Mohamed Lamkanfi

The NLRP3 Inflammasome Protects against Loss of Epithelial Integrity and Mortality during Experimental Colitis

Decreased expression of the Nlrp3 protein is associated with susceptibility to Crohns disease. However, the role of Nlrp3 in colitis has not been characterized. Nlrp3 interacts with the adaptor protein ASC to activate caspase-1 in inflammasomes, which are protein complexes responsible for the maturation and secretion of interleukin-1beta (IL-1beta) and IL-18. Here, we showed that mice deficient for Nlrp3 or ASC and caspase-1 were highly susceptible to dextran sodium sulfate (DSS)-induced colitis. Defective inflammasome activation led to loss of epithelial integrity, resulting in systemic dispersion of commensal bacteria, massive leukocyte infiltration, and increased chemokine production in the colon. This process was a consequence of a decrease in IL-18 in mice lacking components of the Nlrp3 inflammasome, resulting in higher mortality rates. Thus, the Nlrp3 inflammasome is critically involved in the maintenance of intestinal homeostasis and protection against colitis.

Caspases in cell survival, proliferation and differentiation

Caspases, a family of evolutionarily, conserved cysteinyl proteases, mediate both apoptosis and inflammation through aspartate-specific cleavage of a wide number of cellular substrates. Most substrates of apoptotic caspases have been conotated with cellular dismantling, while inflammatory caspases mediate the proteolytic activation of inflammatory cytokines. Through detailed functional analysis of conditional caspase-deficient mice or derived cells, caspase biology has been extended to cellular responses such as cell differentiation, proliferation and NF-κB activation. Here, we discuss recent data indicating that non-apoptotic functions of caspases involve proteolysis exerted by their catalytic domains as well as non-proteolytic functions exerted by their prodomains. Homotypic oligomerization motifs in the latter mediate the recruitment of adaptors and effectors that modulate NF-κB activation. The non-apoptotic functions of caspases suggest that they may become activated independently of – or without – inducing an apoptotic cascade. Moreover, the existence of non-catalytic caspase-like molecules such as human caspase-12, c-FLIP and CARD-only proteins further supports the non-proteolytic functions of caspases in the regulation of cell survival, proliferation, differentiation and inflammation.

Redundant roles for inflammasome receptors NLRP3 and NLRC4 in host defense against Salmonella

Intracellular pathogens and endogenous danger signals in the cytosol engage NOD-like receptors (NLRs), which assemble inflammasome complexes to activate caspase-1 and promote the release of proinflammatory cytokines IL-1β and IL-18. However, the NLRs that respond to microbial pathogens in vivo are poorly defined. We show that the NLRs NLRP3 and NLRC4 both activate caspase-1 in response to Salmonella typhimurium. Responding to distinct bacterial triggers, NLRP3 and NLRC4 recruited ASC and caspase-1 into a single cytoplasmic focus, which served as the site of pro–IL-1β processing. Consistent with an important role for both NLRP3 and NLRC4 in innate immune defense against S. typhimurium, mice lacking both NLRs were markedly more susceptible to infection. These results reveal unexpected redundancy among NLRs in host defense against intracellular pathogens in vivo.

Manipulation of Host Cell Death Pathways during Microbial Infections

Viral and microbial infections often elicit programmed cell death as part of the host defense system or as a component of the survival strategy of the pathogen. It is thus not surprising that pathogens have evolved an array of toxins and virulence factors to modulate host cell death pathways. Apoptosis, necrosis, and pyroptosis constitute the three major cell death modes for elimination of infected cells. Herein, we discuss the signaling pathways underlying the principal host cell death mechanisms and provide an overview of the strategies employed by viral and microbial pathogens to manipulate these cell death processes.

The mitochondrial serine protease HtrA2/Omi: an overview

The HtrA family refers to a group of related oligomeric serine proteases that combine a trypsin-like protease domain with at least one PDZ interaction domain. Mammals encode four HtrA proteases, named HtrA1–4. The protease activity of the HtrA member HtrA2/Omi is required for mitochondrial homeostasis in mice and humans and inactivating mutations associated with neurodegenerative disorders such as Parkinsons disease. Moreover, HtrA2/Omi is released in the cytosol, where it contributes to apoptosis through both caspase-dependent and -independent pathways. Here, we review the current knowledge of HtrA2/Omi biology and discuss the signaling pathways that underlie its mitochondrial and apoptotic functions from an evolutionary perspective.

NLRP6 negatively regulates innate immunity and host defence against bacterial pathogens

Members of the intracellular nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) family contribute to immune responses through activation of nuclear factor-κB (NF-κB), type I interferon and inflammasome signalling. Mice lacking the NLR family member NLRP6 were recently shown to be susceptible to colitis and colorectal tumorigenesis, but the role of NLRP6 in microbial infections and the nature of the inflammatory signalling pathways regulated by NLRP6 remain unclear. Here we show that Nlrp6-deficient mice are highly resistant to infection with the bacterial pathogens Listeria monocytogenes, Salmonella typhimurium and Escherichia coli. Infected Nlrp6-deficient mice had increased numbers of monocytes and neutrophils in circulation, and NLRP6 signalling in both haematopoietic and radioresistant cells contributed to increased susceptibility. Nlrp6 deficiency enhanced activation of mitogen-activated protein kinase (MAPK) and the canonical NF-κB pathway after Toll-like receptor ligation, but not cytosolic NOD1/2 ligation, in vitro. Consequently, infected Nlrp6-deficient cells produced increased levels of NF-κB- and MAPK-dependent cytokines and chemokines. Thus, our results reveal NLRP6 as a negative regulator of inflammatory signalling, and demonstrate a role for this NLR in impeding clearance of both Gram-positive and -negative bacterial pathogens.

