Gasdermin D licenses MHCII induction to maintain food tolerance in small intestine.

The intestinal epithelial cells (IECs) constitute the primary barrier between host cells and numerous foreign antigens; it is unclear how IECs induce the protective immunity against pathogens while maintaining the immune tolerance to food. Here, we found IECs accumulate a less recognized 13-kD N-terminal fragment of GSDMD that is cleaved by caspase-3/7 in response to dietary antigens. Unlike the 30-kD GSDMD cleavage fragment that executes pyroptosis, the IEC-accumulated GSDMD cleavage fragment translocates to the nucleus and induces the transcription of CIITA and MHCII molecules, which in turn induces the Tr1 cells in upper small intestine. Mice treated with a caspase-3/7 inhibitor, mice with GSDMD mutation resistant to caspase-3/7 cleavage, mice with MHCII deficiency in IECs, and mice with Tr1 deficiency all displayed a disrupted food tolerance phenotype. Our study supports that differential cleavage of GSDMD can be understood as a regulatory hub controlling immunity versus tolerance in the small intestine.

DAMPs and DAMP-sensing receptors in inflammation and diseases.

Damage-associated molecular patterns (DAMPs) are endogenous danger molecules produced in cellular damage or stress, and they can activate the innate immune system. DAMPs contain multiple types of molecules, including nucleic acids, proteins, ions, glycans, and metabolites. Although these endogenous molecules do not trigger immune response under steady-state condition, they may undergo changes in distribution, physical or chemical property, or concentration upon cellular damage or stress, and then they become DAMPs that can be sensed by innate immune receptors to induce inflammatory response. Thus, DAMPs play an important role in inflammation and inflammatory diseases. In this review, we summarize the conversion of homeostatic molecules into DAMPs; the diverse nature and classification, cellular origin, and sensing of DAMPs; and their role in inflammation and related diseases. Furthermore, we discuss the clinical strategies to treat DAMP-associated diseases via targeting DAMP-sensing receptors.

Bile acids modified by the intestinal microbiota promote colorectal cancer growth by suppressing CD8+ T cell effector functions.

Concentrations of the secondary bile acid, deoxycholic acid (DCA), are aberrantly elevated in colorectal cancer (CRC) patients, but the consequences remain poorly understood. Here, we screened a library of gut microbiota-derived metabolites and identified DCA as a negative regulator for CD8+ T cell effector function. Mechanistically, DCA suppressed CD8+ T cell responses by targeting plasma membrane Ca2+ ATPase (PMCA) to inhibit Ca2+-nuclear factor of activated T cells (NFAT)2 signaling. In CRC patients, CD8+ T cell effector function negatively correlated with both DCA concentration and expression of a bacterial DCA biosynthetic gene. Bacteria harboring DCA biosynthetic genes suppressed CD8+ T cells effector function and promoted tumor growth in mice. This effect was abolished by disrupting bile acid metabolism via bile acid chelation, genetic ablation of bacterial DCA biosynthetic pathway, or specific bacteriophage. Our study demonstrated causation between microbial DCA metabolism and anti-tumor CD8+ T cell response in CRC, suggesting potential directions for anti-tumor therapy.

Commensal viruses maintain intestinal intraepithelial lymphocytes via noncanonical RIG-I signaling.

Much attention has focused on commensal bacteria in health and disease, but the role of commensal viruses is understudied. Although metagenomic analysis shows that the intestine of healthy humans and animals harbors various commensal viruses and the dysbiosis of these viruses can be associated with inflammatory diseases, there is still a lack of causal data and underlying mechanisms to understand the physiological role of commensal viruses in intestinal homeostasis. In the present study, we show that commensal viruses are essential for the homeostasis of intestinal intraepithelial lymphocytes (IELs). Mechanistically, the cytosolic viral RNA-sensing receptor RIG-I in antigen-presenting cells can recognize commensal viruses and maintain IELs via a type I interferon-independent, but MAVS-IRF1-IL-15 axis-dependent, manner. The recovery of IELs by interleukin-15 administration reverses the susceptibility of commensal virus-depleted mice to dextran sulfate sodium-induced colitis. Collectively, our results indicate that commensal viruses maintain the IELs and consequently sustain intestinal homeostasis via noncanonical RIG-I signaling.

