CARD-only proteins regulate in vivo inflammasome responses and ameliorate gout.

Inflammatory responses are crucial for controlling infections and initiating tissue repair. However, excessive and uncontrolled inflammation causes inflammatory disease. Processing and release of the pro-inflammatory cytokines interleukin-1ß (IL-1ß) and IL-18 depend on caspase-1 activation within inflammasomes. Assembly of inflammasomes is initiated upon activation of cytosolic pattern recognition receptors (PRRs), followed by sequential polymerization of pyrin domain (PYD)-containing and caspase recruitment domain (CARD)-containing proteins mediated by homotypic PYD and CARD interactions. Small PYD- or CARD-only proteins (POPs and COPs, respectively) evolved in higher primates to target these crucial interactions to limit inflammation. Here, we show the ability of COPs to regulate inflammasome activation by modulating homotypic CARD-CARD interactions in vitro and in vivo. CARD16, CARD17, and CARD18 displace crucial CARD interactions between caspase-1 proteins through competitive binding and ameliorate uric acid crystal-mediated NLRP3 inflammasome activation and inflammatory disease. COPs therefore represent an important family of inflammasome regulators and ameliorate inflammatory disease.

NLRP3 licenses NLRP11 for inflammasome activation in human macrophages.

Intracellular sensing of stress and danger signals initiates inflammatory innate immune responses by triggering inflammasome assembly, caspase-1 activation and pyroptotic cell death as well as the release of interleukin 1ß (IL-1ß), IL-18 and danger signals. NLRP3 broadly senses infectious patterns and sterile danger signals, resulting in the tightly coordinated and regulated assembly of the NLRP3 inflammasome, but the precise mechanisms are incompletely understood. Here, we identified NLRP11 as an essential component of the NLRP3 inflammasome in human macrophages. NLRP11 interacted with NLRP3 and ASC, and deletion of NLRP11 specifically prevented NLRP3 inflammasome activation by preventing inflammasome assembly, NLRP3 and ASC polymerization, caspase-1 activation, pyroptosis and cytokine release but did not affect other inflammasomes. Restored expression of NLRP11, but not NLRP11 lacking the PYRIN domain (PYD), restored inflammasome activation. NLRP11 was also necessary for inflammasome responses driven by NLRP3 mutations that cause cryopyrin-associated periodic syndrome (CAPS). Because NLRP11 is not expressed in mice, our observations emphasize the specific complexity of inflammasome regulation in humans.

SARS-CoV-2 Nsp5 Demonstrates Two Distinct Mechanisms Targeting RIG-I and MAVS To Evade the Innate Immune Response.

Newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a global pandemic with astonishing mortality and morbidity. The high replication and transmission of SARS-CoV-2 are remarkably distinct from those of previous closely related coronaviruses, and the underlying molecular mechanisms remain unclear. The innate immune defense is a physical barrier that restricts viral replication. We report here that the SARS-CoV-2 Nsp5 main protease targets RIG-I and mitochondrial antiviral signaling (MAVS) protein via two distinct mechanisms for inhibition. Specifically, Nsp5 cleaves off the 10 most-N-terminal amino acids from RIG-I and deprives it of the ability to activate MAVS, whereas Nsp5 promotes the ubiquitination and proteosome-mediated degradation of MAVS. As such, Nsp5 potently inhibits interferon (IFN) induction by double-stranded RNA (dsRNA) in an enzyme-dependent manner. A synthetic small-molecule inhibitor blunts the Nsp5-mediated destruction of cellular RIG-I and MAVS and processing of SARS-CoV-2 nonstructural proteins, thus restoring the innate immune response and impeding SARS-CoV-2 replication. This work offers new insight into the immune evasion strategy of SARS-CoV-2 and provides a potential antiviral agent to treat CoV disease 2019 (COVID-19) patients. IMPORTANCE The ongoing COVID-19 pandemic is caused by SARS-CoV-2, which is rapidly evolving with better transmissibility. Understanding the molecular basis of the SARS-CoV-2 interaction with host cells is of paramount significance, and development of antiviral agents provides new avenues to prevent and treat COVID-19 diseases. This study describes a molecular characterization of innate immune evasion mediated by the SARS-CoV-2 Nsp5 main protease and subsequent development of a small-molecule inhibitor.

NLRP7: From inflammasome regulation to human disease.

