IL-6 and IL-12. Loss of Pellino2 does not affect TLR9-induced activation of NF-κB or MAPKs, pathways that drive expression of IL-6 and IL-12. Furthermore, DCs from Pellino2-deficient mice show impaired production of type I IFN following endosomal TLR9 activation, and it partly mediates a feed-forward loop of IFN-ß that promotes IL-12 production in DCs. We also observe that Pellino2 in murine DCs is downregulated following TLR9 stimulation, and its overexpression induces upregulation of both IFN-ß and IL-12, demonstrating the sufficiency of Pellino2 in driving these responses. This suggests that Pellino2 is critical for executing TLR9 signaling, with its expression being tightly regulated to prevent excessive inflammatory response. Overall, this study highlights a (to our knowledge) novel role for Pellino2 in regulating DC functions and further supports important roles for Pellino proteins in mediating and controlling immunity.

Obesity Reduces mTORC1 Activity in Mucosal-Associated Invariant T Cells, Driving Defective Metabolic and Functional Responses.

Obesity underpins the development of numerous chronic diseases, such as type II diabetes mellitus. It is well established that obesity negatively alters immune cell frequencies and functions. Mucosal-associated invariant T (MAIT) cells are a population of innate T cells, which we have previously reported are dysregulated in obesity, with altered circulating and adipose tissue frequencies and a reduction in their IFN-γ production, which is a critical effector function of MAIT cells in host defense. Hence, there is increased urgency to characterize the key molecular mechanisms that drive MAIT cell effector functions and to identify those which are impaired in the obesity setting. In this study, we found that MAIT cells significantly upregulate their rates of glycolysis upon activation in an mTORC1-dependent manner, and this is essential for MAIT cell IFN-γ production. Furthermore, we show that mTORC1 activation is dependent on amino acid transport via SLC7A5. In obese patients, using RNA sequencing, Seahorse analysis, and a series of in vitro experiments, we demonstrate that MAIT cells isolated from obese adults display defective glycolytic metabolism, mTORC1 signaling, and SLC7A5 aa transport. Collectively, our data detail the intrinsic metabolic pathways controlling MAIT cell cytokine production and highlight mTORC1 as an important metabolic regulator that is impaired in obesity, leading to altered MAIT cell responses.

Molecular and physiological roles of Pellino E3 ubiquitin ligases in immunity.

The sensing of foreign agents by the innate and adaptive immune system triggers complex signal transduction cascades that culminate in expression of gene patterns that facilitate host protection from the invading agent. Post-translational modification of intracellular signaling proteins in these pathways is a key regulatory mechanism with ubiquitination being one of the important processes that controls levels and activities of signaling molecules. E3 ubiquitin ligases are the determining enzymes in dictating the ubiquitination status of individual proteins. Among these hundred E3 ubiquitin ligases are a family of Pellino proteins that are emerging to be important players in immunity and beyond. Herein, we review the roles of the Pellino E3 ubiquitin ligases in innate and adaptive immunity. We discuss their early discovery and characterization and how this has been aided by the highly conserved nature of innate immune signaling across evolution. We describe the molecular roles of Pellino proteins in immune signaling with particular emphasis on their involvement in pathogen recognition receptor (PRR) signaling. The growing appreciation of the importance of Pellino proteins in a wide range of immune-mediated diseases are also evaluated.

Differential modulation of Helicobacter pylori lipopolysaccharide-mediated TLR2 signaling by individual Pellino proteins.

