Biology subjects

Mitchell, P. S.

Publications and source records attributed to Mitchell, P. S..

8 recordsLinked to original sources

bioRxiv2026

TAK1 integrates the NLRP1 inflammasome into the innate immune response to double-stranded RNA

Innate immune recognition of double-stranded RNA (dsRNA) by germline-encoded receptors initiates antiviral defenses, including type I interferon (IFN) production. The inflammasome-forming sensor NLRP1 binds and is activated by dsRNA in a mitogen-activated protein kinase (MAPK) p38-dependent manner. How dsRNA initiates these events to induce NLRP1 inflammasome activation is unclear. Here we demonstrate that both exogenous and cellular dsRNA triggers NLRP1 inflammasome activation downstream of RIG-I/MDA5-MAVS and/or TLR3-TRIF signaling but is independent of type I IFN. In immortalized and primary human keratinocytes, we find that NLRP1 inflammasome activation by dsRNA, including during viral infection, requires the MAPK kinase kinase TAK1. Mechanistically, TAK1-dependent phosphorylation of the NLRP1 N-terminal disordered region is necessary and sufficient for inflammasome activation. Collectively, we reveal TAK1 as a novel activator of the NLRP1 inflammasome, functioning as a critical signaling hub linking NLRP1 to inflammatory responses in the context of viral infection and autoimmunity.

immunology↗

bioRxiv2025

Shigella OspF blocks rapid p38-dependent priming of the NAIP-NLRC4 inflammasome

The NAIP-NLRC4 inflammasome senses pathogenic bacteria by recognizing the cytosolic presence of bacterial proteins such as flagellin and type III secretion system (T3SS) subunits. In mice, the NAIP-NLRC4 inflammasome provides robust protection against bacterial pathogens that infect intestinal epithelial cells, including the gastrointestinal pathogen Shigella flexneri. By contrast, humans are highly susceptible to Shigella, despite the ability of human NAIP-NLRC4 to robustly detect Shigella T3SS proteins. Why the NAIP-NLRC4 inflammasome protects mice but not humans against Shigella infection remains unclear. We previously found that human THP-1 cells infected with Shigella lose responsiveness to NAIP-NLRC4 stimuli, while retaining sensitivity to other inflammasome agonists. Using mT3Sf, a "minimal Shigella" system, to express individual secreted Shigella effector proteins, we found that the OspF effector specifically suppresses NAIP-NLRC4-dependent cell death during infection. OspF was previously characterized as a phosphothreonine lyase that inactivates p38 and ERK MAP kinases. We found that p38 was critical for rapid priming of NAIP-NLRC4 activity, particularly in cells with low NAIP-NLRC4 expression. Overall, our results provide a mechanism by which Shigella evades inflammasome activation in humans, and describe a new mechanism for rapid priming of the NAIP-NLRC4 inflammasome.

immunology↗

bioRxiv2024

CARD8 inflammasome activation during HIV-1 cell-to-cell transmission

Our previous work demonstrated that CARD8 detects HIV-1 infection by sensing the enzymatic activity of the HIV protease, resulting in CARD8-dependent inflammasome activation (Kulsuptrakul et al., 2023). CARD8 harbors a motif in its N-terminus that functions as a HIV protease substrate mimic, permitting innate immune recognition of HIV-1 protease activity, which when cleaved by HIV protease triggers CARD8 inflammasome activation. Here, we sought to understand CARD8 responses in the context of HIV-1 cell-to-cell transmission via a viral synapse. We observed that cell-to-cell transmission of HIV-1 between infected T cells and primary human monocyte-derived macrophages induces CARD8 inflammasome activation in a manner that is dependent on viral protease activity and largely independent of the NLRP3 inflammasome. Additionally, to further evaluate the viral determinants of CARD8 sensing, we tested a panel of HIV protease inhibitor resistant clones to establish how variation in HIV protease affects CARD8 activation. We identified mutant HIV-1 proteases that differentially cleave and activate CARD8 compared to wildtype HIV-1, thus indicating that natural variation in HIV protease affects not only the cleavage of the viral Gag-Pol polyprotein but also likely impacts innate sensing and inflammation.

immunology↗

bioRxiv2023

Intracellular replication of Pseudomonas aeruginosa in epithelial cells requires suppression of the caspase-4 inflammasome