Nucleotide-binding oligomerization domain (NOD) and leucine-rich repeat (LRR)-containing receptors or NOD-like receptors (NLRs) are cytosolic pattern recognition receptors, which sense conserved microbial patterns and host-derived danger signals to elicit innate immune responses. The activation of several prototypic NLRs, including NLR and pyrin domain (PYD) containing (NLRP) 1, NLRP3 and NLR and caspase recruitment domain (CARD) containing (NLRC) 4, results in the assembly of inflammasomes, which are large, cytoplasmic multiprotein signalling platforms responsible for the maturation and release of the pro-inflammatory cytokines IL-1ß and IL-18, and for the induction of a specialized form of inflammatory cell death called pyroptosis. However, the function of other members of the NLR family, including NLRP7, are less well understood. NLRP7 has been linked to innate immune signalling, but its precise role is still controversial as it has been shown to positively and negatively affect inflammasome responses. Inflammasomes are essential for homeostasis and host defence, but inappropriate inflammasome responses due to hereditary mutations and somatic mosaicism in inflammasome components and defective regulation have been linked to a broad spectrum of human diseases. A compelling connection between NLRP7 mutations and reproductive diseases, and in particular molar pregnancy, has been established. However, the molecular mechanisms by which NLRP7 mutations contribute to reproductive diseases are largely unknown. In this review, we focus on NLRP7 and discuss the current evidence of its role in inflammasome regulation and its implication in human reproductive diseases.

Recruitment of pro-IL-1α to mitochondrial cardiolipin, via shared LC3 binding domain, inhibits mitophagy and drives maximal NLRP3 activation.

The balance between NLRP3 inflammasome activation and mitophagy is essential for homeostasis and cellular health, but this relationship remains poorly understood. Here we found that interleukin-1α (IL-1α)-deficient macrophages have reduced caspase-1 activity and diminished IL-1ß release, concurrent with reduced mitochondrial damage, suggesting a role for IL-1α in regulating this balance. LPS priming of macrophages induced pro-IL-1α translocation to mitochondria, where it directly interacted with mitochondrial cardiolipin (CL). Computational modeling revealed a likely CL binding motif in pro-IL-1α, similar to that found in LC3b. Thus, binding of pro-IL-1α to CL in activated macrophages may interrupt CL-LC3b-dependent mitophagy, leading to enhanced Nlrp3 inflammasome activation and more robust IL-1ß production. Mutation of pro-IL-1α residues predicted to be involved in CL binding resulted in reduced pro-IL-1α-CL interaction, a reduction in NLRP3 inflammasome activity, and increased mitophagy. These data identify a function for pro-IL-1α in regulating mitophagy and the potency of NLRP3 inflammasome activation.

Post-translational Control of Innate Immune Signaling Pathways by Herpesviruses.

Herpesviruses constitute a large family of disease-causing DNA viruses. Each herpesvirus strain is capable of infecting particular organisms with a specific cell tropism. Upon infection, pattern recognition receptors (PRRs) recognize conserved viral features to trigger signaling cascades that culminate in the production of interferons and pro-inflammatory cytokines. To invoke a proper immune response while avoiding collateral tissue damage, signaling proteins involved in these cascades are tightly regulated by post-translational modifications (PTMs). Herpesviruses have developed strategies to subvert innate immune signaling pathways in order to ensure efficient viral replication and achieve persistent infection. The ability of these viruses to control the proteins involved in these signaling cascades post-translationally, either directly via virus-encoded enzymes or indirectly through the deregulation of cellular enzymes, has been widely reported. This ability provides herpesviruses with a powerful tool to shut off or restrict host antiviral and inflammatory responses. In this review, we highlight recent findings on the herpesvirus-mediated post-translational control along PRR-mediated signaling pathways.

Interplay between Cellular Metabolism and Cytokine Responses during Viral Infection.

Metabolism and immune responses are two fundamental biological processes that serve to protect hosts from viral infection. As obligate intracellular pathogens, viruses have evolved diverse strategies to activate metabolism, while inactivating immune responses to achieve maximal reproduction or persistence within their hosts. The two-way virus-host interaction with metabolism and immune responses choreograph cytokine production via reprogramming metabolism of infected cells/hosts. In return, cytokines can affect the metabolism of virus-infected and bystander cells to impede viral replication processes. This review aims to summarize our current understanding of the cross-talk between metabolic reprogramming and cytokine responses, and to highlight future potential research topics. Although the focus is placed on viral pathogens, relevant findings from other microbes are integrated to provide an overall picture, particularly when corresponding information on viral infection is lacking.

Exosomes: From Functions in Host-Pathogen Interactions and Immunity to Diagnostic and Therapeutic Opportunities.

Since their first description in the 1980s, exosomes, small endosomal-derived extracellular vesicles, have been involved in innate and adaptive immunity through modulating immune responses and mediating antigen presentation. Increasing evidence has reported the role of exosomes in host-pathogen interactions and particularly in the activation of antimicrobial immune responses. The growing interest concerning exosomes in infectious diseases, their accessibility in various body fluids, and their capacity to convey a rich content (e.g., proteins, lipids, and nucleic acids) to distant recipient cells led the scientific community to consider the use of exosomes as potential new diagnostic and therapeutic tools. In this review, we summarize current understandings of exosome biogenesis and their composition and highlight the function of exosomes as immunomodulators in pathological states such as in infectious disorders. The potential of using exosomes as diagnostic and therapeutic tools is also discussed.