BACKGROUND: Eradication rates for current H. pylori therapies have fallen in recent years, in line with the emergence of antibiotic resistant infections. The development of therapeutic alternatives to antibiotics, such as immunomodulatory therapy and vaccines, requires a more lucid understanding of host-pathogen interactions, including the relationships between the organism and the innate immune response. Pellino proteins are emerging as key regulators of immune signaling, including the Toll-like receptor pathways known to be regulated by H. pylori. The aim of this study was to characterize the role of Pellino proteins in the innate immune response to H. pylori lipopolysaccharide. MATERIALS AND METHODS: Gain-of-function and loss-of-function approaches were utilized to elucidate the role of individual Pellino proteins in the Toll-like receptor 2-mediated response to H. pylori LPS by monitoring NF-ĸB activation and the induction of proinflammatory chemokines. Expression of Pellino family members was investigated in gastric epithelial cells and gastric tissue biopsy material. RESULTS: Pellino1 and Pellino2 positively regulated Toll-like receptor 2-driven responses to H. pylori LPS, whereas Pellino3 exerted a negative modulatory role. Expression of Pellino1 was significantly higher than Pellino3 in gastric epithelial cells and gastric tissue. Furthermore, Pellino1 expression was further augmented in gastric epithelial cells in response to infection with H. pylori or stimulation with H. pylori LPS. CONCLUSIONS: The combination of low Pellino3 levels together with high and inducible Pellino1 expression may be an important determinant of the degree of inflammation triggered upon Toll-like receptor 2 engagement by H. pylori and/or its components, contributing to H. pylori-associated pathogenesis by directing the incoming signal toward an NF-kB-mediated proinflammatory response.

Epstein-Barr virus large tegument protein BPLF1 contributes to innate immune evasion through interference with toll-like receptor signaling.

Viral infection triggers an early host response through activation of pattern recognition receptors, including Toll-like receptors (TLR). TLR signaling cascades induce production of type I interferons and proinflammatory cytokines involved in establishing an anti-viral state as well as in orchestrating ensuing adaptive immunity. To allow infection, replication, and persistence, (herpes)viruses employ ingenious strategies to evade host immunity. The human gamma-herpesvirus Epstein-Barr virus (EBV) is a large, enveloped DNA virus persistently carried by more than 90% of adults worldwide. It is the causative agent of infectious mononucleosis and is associated with several malignant tumors. EBV activates TLRs, including TLR2, TLR3, and TLR9. Interestingly, both the expression of and signaling by TLRs is attenuated during productive EBV infection. Ubiquitination plays an important role in regulating TLR signaling and is controlled by ubiquitin ligases and deubiquitinases (DUBs). The EBV genome encodes three proteins reported to exert in vitro deubiquitinase activity. Using active site-directed probes, we show that one of these putative DUBs, the conserved herpesvirus large tegument protein BPLF1, acts as a functional DUB in EBV-producing B cells. The BPLF1 enzyme is expressed during the late phase of lytic EBV infection and is incorporated into viral particles. The N-terminal part of the large BPLF1 protein contains the catalytic site for DUB activity and suppresses TLR-mediated activation of NF-κB at, or downstream of, the TRAF6 signaling intermediate. A catalytically inactive mutant of this EBV protein did not reduce NF-κB activation, indicating that DUB activity is essential for attenuating TLR signal transduction. Our combined results show that EBV employs deubiquitination of signaling intermediates in the TLR cascade as a mechanism to counteract innate anti-viral immunity of infected hosts.

Identification of the flagellin glycosylation system in Burkholderia cenocepacia and the contribution of glycosylated flagellin to evasion of human innate immune responses.

Burkholderia cenocepacia is an opportunistic pathogen threatening patients with cystic fibrosis. Flagella are required for biofilm formation, as well as adhesion to and invasion of epithelial cells. Recognition of flagellin via the Toll-like receptor 5 (TLR5) contributes to exacerbate B. cenocepacia-induced lung epithelial inflammatory responses. In this study, we report that B. cenocepacia flagellin is glycosylated on at least 10 different sites with a single sugar, 4,6-dideoxy-4-(3-hydroxybutanoylamino)-D-glucose. We have identified key genes that are required for flagellin glycosylation, including a predicted glycosyltransferase gene that is linked to the flagellin biosynthesis cluster and a putative acetyltransferase gene located within the O-antigen lipopolysaccharide cluster. Another O-antigen cluster gene, rmlB, which is required for flagellin glycan and O-antigen biosynthesis, was essential for bacterial viability, uncovering a novel target against Burkholderia infections. Using glycosylated and nonglycosylated purified flagellin and a cell reporter system to assess TLR5-mediated responses, we also show that the presence of glycan in flagellin significantly impairs the inflammatory response of epithelial cells. We therefore suggest that flagellin glycosylation reduces recognition of flagellin by host TLR5, providing an evasive strategy to infecting bacteria.

