Karine Doiron

Control of intestinal homeostasis, colitis, and colitis-associated colorectal cancer by the inflammatory caspases.

Inflammatory caspases are essential effectors of inflammation and cell death. Here, we investigated their roles in colitis and colorectal cancer and report a bimodal regulation of intestinal homeostasis, inflammation and tumorigenesis by caspases-1 and -12. Casp1(-/-) mice exhibited defects in mucosal tissue repair and succumbed rapidly after dextran sulfate sodium administration. This phenotype was rescued by administration of exogenous interleukin-18 and was partially reproduced in mice deficient in the inflammasome adaptor ASC. Casp12(-/-) mice, in which the inflammasome is derepressed, were resistant to acute colitis and showed signs of enhanced repair. Together with their increased inflammatory response, the enhanced repair response of Casp12(-/-) mice rendered them more susceptible to colorectal cancer induced by azoxymethane (AOM)+DSS. Taken together, our results indicate that the inflammatory caspases are critical in the induction of inflammation in the gut after injury, which is necessary for tissue repair and maintenance of immune tolerance.

Cellular Inhibitors of Apoptosis cIAP1 and cIAP2 Are Required for Innate Immunity Signaling by the Pattern Recognition Receptors NOD1 and NOD2

Cellular inhibitor of apoptosis proteins (cIAPs) block apoptosis, but their physiological functions are still under investigation. Here, we report that cIAP1 and cIAP2 are E3 ubiquitin ligases that are required for receptor-interacting protein 2 (RIP2) ubiquitination and for nucleotide-binding and oligomerization (NOD) signaling. Macrophages derived from Birc2(-/-) or Birc3(-/-) mice, or colonocytes depleted of cIAP1 or cIAP2 by RNAi, were defective in NOD signaling and displayed sharp attenuation of cytokine and chemokine production. This blunted response was observed in vivo when Birc2(-/-) and Birc3(-/-) mice were challenged with NOD agonists. Defects in NOD2 signaling are associated with Crohns disease, and muramyl dipeptide (MDP) activation of NOD2 signaling protects mice from experimental colitis. Here, we show that administration of MDP protected wild-type but not Ripk2(-/-) or Birc3(-/-) mice from colitis, confirming the role of the cIAPs in NOD2 signaling in vivo. This discovery provides therapeutic opportunities in the treatment of NOD-dependent immunologic and inflammatory diseases.

The Caspase-1 Digestome Identifies the Glycolysis Pathway as a Target during Infection and Septic Shock

Caspase-1 is an essential effector of inflammation, pyroptosis, and septic shock. Few caspase-1 substrates have been identified to date, and these substrates do not account for its wide range of actions. To understand the function of caspase-1, we initiated the systematic identification of its cellular substrates. Using the diagonal gel proteomic approach, we identified 41 proteins that are directly cleaved by caspase-1. Among these were chaperones, cytoskeletal and translation machinery proteins, and proteins involved in immunity. A series of unexpected proteins along the glycolysis pathway were also identified, including aldolase, triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, α-enolase, and pyruvate kinase. With the exception of the latter, the identified glycolysis enzymes were specifically cleaved in vitro by recombinant caspase-1, but not caspase-3. The enzymatic activity of wild-type glyceraldehyde-3-phosphate dehydrogenase, but not a non-cleavable mutant, was dampened by caspase-1 processing. In vivo, stimuli that fully activated caspase-1, including Salmonella typhimurium infection and septic shock, caused a pronounced processing of these proteins in the macrophage and diaphragm muscle, respectively. Notably, these stimuli inhibited glycolysis in wild-type cells compared with caspase-1-deficient cells. The systematic characterization of caspase-1 substrates identifies the glycolysis pathway as a caspase-1 target and provides new insights into its function during pyroptosis and septic shock.

Cellular inhibitors of apoptosis proteins cIAP1 and cIAP2 are required for efficient caspase-1 activation by the inflammasome.