Activation of the EIF2AK4-EIF2A/eIF2α-ATF4 pathway triggers autophagy response to Crohn disease-associated adherent-invasive Escherichia coli infection.

The intestinal mucosa of Crohn disease (CD) patients is abnormally colonized by adherent-invasive E. coli (AIEC). Upon AIEC infection, autophagy is induced in host cells to restrain bacterial intracellular replication. The underlying mechanism, however, remains unknown. Here, we investigated the role of the EIF2AK4-EIF2A/eIF2α-ATF4 pathway in the autophagic response to AIEC infection. We showed that infection of human intestinal epithelial T84 cells with the AIEC reference strain LF82 activated the EIF2AK4-EIF2A-ATF4 pathway, as evidenced by increased phospho-EIF2AK4, phospho-EIF2A and ATF4 levels. EIF2AK4 depletion inhibited autophagy activation in response to LF82 infection, leading to increased LF82 intracellular replication and elevated pro-inflammatory cytokine production. Mechanistically, EIF2AK4 depletion suppressed the LF82-induced ATF4 binding to promoters of several autophagy genes including MAP1LC3B, BECN1, SQSTM1, ATG3 and ATG7, and this subsequently inhibited transcription of these genes. LF82 infection of wild-type (WT), but not eif2ak4(-/-), mice activated the EIF2AK4-EIF2A-ATF4 pathway, inducing autophagy gene transcription and autophagy response in enterocytes. Consequently, eif2ak4(-/-) mice exhibited increased intestinal colonization by LF82 bacteria and aggravated inflammation compared to WT mice. Activation of the EIF2AK4-EIF2A-ATF4 pathway was observed in ileal biopsies from patients with noninflamed CD, and this was suppressed in inflamed CD, suggesting that a defect in the activation of this pathway could be one of the mechanisms contributing to active disease. In conclusion, we show that activation of the EIF2AK4-EIF2A-ATF4 pathway upon AIEC infection serves as a host defense mechanism to induce functional autophagy to control AIEC intracellular replication.

Exosomes Released from Cells Infected with Crohn's Disease-associated Adherent-Invasive Escherichia coli Activate Host Innate Immune Responses and Enhance Bacterial Intracellular Replication.

BACKGROUND: Crohn's disease is a chronic inflammatory bowel disease, of which the etiology involves environmental, genetic, and microbial factors. A high prevalence of adherent-invasive Escherichia coli, named AIEC, has been reported in the intestinal mucosa of patients with Crohn's disease. Exosomes are extracellular vesicles that function in intercellular communication and have been implicated in host responses to intracellular pathogens. We investigated the potential involvement of exosomes in host response to AIEC infection. METHODS: Human intestinal epithelial T84 cells, THP-1 macrophages, and CEABAC10 transgenic mice were infected with the AIEC reference strain LF82 or the nonpathogenic E. coli K-12 MG1655 strain. Exosomes were purified using the ExoQuick reagent. RESULTS: LF82 infection induced the release of exosomes by T84 and THP-1 cells. Compared with exosomes released from the uninfected or MG1655-infected T84 cells, those released from LF82-infected cells activated nuclear factor-kappa B, mitogen-activated protein kinases p38, and c-Jun N-terminal kinase and increased the secretion of proinflammatory cytokines in naive THP-1 macrophages. LF82 infection of THP-1 macrophages also induced the release of exosomes that triggered a proinflammatory response in recipient THP-1 cells. Importantly, stimulation of T84 or THP-1 cells with exosomes released from LF82-infected cells increased LF82 intracellular replication compared with stimulation with exosomes secreted by uninfected cells. Exosomes purified from intestinal lumen of CEABAC10 transgenic mice infected with LF82 increased proinflammatory responses in murine RAW 264.7 macrophages compared with those from uninfected or MG1655-infected mice. CONCLUSIONS: Exosomes are new mediators of host-AIEC interaction with their capacity to activate innate immune responses and subvert the control of AIEC replication.

Analysis of the σE regulon in Crohn's disease-associated Escherichia coli revealed involvement of the waaWVL operon in biofilm formation.