Lipopolysaccharide-induced sepsis induces long-lasting affective changes in the mouse.

Post-septic encephalopathy is a poorly understood condition in survivors of sepsis that is characterised by cognitive and affective impairments. In this study we have sought to better understand this condition by undertaking a comprehensive behavioural and cognitive assessment of mice who had previously survived sepsis. Mice were treated with lipopolysaccharide (LPS; 5mg/kg) and one month after this assessed on a battery of tests. Post-septic animals were found to display significantly more immobility in the tail suspension test and show a significantly decreased sucrose preference. Acute fluoxetine treatment reversed the increase in immobility in the tail suspension test in post-septic animals. Post-septic animals also showed less overall exploratory behaviour in the novel object recognition task and also showed increased anxiety-like behaviour in the elevated plus maze. Post-septic mice did not show signs of cognitive impairment, as assessed in the Morris watermaze, the 8-arm radial maze or on preference for the novel object in the novel object recognition task. Immunohistochemical analysis revealed significant upregulation of the microglial marker CD-11b, F4/80 and IBA-1 in the hippocampus of post-septic animals, as well as significant downregulation of the plasticity-related immediate early gene products ARC and EGR1. We also observed a decrease in neural stem cell proliferation in the dentate gyrus of post-septic animals as judged by BrdU incorporation. Co-treatment with the NF-κB pathway inhibitor PDTC attenuated the long-lasting effects of LPS on most of the affected parameters, but not on neural stem cell proliferation. These results show that LPS-induced sepsis in the mouse is followed by long-lasting increases in depressive- and anxiety-like behaviours, as well as by changes in neuroinflammatory- and neural plasticity-associated factors, and that attenuation of the severity of sepsis by PDTC attenuates many of these effects.

The synthetic cannabinoid R(+)WIN55,212-2 augments interferon-ß expression via peroxisome proliferator-activated receptor-α.

We have demonstrated that R(+)WIN55,212-2, a synthetic cannabinoid that possesses cannabimimetic properties, acts as a novel regulator of Toll-like receptor 3 (TLR3) signaling to interferon (IFN) regulatory factor 3 (IRF3) activation and IFN-ß expression, and this is critical for manifesting its protective effects in a murine multiple sclerosis model. Here we investigated the role of peroxisome proliferator-activated receptor-α (PPARα) in mediating the effects of R(+)WIN55,212-2 on this pathway. Data herein demonstrate that the TLR3 agonist poly(I:C) promotes IFN-ß expression and R(+)WIN55,212-2 enhances TLR3-induced IFN-ß expression in a stereoselective manner via PPARα. R(+)WIN55,212-2 promotes increased transactivation and expression of PPARα. Using the PPARα antagonist GW6471, we demonstrate that R(+)WIN55,212-2 acts via PPARα to activate JNK, activator protein-1, and positive regulatory domain IV to transcriptionally regulate the IFN-ß promoter. Furthermore, GW6471 ameliorated the protective effects of R(+)WIN55,212-2 during the initial phase of experimental autoimmune encephalomyelitis. Overall, these findings define PPARα as an important mediator in manifesting the effects of R(+)WIN55,212-2 on the signaling cascade regulating IFN-ß expression. The study adds to our molecular appreciation of potential therapeutic effects of R(+)WIN55,212-2 in multiple sclerosis.

Mal mediates TLR-induced activation of CREB and expression of IL-10.