Pathogen and danger recognition by the inflammasome activates inflammatory caspases that mediate inflammation and cell death. The cellular inhibitor of apoptosis proteins (cIAPs) function in apoptosis and innate immunity, but their role in modulating the inflammasome and the inflammatory caspases is unknown. Here we report that the cIAPs are critical effectors of the inflammasome and are required for efficient caspase-1 activation. cIAP1, cIAP2, and the adaptor protein TRAF2 interacted with caspase-1-containing complexes and mediated the activating nondegradative K63-linked polyubiquitination of caspase-1. Deficiency in cIAP1 (encoded by Birc2) or cIAP2 (Birc3) impaired caspase-1 activation after spontaneous or agonist-induced inflammasome assembly, and Birc2(-/-) or Birc3(-/-) mice or mice administered with an IAP antagonist had a dampened response to inflammasome agonists and were resistant to peritonitis. Our results describe a role for the cIAPs in innate immunity and further demonstrate the evolutionary conservation between cell death and inflammation mechanisms.

Caspase-12 modulates NOD signaling and regulates antimicrobial peptide production and mucosal immunity.

Bacterial sensing by intracellular Nod proteins and other Nod-like receptors (NLRs) activates signaling pathways that mediate inflammation and pathogen clearance. Nod1 and Nod2 associate with the kinase Rip2 to stimulate NF-kappaB signaling. Other cytosolic NLRs assemble caspase-1-activating multiprotein complexes termed inflammasomes. Caspase-12 modulates the caspase-1 inflammasome, but unlike other NLRs, Nod1 and Nod2 have not been linked to caspases, and mechanisms regulating the Nod-Rip2 complex are less clear. We report that caspase-12 dampens mucosal immunity to bacterial infection independent of its effects on caspase-1. Caspase-12 deficiency enhances production of antimicrobial peptides, cytokines, and chemokines to entric pathogens, an effect dependent on bacterial type III secretion and the Nod pathway. Mechanistically, caspase-12 binds to Rip2, displacing Traf6 from the signaling complex, inhibiting its ubiquitin ligase activity, and blunting NF-kappaB activation. Nod activation and resulting antimicrobial peptide production constitute an early innate defense mechanism, and caspase-12 inhibits this mucosal antimicrobial response.

Non-apoptotic role of BID in inflammation and innate immunity

Innate immunity is a fundamental defence response that depends on evolutionarily conserved pattern recognition receptors for sensing infections or danger signals. Nucleotide-binding and oligomerization domain (NOD) proteins are cytosolic pattern-recognition receptors of paramount importance in the intestine, and their dysregulation is associated with inflammatory bowel disease. They sense peptidoglycans from commensal microorganisms and pathogens and coordinate signalling events that culminate in the induction of inflammation and anti-microbial responses. However, the signalling mechanisms involved in this process are not fully understood. Here, using genome-wide RNA interference, we identify candidate genes that modulate the NOD1 inflammatory response in intestinal epithelial cells. Our results reveal a significant crosstalk between innate immunity and apoptosis and identify BID, a BCL2 family protein, as a critical component of the inflammatory response. Colonocytes depleted of BID or macrophages from Bid−/− mice are markedly defective in cytokine production in response to NOD activation. Furthermore, Bid−/− mice are unresponsive to local or systemic exposure to NOD agonists or their protective effect in experimental colitis. Mechanistically, BID interacts with NOD1, NOD2 and the IκB kinase (IKK) complex, impacting NF-κB and extracellular signal-regulated kinase (ERK) signalling. Our results define a novel role of BID in inflammation and immunity independent of its apoptotic function, furthering the mounting evidence of evolutionary conservation between the mechanisms of apoptosis and immunity.

Caspase-12 controls West Nile virus infection via the viral RNA receptor RIG-I

Caspase-12 has been shown to negatively modulate inflammasome signaling during bacterial infection. Its function in viral immunity, however, has not been characterized. We now report an important role for caspase-12 in controlling viral infection via the pattern-recognition receptor RIG-I. After challenge with West Nile virus (WNV), caspase-12-deficient mice had greater mortality, higher viral burden and defective type I interferon response compared with those of challenged wild-type mice. In vitro studies of primary neurons and mouse embryonic fibroblasts showed that caspase-12 positively modulated the production of type I interferon by regulating E3 ubiquitin ligase TRIM25–mediated ubiquitination of RIG-I, a critical signaling event for the type I interferon response to WNV and other important viral pathogens.