GPR34-mediated sensing of lysophosphatidylserine released by apoptotic neutrophils activates type 3 innate lymphoid cells to mediate tissue repair.

Neutrophils migrate rapidly to damaged tissue and play critical roles in host defense and tissue homeostasis. Here we investigated the mechanisms whereby neutrophils participate in tissue repair. In an intestinal epithelia injury model, neutrophil depletion exacerbated colitis and associated with reduced interleukin (IL)-22 and limited activation of type 3 innate lymphoid cells (ILC3s). Co-culture with neutrophils activated ILC3s in a manner dependent on neutrophil apoptosis. Metabolomic analyses revealed that lysophosphatidylserine (LysoPS) from apoptotic neutrophils directly stimulated ILC3 activation. ILC3-specific deletion of Gpr34, encoding the LysoPS receptor GPR34, or inhibition of downstream PI3K-AKT or ERK suppressed IL-22 production in response to apoptotic neutrophils. Gpr34-/- mice exhibited compromised ILC3 activation and tissue repair during colon injury, and neutrophil depletion abrogated these defects. GPR34 deficiency in ILC3s limited IL-22 production and tissue repair in vivo in settings of colon and skin injury. Thus, GPR34 is an ILC3-expressed damage-sensing receptor that triggers tissue repair upon recognition of dying neutrophils.

Dopamine controls systemic inflammation through inhibition of NLRP3 inflammasome.

Inflammasomes are involved in diverse inflammatory diseases, so the activation of inflammasomes needs to be tightly controlled to prevent excessive inflammation. However, the endogenous regulatory mechanisms of inflammasome activation are still unclear. Here, we report that the neurotransmitter dopamine (DA) inhibits NLRP3 inflammasome activation via dopamine D1 receptor (DRD1). DRD1 signaling negatively regulates NLRP3 inflammasome via a second messenger cyclic adenosine monophosphate (cAMP), which binds to NLRP3 and promotes its ubiquitination and degradation via the E3 ubiquitin ligase MARCH7. Importantly, in vivo data show that DA and DRD1 signaling prevent NLRP3 inflammasome-dependent inflammation, including neurotoxin-induced neuroinflammation, LPS-induced systemic inflammation, and monosodium urate crystal (MSU)-induced peritoneal inflammation. Taken together, our results reveal an endogenous mechanism of inflammasome regulation and suggest DRD1 as a potential target for the treatment of NLRP3 inflammasome-driven diseases.

Kinases Mst1 and Mst2 positively regulate phagocytic induction of reactive oxygen species and bactericidal activity.

Mitochondria need to be juxtaposed to phagosomes for the synergistic production of ample reactive oxygen species (ROS) in phagocytes to kill pathogens. However, how phagosomes transmit signals to recruit mitochondria has remained unclear. Here we found that the kinases Mst1 and Mst2 functioned to control ROS production by regulating mitochondrial trafficking and mitochondrion-phagosome juxtaposition. Mst1 and Mst2 activated the GTPase Rac to promote Toll-like receptor (TLR)-triggered assembly of the TRAF6-ECSIT complex that is required for the recruitment of mitochondria to phagosomes. Inactive forms of Rac, including the human Rac2(D57N) mutant, disrupted the TRAF6-ECSIT complex by sequestering TRAF6 and substantially diminished ROS production and enhanced susceptibility to bacterial infection. Our findings demonstrate that the TLR-Mst1-Mst2-Rac signaling axis is critical for effective phagosome-mitochondrion function and bactericidal activity.

RNA viruses promote activation of the NLRP3 inflammasome through a RIP1-RIP3-DRP1 signaling pathway.

The NLRP3 inflammasome functions as a crucial component of the innate immune system in recognizing viral infection, but the mechanism by which viruses activate this inflammasome remains unclear. Here we found that inhibition of the serine-threonine kinases RIP1 (RIPK1) or RIP3 (RIPK3) suppressed RNA virus-induced activation of the NLRP3 inflammasome. Infection with an RNA virus initiated assembly of the RIP1-RIP3 complex, which promoted activation of the GTPase DRP1 and its translocation to mitochondria to drive mitochondrial damage and activation of the NLRP3 inflammasome. Notably, the RIP1-RIP3 complex drove the NLRP3 inflammasome independently of MLKL, an essential downstream effector of RIP1-RIP3-dependent necrosis. Together our results reveal a specific role for the RIP1-RIP3-DRP1 pathway in RNA virus-induced activation of the NLRP3 inflammasome and establish a direct link between inflammation and cell-death signaling pathways.