TLRs initiate immune responses by direct detection of molecular motifs that distinguish invading microbes from host cells. Five intracellular adaptor proteins, each containing a Toll/IL-1R (TIR) domain, are used by TLRs and play key roles in dictating gene expression patterns that are tailored to the invader. Such gene expression is mediated by transcription factors, and although TIR adaptor-induced activation of NF-κB and the IFN regulatory factors have been intensively studied, there is a dearth of information on the role of TIR adaptors in regulating CREB. In this paper, we describe a role for the TIR adaptor Mal in enhancing activation of CREB. Mal-deficient murine bone marrow-derived macrophages show a loss in responsiveness to TLR2 and TLR4 ligands with respect to activation of CREB. Mal-deficient cells also fail to express the CREB-responsive genes IL-10 and cyclooxygenase 2 in response to Pam(2)Cys-Ser-(Lys)4 and LPS. We reveal that Mal-mediated activation of CREB is dependent on Pellino3 and TNFR-associated factor 6, because CREB activation is greatly diminished in Pellino3 knockdown cells and TNFR-associated factor 6-deficient cells. We also demonstrate the importance of p38 MAPK in this pathway with the p38 inhibitor SB203580 abolishing activation of CREB in murine macrophages. MAPK-activated protein kinase 2 (MK2), a substrate for p38 MAPK, is the likely downstream mediator of p38 MAPK in this pathway, because Mal is shown to activate MK2 and inhibition of MK2 decreases TLR4-induced activation of CREB. Overall, these studies demonstrate a new role for Mal as a key upstream regulator of CREB and as a contributor to the expression of both pro- and anti-inflammatory genes.

ECSIT Is a Critical Factor for Controlling Intestinal Homeostasis and Tumorigenesis through Regulating the Translation of YAP Protein.

The intestinal epithelium is the fastest renewing tissue in mammals and its regenerative process must be tightly controlled to minimize the risk of dysfunction and tumorigenesis. The orderly expression and activation of Yes-associated protein (YAP) are the key steps in driving intestinal regeneration and crucial for intestinal homeostasis. However, the regulatory mechanisms controlling this process remain largely unknown. Here, it is discovered that evolutionarily conserved signaling intermediate in Toll pathways (ECSIT), a multi-functional protein, is enriched along the crypt-villus axis. Intestinal cell-specific ablation of ECSIT results in the dysregulation of intestinal differentiation unexpectedly accompanied with enhanced YAP protein dependent on translation, thus transforming intestinal cells to early proliferative stem "-like" cells and augmenting intestinal tumorigenesis. Loss of ECSIT leads to metabolic reprogramming in favor of amino acid-based metabolism, which results in demethylation of genes encoding the eukaryotic initiation factor 4F pathway and their increased expression that further promotes YAP translation initiation culminating in intestinal homeostasis imbalance and tumorigenesis. It is also shown that the expression of ECSIT is positively correlated with the survival of patients with colorectal cancer. Together, these results demonstrate the important role of ECSIT in regulating YAP protein translation to control intestinal homeostasis and tumorigenesis.

Identification of the synthetic cannabinoid R(+)WIN55,212-2 as a novel regulator of IFN regulatory factor 3 activation and IFN-beta expression: relevance to therapeutic effects in models of multiple sclerosis.

ß-Interferons (IFN-ßs) represent one of the first line treatments for relapsing-remitting multiple sclerosis, slowing disease progression while reducing the frequency of relapses. Despite this, more effective, well tolerated therapeutic strategies are needed. Cannabinoids palliate experimental autoimmune encephalomyelitis (EAE) symptoms and have therapeutic potential in MS patients although the precise molecular mechanism for these effects is not understood. Toll-like receptor (TLR) signaling controls innate immune responses and TLRs are implicated in MS. Here we demonstrate that the synthetic cannabinoid R(+)WIN55,212-2 is a novel regulator of TLR3 and TLR4 signaling by inhibiting the pro-inflammatory signaling axis triggered by TLR3 and TLR4, whereas selectively augmenting TLR3-induced activation of IFN regulatory factor 3 (IRF3) and expression of IFN-ß. We present evidence that R(+)WIN55,212-2 strongly promotes the nuclear localization of IRF3. The potentiation of IFN-ß expression by R(+)WIN55,212-2 is critical for manifesting its protective effects in the murine MS model EAE as evidenced by its reduced therapeutic efficacy in the presence of an anti-IFN-ß antibody. R(+)WIN55,212-2 also induces IFN-ß expression in MS patient peripheral blood mononuclear cells, whereas down-regulating inflammatory signaling in these cells. These findings identify R(+)WIN55,212-2 as a novel regulator of TLR3 signaling to IRF3 activation and IFN-ß expression and highlights a new mechanism that may be open to exploitation in the development of new therapeutics for the treatment of MS.