Targeted Peptidecentric Proteomics Reveals Caspase-7 as a Substrate of the Caspase-1 Inflammasomes

The aspartate-specific cysteine protease caspase-1 is activated by the inflammasomes and is responsible for the proteolytic maturation of the cytokines IL-1β and IL-18 during infection and inflammation. To discover new caspase-1 substrates, we made use of a proteome-wide gel-free differential peptide sorting methodology that allows unambiguous localization of the processing site in addition to identification of the substrate. Of the 1022 proteins that were identified, 20 were found to be specifically cleaved after Asp in the setup incubated with recombinant caspase-1. Interestingly, caspase-7 emerged as one of the identified caspase-1 substrates. Moreover half of the other identified cleavage events occurred at sites closely resembling the consensus caspase-7 recognition sequence DEVD, suggesting caspase-1-mediated activation of endogenous caspase-7 in this setup. Consistently recombinant caspase-1 cleaved caspase-7 at the canonical activation sites Asp23 and Asp198, and recombinant caspase-7 processed a subset of the identified substrates. In vivo, caspase-7 activation was observed in conditions known to induce activation of caspase-1, including Salmonella infection and microbial stimuli combined with ATP. Interestingly Salmonella- and lipopolysaccharide + ATP-induced activation of caspase-7 was abolished in macrophages deficient in caspase-1, the pattern recognition receptors Ipaf and Cryopyrin, and the inflammasome adaptor ASC, demonstrating an upstream role for the caspase-1 inflammasomes in caspase-7 activation in vivo. In contrast, caspase-1 and the inflammasomes were not required for caspase-3 activation. In conclusion, we identified 20 new substrates activated downstream of caspase-1 and validated caspase-1-mediated caspase-7 activation in vitro and in knock-out macrophages. These results demonstrate for the first time the existence of a nucleotide binding and oligomerization domain-like receptor/caspase-1/caspase-7 cascade and the existence of distinct activation mechanisms for caspase-3 and -7 in response to microbial stimuli and bacterial infection.

FADD and Caspase-8 Mediate Priming and Activation of the Canonical and Noncanonical Nlrp3 Inflammasomes

The Nlrp3 inflammasome is critical for host immunity, but the mechanisms controlling its activation are enigmatic. In this study, we show that loss of FADD or caspase-8 in a RIP3-deficient background, but not RIP3 deficiency alone, hampered transcriptional priming and posttranslational activation of the canonical and noncanonical Nlrp3 inflammasome. Deletion of caspase-8 in the presence or absence of RIP3 inhibited caspase-1 and caspase-11 activation by Nlrp3 stimuli but not the Nlrc4 inflammasome. In addition, FADD deletion prevented caspase-8 maturation, positioning FADD upstream of caspase-8. Consequently, FADD- and caspase-8–deficient mice had impaired IL-1β production when challenged with LPS or infected with the enteropathogen Citrobacter rodentium. Thus, our results reveal FADD and caspase-8 as apical mediators of canonical and noncanonical Nlrp3 inflammasome priming and activation.

Receptor interacting protein kinase 2–mediated mitophagy regulates inflammasome activation during virus infection

NOD2 receptor and the cytosolic protein kinase RIPK2 regulate NF-κB and MAP kinase signaling during bacterial infections, but the role of this immune axis during viral infections has not been addressed. We demonstrate that Nod2−/− and Ripk2−/− mice are hypersusceptible to infection with influenza A virus. Ripk2−/− cells exhibited defective autophagy of mitochondria (mitophagy), leading to enhanced mitochondrial production of superoxide and accumulation of damaged mitochondria, which resulted in greater activation of the NLRP3 inflammasome and production of IL-18. RIPK2 regulated mitophagy in a kinase-dependent manner by phosphorylating the mitophagy inducer ULK1. Accordingly, Ulk1−/− cells exhibited enhanced mitochondrial production of superoxide and activation of caspase-1. These results demonstrate a role for NOD2-RIPK2 signaling in protection against virally triggered immunopathology by negatively regulating activation of the NLRP3 inflammasome and production of IL-18 via ULK1-dependent mitophagy.

Toll or Interleukin-1 Receptor (TIR) Domain-containing Adaptor Inducing Interferon-β (TRIF)-mediated Caspase-11 Protease Production Integrates Toll-like Receptor 4 (TLR4) Protein- and Nlrp3 Inflammasome-mediated Host Defense against Enteropathogens

Background: C. rodentium and E. coli induce noncanonical Nlrp3 inflammasome activation through caspase-11. Results: TLR4-TRIF are important for caspase-11 expression, caspase-1 activation, and downstream IL-1β and IL-18 production. Conclusion: TLR4-TRIF axis plays an important role in the up-regulation of caspase-11 and activation of noncanonical inflammasome. Significance: Our study identifies novel molecules upstream of caspase-11 that are involved in activation of noncanonical inflammasome. Enteric pathogens represent a major cause of morbidity and mortality worldwide. Toll-like receptor (TLR) and inflammasome signaling are critical for host responses against these pathogens, but how these pathways are integrated remains unclear. Here, we show that TLR4 and the TLR adaptor TRIF are required for inflammasome activation in macrophages infected with the enteric pathogens Escherichia coli and Citrobacter rodentium. In contrast, TLR4 and TRIF were dispensable for Salmonella typhimurium-induced caspase-1 activation. TRIF regulated expression of caspase-11, a caspase-1-related protease that is critical for E. coli- and C. rodentium-induced inflammasome activation, but dispensable for inflammasome activation by S. typhimurium. Thus, TLR4- and TRIF-induced caspase-11 synthesis is critical for noncanonical Nlrp3 inflammasome activation in macrophages infected with enteric pathogens.