Gender differences in expression of the human caspase-12 long variant determines susceptibility to Listeria monocytogenes infection

Inflammatory caspases are important effectors of innate immunity. Caspase-12, of the inflammatory caspase subfamily, is expressed in all mammals tested to date, but has acquired deleterious mutation in humans. A single-nucleotide polymorphism introduces a premature stop codon in caspase-12 in the majority of the population. However, in 20% of African descendants, caspase-12 is expressed and sensitizes to infections and sepsis. Here, we examined the modalities by which human caspase-12 confers susceptibility to infection. We have generated a fully humanized mouse that expresses the human caspase-12 rare variant (Csp-12L) in a mouse casp-12−/− background. Characterization of the humanized mouse uncovered sex differences in Csp-12L expression and gender disparity in innate immunity to Listeria monocytogenes infection. The Csp-12L transgene completely reversed the knockout resistance-to-infection phenotype in casp-12−/− males. In contrast, it had a marginal effect on the response of female mice. We found that estrogen levels modulated the expression of caspase-12. Csp-12L was expressed in male mice but its expression was repressed in female mice. Administration of 17-β-estradiol (E2) to humanized male mice had a direct suppressive effect on Csp-12L expression and conferred relative resistance to infection. Chromatin immunoprecipitation experiments revealed that caspase-12 is a direct transcriptional target of the estrogen receptor alpha (ERα) and mapped the estrogen response element (ERE) to intron 7 of the gene. We propose that estrogen-mediated inhibition of Csp-12L expression is a built-in mechanism that has evolved to protect females from infection.

Restriction of Legionella pneumophila Replication in Macrophages Requires Concerted Action of the Transcriptional Regulators Irf1 and Irf8 and Nod-Like Receptors Naip5 and Nlrc4

ABSTRACT The unique permissiveness of A/J mouse macrophages for replication of Legionella pneumophila is caused by a deficiency in the Nod-like receptor (NLR) protein and intracellular sensor for L. pneumophila flagellin (Naip5). The signaling pathways and proteins activated by Naip5 sensing in macrophages were investigated. Transcript profiling of macrophages from susceptible A/J mice and from resistant A/J mice harboring a transgenic wild-type copy of Naip5 at 4 h following L. pneumophila infection suggested that two members of the Irf transcriptional regulator family, Irf1 and Irf8, are regulated in response to Naip5 sensing of L. pneumophila. We show that macrophages having defective alleles of either Irf1 (Irf1−/−) or its heterodimerization partner gene Irf8 (Irf8R294C) become permissive for L. pneumophila replication, indicating that both the Irf1 and Irf8 proteins are essential for macrophage defense against L. pneumophila. Moreover, macrophages doubly heterozygous (Naip5AJ/WT Irf8R294C/WT or Nlrc4−/+Irf8R294C/WT) for combined loss-of-function mutations in Irf8 and in either Naip5 or Nlrc4 are highly susceptible to L. pneumophila, indicating that there is a strong genetic interaction between Irf8 and the NLR protein family in the macrophage response to L. pneumophila. Legionella-containing phagosomes (LCPs) formed in permissive Irf1−/− or Irf8R294C macrophages behave like LCPs formed in Naip5-insufficient and Nlrc4-deficient macrophages which fail to acidify. These results suggest that, in addition to Naip5 and Nlrc4, Irf1 and Irf8 play a critical role in the early response of macrophages to infection with L. pneumophila, including antagonizing the ability of L. pneumophila to block phagosome acidification. They also suggest that flagellin sensing by the NLR proteins Naip5 and Nlrc4 may be coupled to Irf1-Irf8-mediated transcriptional activation of key effector genes essential for macrophage resistance to L. pneumophila infection.

The mitochondrial protease HtrA2 restricts the NLRP3 and AIM2 inflammasomes.

Activation of the inflammasome pathway is crucial for effective intracellular host defense. The mitochondrial network plays an important role in inflammasome regulation but the mechanisms linking mitochondrial homeostasis to attenuation of inflammasome activation are not fully understood. Here, we report that the Parkinson’s disease-associated mitochondrial serine protease HtrA2 restricts the activation of ASC-dependent NLRP3 and AIM2 inflammasomes, in a protease activity-dependent manner. Consistently, disruption of the protease activity of HtrA2 results in exacerbated NLRP3 and AIM2 inflammasome responses in macrophages ex vivo and systemically in vivo. Mechanistically, we show that the HtrA2 protease activity regulates autophagy and controls the magnitude and duration of inflammasome signaling by preventing prolonged accumulation of the inflammasome adaptor ASC. Our findings identify HtrA2 as a non-redundant mitochondrial quality control effector that keeps NLRP3 and AIM2 inflammasomes in check.