Cryo-EM structures of human GPR34 enable the identification of selective antagonists.

GPR34 is a functional G-protein-coupled receptor of Lysophosphatidylserine (LysoPS), and has pathogenic roles in numerous diseases, yet remains poorly targeted. We herein report a cryo-electron microscopy (cryo-EM) structure of GPR34 bound with LysoPS (18:1) and Gi protein, revealing a unique ligand recognition mode with the negatively charged head group of LysoPS occupying a polar cavity formed by TM3, 6 and 7, and the hydrophobic tail of LysoPS residing in a lateral open hydrophobic groove formed by TM3-5. Virtual screening and subsequent structural optimization led to the identification of a highly potent and selective antagonist (YL-365). Design of fusion proteins allowed successful determination of the challenging cryo-EM structure of the inactive GPR34 complexed with YL-365, which revealed the competitive binding of YL-365 in a portion of the orthosteric binding pocket of GPR34 and the antagonist-binding-induced allostery in the receptor, implicating the inhibition mechanism of YL-365. Moreover, YL-365 displayed excellent activity in a neuropathic pain model without obvious toxicity. Collectively, this study offers mechanistic insights into the endogenous agonist recognition and antagonist inhibition of GPR34, and provides proof of concept that targeting GPR34 represents a promising strategy for disease treatment.

Control of Inflammasome Activation by Phosphorylation.

Inflammasomes are cytosolic protein complexes composed of innate immune sensors, the adaptor protein ASC, and the cysteine protease caspase-1. In response to microbial infection or 'danger signals', inflammasomes play critical roles in host defense or contribute to the pathogenesis of various inflammatory diseases. Recent studies have provided abundant evidence for a vital role of phosphorylation in the regulation of inflammasome assembly and activation. This review integrates previous observations and discoveries for inflammasome regulation by protein phosphorylation with the most recent findings. Additionally, the timely application and clinical prospects in the treatment of inflammatory diseases, by targeting related protein kinases or phosphatases, are also discussed.

Thioredoxin-interacting protein links oxidative stress to inflammasome activation.

The NLRP3 inflammasome has a major role in regulating innate immunity. Deregulated inflammasome activity is associated with several inflammatory diseases, yet little is known about the signaling pathways that lead to its activation. Here we show that NLRP3 interacted with thioredoxin (TRX)-interacting protein (TXNIP), a protein linked to insulin resistance. Inflammasome activators such as uric acid crystals induced the dissociation of TXNIP from thioredoxin in a reactive oxygen species (ROS)-sensitive manner and allowed it to bind NLRP3. TXNIP deficiency impaired activation of the NLRP3 inflammasome and subsequent secretion of interleukin 1beta (IL-1beta). Akin to Txnip(-/-) mice, Nlrp3(-/-) mice showed improved glucose tolerance and insulin sensitivity. The participation of TXNIP in the NLRP3 inflammasome activation may provide a mechanistic link to the observed involvement of IL-1beta in the pathogenesis of type 2 diabetes.

Omega-3 fatty acids prevent inflammation and metabolic disorder through inhibition of NLRP3 inflammasome activation.

Omega-3 fatty acids (ω-3 FAs) have potential anti-inflammatory activity in a variety of inflammatory human diseases, but the mechanisms remain poorly understood. Here we show that stimulation of macrophages with ω-3 FAs, including eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and other family members, abolished NLRP3 inflammasome activation and inhibited subsequent caspase-1 activation and IL-1ß secretion. In addition, G protein-coupled receptor 120 (GPR120) and GPR40 and their downstream scaffold protein ß-arrestin-2 were shown to be involved in inflammasome inhibition induced by ω-3 FAs. Importantly, ω-3 FAs also prevented NLRP3 inflammasome-dependent inflammation and metabolic disorder in a high-fat-diet-induced type 2 diabetes model. Our results reveal a mechanism through which ω-3 FAs repress inflammation and prevent inflammation-driven diseases and suggest the potential clinical use of ω-3 FAs in gout, autoinflammatory syndromes, or other NLRP3 inflammasome-driven inflammatory diseases.

