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.

In vitro binding assay and co-immunoprecipitation experimentWe prepared purified S-tagged recombinant LTA and T7-tagged galectin-2 derived from E. coli using the pET system (Novagen), and combined them. The co-immunoprecipitation experiments were performed using a monoclonal antibody against LTA (R&D Systems) coupled to HiTrapTM NHS-activated Sepharose HP (Amersham). We visualized the immune complex using T7 tag antibody (Stratagene) and horseradish peroxidase (HRP) conjugated with anti-mouse IgG antibody. For coimmunoprecipitation in mammalian cells, we transfected expression plasmids of Flag or S-tagged LTA, galectin-2 and LacZ (as a negative control) into COS7 cells (HSRRB; JCRB9127) or HeLa cells using Fugene. Immunoprecipitations were done in lysis buffer (20 mM Tris pH 7.5, with 150 mM NaCl, 0.1 % Nonident P-40). Twenty-four hours after transfection, cells were lysed, and immunoprecipitations were performed using anti-Flag tag M2 agarose (Sigma). We visualized the immune complex using HRP-conjugated S-protein (Novagen), anti-Flag M2 peroxidase conjugate (Sigma) or mouse monoclonal antibody against human a-tubulin (Molecular Probes) and HRP-conjugated anti-mouse IgG antibody. Confocal microscopyPolyclonal anti-human galectin-2 antisera were raised in rabbits using recombinant protein synthesized in E. coli. The antisera showed no cross-reactivity to structurally related molecules galectin-1 and galectin-3, analysed by western blot. Polyclonal antigalectin-2 antisera and either goat anti-human LTA IgG (R&D Systems) or mouse antihuman a-tubulin monoclonal IgM antibodies were used with Alexa secondary antibodies (Molecular Probes). U937 cells (HSRRB; JCRB9021) were stimulated for 30 min with phorbol myristate acetate (PMA) (20 ng ml 21 ) and fixed. They were subsequently incubated with the corresponding primary antibodies in phosphatebuffered saline containing 3% bovine serum albumin, and the corresponding Alexa secondary antibodies. siRNA and over-expression experimentsThe target sequences for galectin-2 (5 0 -AATCCACCATTGTCTGCAACT-3 0 ) were cloned into pSilencer 2.0-U6 siRNA vector (Ambion). For the over-expression experiment, the galectin-2 was cloned into pFlag-CMV5a vector. After transfection, Jurkat cells were stimulated with PMA (20 ng ml 21 ) for 24 h, and cells and supernatants were collected separately. LTA concentration was measured using an LTA-specific ELISA system (R&D Systems), and normalized by comparison with total protein concentration. The mRNA quantification procedure has been described previously 2 . Luciferase assayA DNA fragment, corresponding to nucleotides 3,188 to 3,404 of intron-1 of LGALS2, was cloned into pGL3-enhancer vector (Promega) in the downstream of SV40 enhancer in the 5 0 to 3 0 orientation. After 24 h transfection, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega). ImmunohistochemistryTissue samples were obtained from 16 patients with MI by elective directional coronary atherectomy. Immunohistochemical protocols were carried out a...

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 Crohn's 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.

Caspases function in both apoptosis and inflammatory cytokine processing and thereby have a role in resistance to sepsis. Here we describe a novel role for a caspase in dampening responses to bacterial infection. We show that in mice, gene-targeted deletion of caspase-12 renders animals resistant to peritonitis and septic shock. The resulting survival advantage was conferred by the ability of the caspase-12-deficient mice to clear bacterial infection more efficiently than wild-type littermates. Caspase-12 dampened the production of the pro-inflammatory cytokines interleukin (IL)-1beta, IL-18 (interferon (IFN)-gamma inducing factor) and IFN-gamma, but not tumour-necrosis factor-alpha and IL-6, in response to various bacterial components that stimulate Toll-like receptor and NOD pathways. The IFN-gamma pathway was crucial in mediating survival of septic caspase-12-deficient mice, because administration of neutralizing antibodies to IFN-gamma receptors ablated the survival advantage that otherwise occurred in these animals. Mechanistically, caspase-12 associated with caspase-1 and inhibited its activity. Notably, the protease function of caspase-12 was not necessary for this effect, as the catalytically inactive caspase-12 mutant Cys299Ala also inhibited caspase-1 and IL-1beta production to the same extent as wild-type caspase-12. In this regard, caspase-12 seems to be the cFLIP counterpart for regulating the inflammatory branch of the caspase cascade. In mice, caspase-12 deficiency confers resistance to sepsis and its presence exerts a dominant-negative suppressive effect on caspase-1, resulting in enhanced vulnerability to bacterial infection and septic mortality.