UNLABELLED: Ileal lesions of patients with Crohn's disease are colonized by adherent-invasive Escherichia coli (AIEC), which is able to adhere to and to invade intestinal epithelial cells (IEC), to replicate within macrophages, and to form biofilms on the surface of the intestinal mucosa. Previous analyses indicated the involvement of the σ(E) pathway in AIEC-IEC interaction, as well as in biofilm formation, with σ(E) pathway inhibition leading to an impaired ability of AIEC to colonize the intestinal mucosa and to form biofilms. The aim of this study was to characterize the σ(E) regulon of AIEC strain LF82 in order to identify members involved in AIEC phenotypes. Using comparative in silico analysis of the σ(E) regulon, we identified the waaWVL operon as a new member of the σ(E) regulon in reference AIEC strain LF82. We determined that the waaWVL operon is involved in AIEC lipopolysaccharide structure and composition, and the waaWVL operon was found to be essential for AIEC strains to produce biofilm and to colonize the intestinal mucosa. IMPORTANCE: An increased prevalence of adherent-invasive Escherichia coli (AIEC) bacteria was previously observed in the intestinal mucosa of Crohn's disease (CD) patients, and clinical observations revealed bacterial biofilms associated with the mucosa of CD patients. Here, analysis of the σ(E) regulon in AIEC and commensal E. coli identified 12 genes controlled by σ(E) only in AIEC. Among them, WaaWVL factors were found to play an essential role in biofilm formation and mucosal colonization by AIEC. In addition to identifying molecular tools that revealed a pathogenic population of E. coli colonizing the mucosa of CD patients, these results indicate that targeting the waaWVL operon could be a potent therapeutic strategy to interfere with the ability of AIEC to form biofilms and to colonize the gut mucosa.

Infectious etiopathogenesis of Crohn's disease.

Important advances during the last decade have been made in understanding the complex etiopathogenesis of Crohn's disease (CD). While many gaps in our knowledge still exist, it has been suggested that the etiology of CD is multifactorial including genetic, environmental and infectious factors. The most widely accepted theory states that CD is caused by an aggressive immune response to infectious agents in genetically predisposed individuals. The rise of genome-wide association studies allowed the identification of loci and genetic variants in several components of host innate and adaptive immune responses to microorganisms in the gut, highlighting an implication of intestinal microbiota in CD etiology. Moreover, numerous independent studies reported a dysbiosis, i.e., a modification of intestinal microbiota composition, with an imbalance between the abundance of beneficial and harmful bacteria. Although microorganisms including viruses, yeasts, fungi and bacteria have been postulated as potential CD pathogens, based on epidemiological, clinicopathological, genetic and experimental evidence, their precise role in this disease is not clearly defined. This review summarizes the current knowledge of the infectious agents associated with an increased risk of developing CD. Therapeutic approaches to modulate the intestinal dysbiosis and to target the putative CD-associated pathogens, as well as their potential mechanisms of action are also discussed.

Crohn's disease-associated adherent invasive Escherichia coli modulate levels of microRNAs in intestinal epithelial cells to reduce autophagy.

BACKGROUND & AIMS: Levels of microRNAs are altered in intestinal tissues of patients with Crohn's disease (CD). The adherent-invasive Escherichia coli (AIEC), which colonize the ileal mucosa of patients with CD, adhere to and invade intestinal epithelial cells. We investigated the mechanism by which AIEC infection alters the expression of microRNAs and the host immune response. METHODS: Levels of microRNAs in human intestinal epithelial T84 cells and in mouse enterocytes were measured using quantitative reverse-transcription polymerase chain reaction. Luciferase assays were used to measure binding of microRNAs to the 3'-untranslated region of messenger RNA targets. Binding of nuclear factor-κB to promoters of genes encoding microRNAs was assessed by chromatin immunoprecipitation assays. Autophagy was measured by immunoblot analyses and immunofluorescent labeling of LC3. Anti-microRNAs were transferred to mice using ileal loops. Biopsy specimens from the terminal ileum of patients with ulcerative colitis (n = 20), CD (n = 20), or individuals without inflammatory bowel disease undergoing surveillance colonoscopies (controls, n = 13) were collected during endoscopic examination. RESULTS: AIEC infection up-regulated levels of microRNA (MIR) 30C and MIR130A in T84 cells and in mouse enterocytes by activating nuclear factor-κB. Up-regulation of these microRNAs reduced the levels of ATG5 and ATG16L1 and inhibited autophagy, leading to increased numbers of intracellular AIEC and an increased inflammatory response. In ileal biopsy samples of patients with CD, there was an inverse correlation between levels of MIR30C and MIR130A and those of ATG5 and ATG16L1, supporting in vitro findings. Inhibition of MIR30C and MIR130A in cultured intestinal epithelial cells and in mouse enterocytes blocked AIEC-induced inhibition of ATG5 and ATG16L1 expression and restored functional autophagy. This resulted in more effective clearance of intracellular AIEC and reduced AIEC-induced inflammation. CONCLUSIONS: Infection with AIEC up-regulates microRNAs to reduce expression of proteins required for autophagy and autophagy response in intestinal epithelial cells. Ileal samples from patients with CD have increased levels of these same microRNAs and reduced levels of ATG5 and ATG16L1.