Orchestration of NLRP3 Inflammasome Activation by Ion Fluxes.

The assembly of the NLRP3 inflammasome can promote the release of IL-1ß/IL-18 and initiate pyroptosis. Accordingly, the dysregulation of NLRP3 inflammasome activation is involved in a variety of human diseases, including gout, diabetes, and Alzheimer's disease. NLRP3 can sense a variety of structurally unrelated pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs) to trigger inflammation, but the unifying mechanism of NLRP3 activation is still poorly understood. Increasing evidence suggests that intracellular ions, such as K+, Ca2+, and Cl-, have a significant role in NLRP3 inflammasome activation. Here, we review the current knowledge about the role of ionic fluxes in NLRP3 inflammasome activation and discuss how disturbances in intracellular ionic levels orchestrate different signaling events upstream of NLRP3.

Loss of Annexin A5 expression attenuates the lipopolysaccharide-induced inflammatory response of rat alveolar macrophages.

Acute lung injury (ALI) is a common respiratory syndrome accompanied with an inflammation response. Annexin A5 (AnxA5) has anti-thrombotic, anti-apoptotic, and anti-inflammatory properties. The current study aims to explore the potential effect of AnxA5 on lipopolysaccharide (LPS)-induced inflammatory response in alveolar macrophages (AMs). Rat AMs (NR8383) were used in this study, and the cell viabilities at 4, 8, and 16 h after LPS administration with gradient concentrations were determined using cell counting kit-8 assay. Cell apoptosis and expressions of messenger RNAs (mRNAs) and protein were determined by flow cytometry, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot, respectively. We found that LPS suppressed the viability of AMs in a dose-dependent manner, and it elevated the expression of AnxA5 in AMs. Inhibition of AnxA5 improved the cell viability compared with the LPS group and could reduce the apoptosis rate in comparison with LPS treatment. The knockdown of AnxA5 suppressed the expressions of tumor necrosis factor-α (TNF-α), interleukin (IL-1ß), and IL-6 at both protein and mRNA levels and regulated the expressions of apoptosis-related molecules (Bax, Bcl-2, and caspase-3). Moreover, the knockdown of AnxA5 improved the expression levels of inhibitory κB (IκB) and nuclear factor E2-related factor 2 (Nrf2) but inhibited the expression of nuclear transcription factor κB (NF-κB), compared with the LPS group. SN50 and ML385 were used to validate this signaling, and the inhibition of AnxA5 suppressed the LPS-induced inflammation, indicating that AnxA5 may be a potential anti-inflammatory target. In addition, NF-κB/Nrf2 signaling pathway may also be involved in the LPS-induced inflammatory response of rat alveolar macrophages.

Discovery and optimization of 4-oxo-2-thioxo-thiazolidinones as NOD-like receptor (NLR) family, pyrin domain-containing protein 3 (NLRP3) inhibitors.

Aberrant activation of NLRP3 inflammasome is present in a subset of acute and chronic inflammatory diseases. The NLRP3 inflammasome has been recognized as an attractive therapeutic target for developing novel and specific anti-inflammatory inhibitors. Cellular structure-activity relationship-guided optimization resulted in the identification of 4-oxo-2-thioxo-thiazolidinone derivative 9 as a selective and direct small-molecule inhibitor of NLRP3 with IC50 of 2.4 µM, possessing favorable ex vivo and in vivo pharmacokinetic properties. Compound 9 may represent a lead for the development of anti-inflammatory therapeutics for treating NLRP3-driven diseases.

NLRP11 disrupts MAVS signalosome to inhibit type I interferon signaling and virus-induced apoptosis.

MAVS signalosome plays an important role in RIG-I-like receptor (RLR)-induced antiviral signaling. Upon the recognition of viral RNAs, RLRs activate MAVS, which further recruits TRAF6 and other signaling proteins to initiate type I interferon (IFN) activation. MAVS signalosome also regulates virus-induced apoptosis to limit viral replication. However, the mechanisms that control the activity of MAVS signalosome are still poorly defined. Here, we report NLRP11, a Nod-like receptor, is induced by type I IFN and translocates to mitochondria to interact with MAVS upon viral infection. Using MAVS as a platform, NLRP11 degrades TRAF6 to attenuate the production of type I IFNs as well as virus-induced apoptosis. Our findings reveal the regulatory role of NLRP11 in antiviral immunity by disrupting MAVS signalosome.