The innate immune system provides first-line defences in response to invading microorganisms and endogenous danger signals by triggering robust inflammatory and antimicrobial responses. However, innate immune sensing of commensal microorganisms in the intestinal tract does not lead to chronic intestinal inflammation in healthy individuals, reflecting the intricacy of the regulatory mechanisms that tame the inflammatory response in the gut. Recent findings suggest that innate immune responses to commensal microorganisms, although once considered to be harmful, are necessary for intestinal homeostasis and immune tolerance. This Review discusses recent findings that identify a crucial role for innate immune effector molecules in protection against colitis and colitis-associated colorectal cancer and the therapeutic implications that ensue.

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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, ␣-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 wildtype 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.Caspases are aspartate-specific cysteine proteases known for their function in regulating programmed cell death and inflammation. Phylogenetically, they are subdivided into the CED3-related enzymes that initiate and execute cell death and the caspase-1-related proteins that process and mature cytokines, viz. IL-1␤, 2 IL-18, and IL-33. Apoptotic caspases execute cell death through the restricted cleavage of key cellular proteins required to maintain cell viability, resulting in the morphological changes observed during apoptosis, including membrane blebbing, nuclear condensation, and cytoskeletal dismantling (1). Caspase-1 is essential during inflammation because of its role in the activation of cytokine signaling pathways. With the exception of its cytokine substrates, very little is known regarding the spectrum of cellular proteins it targets upon full activation. Similar to other caspases, caspase-1 is found in cells as an inactive precursor and is activated in response to inflammatory triggers, including pathogen-derived molecules, as well as danger signals released from infected or dying cells (2). Caspase-1 activation is achieved in a macromolecular complex known as the inflammasome through its recruitment to a scaffolding molecule generally via the adaptor ASC (3). Scaffolding molecules that activate caspase-1 within the inflammasome belong to the cytosolic Nod-like family of pathogen recognition receptors and include Nalp1-3, Ipaf, and Naip5 (4). More recently, a distinct caspase-1 activat...

Nucleotide-binding and oligomerization domain NOD-like receptors (NLRs) are highly conserved cytosolic pattern recognition receptors that perform critical functions in surveying the intracellular environment for the presence of infection, noxious substances, and metabolic perturbations. Sensing of these danger signals by NLRs leads to their oligomerization into large macromolecular scaffolds and the rapid deployment of effector signaling cascades to restore homeostasis. While some NLRs operate by recruiting and activating inflammatory caspases into inflammasomes, others trigger inflammation via alternative routes including the nuclear factor-κB, mitogen-activated protein kinase, and regulatory factor pathways. The critical role of NLRs in development and physiology is demonstrated by their clear implications in human diseases. Mutations in the genes encoding NLRP3 or NLRP12 lead to hereditary periodic fever syndromes, while mutations in CARD15 that encodes NOD2 are linked to Crohn’s disease or Blau’s syndrome. Genome-wide association studies (GWASs) have identified a number of risk alleles encompassing NLR genes in a host of diseases including allergic rhinitis, multiple sclerosis, inflammatory bowel disease, asthma, multi-bacillary leprosy, vitiligo, early-onset menopause, and bone density loss in elderly women. Animal models have allowed the characterization of underlying effector mechanisms in a number of cases. In this review, we highlight the functions of NLRs in health and disease and discuss how the characterization of their molecular mechanisms provides new insights into therapeutic strategies for the management of inflammatory pathologies.