Plant Lectins Activate the NLRP3 Inflammasome To Promote Inflammatory Disorders.

Plant-derived dietary lectins have been reported to be involved in the pathogenesis of several inflammatory diseases, including inflammatory bowel disease, diabetes, rheumatoid arthritis, and celiac disease, but little is known about the molecular mechanisms underlying lectin-induced inflammation. In this study, we showed that plant lectins can induce caspase-1 activation and IL-1ß secretion via the NLRP3 inflammasome. Lectins were internalized and subsequently escaped from the lysosome and then translocated to the endoplasmic reticulum. Endoplasmic reticulum-loaded plant lectins then triggered Ca2+ release and mitochondrial damage, and inhibition of Ca2+ release and mitochondrial reactive oxygen species by chemical inhibitors significantly suppressed NLRP3 inflammasome activation. In vivo, plant lectin-induced inflammation and tissue damage also depended on the NLRP3 inflammasome. Our findings indicate that plant lectins can act as an exogenous "danger signal" that can activate the NLRP3 inflammasome and suggest that dietary lectins might promote inflammatory diseases via the NLRP3 inflammasome.

TLR9 Regulates the NF-κB-NLRP3-IL-1ß Pathway Negatively in Salmonella-Induced NKG2D-Mediated Intestinal Inflammation.

TLRs are key sensors for conserved bacterial molecules and play a critical role in host defense against invading pathogens. Although the roles of TLRs in defense against pathogen infection and in maintaining gut immune homeostasis have been studied, the precise functions of different TLRs in response to pathogen infection in the gut remain elusive. The present study investigated the role of TLR signaling in defense against the Gram-negative bacterial pathogen Salmonella typhimurium The results indicated that TLR9-deficient mice were more susceptible to S. typhimurium infection compared with wild-type and TLR2- or TLR4-deficient mice, as indicated by more severe intestinal damage and the highest bacterial load. TLR9 deficiency in intestinal epithelial cells (IECs) augmented the activation of NF-κB and NLRP3 inflammasomes significantly, resulting in increased secretion of IL-1ß. IL-1ß increased the expression of NKG2D on intestinal intraepithelial lymphocytes and NKG2D ligands on IECs, resulting in higher susceptibility of IECs to cytotoxicity of intestinal intraepithelial lymphocytes and damage to the epithelial barrier. We proposed that TLR9 regulates the NF-κB-NLRP3-IL-1ß pathway negatively in Salmonella-induced NKG2D-mediated intestinal inflammation and plays a critical role in defense against S. typhimurium infection and in the protection of intestinal integrity.

Smart Dual Quenching Strategy Enhances the Detection Sensitivity of Intracellular Furin.

Development of sensitive fluorescence "Turn-On" strategies for imaging enzyme activity in living cells is of disease-diagnostic importance but remains challenging. Herein, by employing a click condensation reaction and rational design of a single quenched probe Cys(StBu)-Lys(Gly-Lys(DABCYL)-Gly-Gly-Arg-Arg-Val-Arg-Gly-FITC)-CBT (1), we developed a "smart" dual quenching strategy and applied it to detect intracellular furin activity with enhanced sensitivity. At physiological conditions, 1 was subjected to reduction-controlled condensation reaction to form 1-NPs and its fluorescence intensity further dropped to 1/2.8 of its original. Upon furin cleavage in vitro, the dual quenched 1-NPs had fluorescence "Turn-On" contrast 11-fold more than that of single quenched control probe FITC-Gly-Arg-Val-Arg-Arg-Gly-Gly-Lys(DABCYL)-Gly-OH (1-P). Live cell imaging results indicated that 1 showed fluorescence "Turn-On" contrast 6.3-fold of that of 1-P for sensing intracellular furin activity. We envision that, by replacing the RVRR substrate with other enzyme-cleavable ones, our versatile "smart" dual quenching strategy could be easily adjusted for the detection (or imaging) of other intracellular enzymes' activity with enhanced sensitivity.