Pathogenesis of Pseudomonas aeruginosa infections can include bacterial survival inside epithelial cells. Previously, we showed this involves multiple roles played by the type three-secretion system (T3SS), and specifically the effector ExoS. This includes ExoS-dependent inhibition of a lytic host cell response that subsequently enables intracellular replication. Here, we studied the underlying cell death response to intracellular P. aeruginosa, comparing wild-type to T3SS mutants varying in capacity to induce cell death and that localize to different intracellular compartments. Results showed that corneal epithelial cell death induced by intracellular P. aeruginosa lacking the T3SS, which remains in vacuoles, correlated with activation of NF-{kappa}B as measured by p65 relocalization and TNF transcription and secretion. Deletion of caspase-4 through CRISPR-Cas9 mutagenesis delayed cell death caused by these intracellular T3SS mutants. Caspase-4 deletion also countered more rapid cell death caused by T3SS effector-null mutants still expressing the TSSS apparatus that traffic to the host cell cytoplasm, and in doing so rescued intracellular replication normally dependent on ExoS. While HeLa cells lacked a lytic death response to T3SS mutants, it was found to be enabled by interferon gamma treatment. Together, these results show that epithelial cells can activate the noncanonical inflammasome pathway to limit proliferation of intracellular P. aeruginosa, not fully dependent on bacterially-driven vacuole escape. Since ExoS inhibits the lytic response, the data implicate targeting of caspase-4, an intracellular pattern recognition receptor, as another contributor to the role of ExoS in the intracellular lifestyle of P. aeruginosa. ImportancePseudomonas aeruginosa can exhibit an intracellular lifestyle within epithelial cells in vivo and in vitro. The type three secretion system (T3SS) effector ExoS contributes via multiple mechanisms, including extending the life of invaded host cells. Here, we aimed to understand the underlying cell death inhibited by ExoS when P. aeruginosa is intracellular. Results showed that intracellular P. aeruginosa lacking T3SS effectors could elicit rapid cell lysis via the non-canonical inflammasome pathway. Caspase-4 contributed to cell lysis even when the intracellular bacteria lacked the entire T33S and were consequently unable to escape vacuoles, representing a naturally occurring subpopulation during wildtype infection. Together, the data show the caspase-4 inflammasome as an epithelial cell defense against intracellular P. aeruginosa, and implicate its targeting as another mechanism by which ExoS preserves the host cell replicative niche.

microbiology↗

bioRxiv2022

Host specific sensing of coronaviruses and picornaviruses by the CARD8 inflammasome

Hosts have evolved diverse strategies to respond to microbial infections, including the detection of pathogen-encoded proteases by inflammasome-forming sensors such as NLRP1 and CARD8. Here, we find that the 3CL protease (3CLpro) encoded by diverse coronaviruses, including SARS-CoV-2, cleaves a rapidly evolving region of human CARD8 and activates a robust inflammasome response. CARD8 is required for cell death and the release of pro-inflammatory cytokines during SARS-CoV-2 infection. We further find that natural variation alters CARD8 sensing of 3CLpro, including 3CLpro-mediated antagonism rather than activation of megabat CARD8. Likewise, we find that a single nucleotide polymorphism (SNP) in humans reduces CARD8s ability to sense coronavirus 3CLpros, and instead enables sensing of 3C proteases (3Cpro) from select picornaviruses. Our findings demonstrate that CARD8 is a broad sensor of viral protease activities and suggests that CARD8 diversity contributes to inter- and intra-species variation in inflammasome-mediated viral sensing and immunopathology.

immunology↗

bioRxiv2020

Diverse viral proteases activate the NLRP1 inflammasome

The NLRP1 inflammasome is a multiprotein complex that is a potent activator of inflammation. Mouse NLRP1B can be activated through proteolytic cleavage by the bacterial Lethal Toxin (LeTx) protease, resulting in degradation of the N-terminal domains of NLRP1B and liberation of the bioactive C-terminal domain, which includes the caspase activation and recruitment domain (CARD). However, a natural pathogen-derived effector that can activate human NLRP1 remains unknown. Here, we use an evolutionary model to identify several proteases from diverse picornaviruses that cleave human NLRP1 within a rapidly evolving region of the protein, leading to host-specific and virus-specific activation of the NLRP1 inflammasome. Our work demonstrates that NLRP1 acts as a "tripwire" to recognize the enzymatic function of a wide range of viral proteases, and suggests that host mimicry of viral polyprotein cleavage sites can be an evolutionary strategy to activate a robust inflammatory immune response.

microbiology↗

bioRxiv2020

NAIP-NLRC4-deficient mice are susceptible to shigellosis

Bacteria of the genus Shigella cause shigellosis, a severe gastrointestinal disease that is a major cause of diarrhea-associated mortality in humans. Shigellosis develops upon oral ingestion of as few as 100 bacteria, but million-fold higher doses fail to cause disease in mice. The lack of a physiologically relevant mouse model of shigellosis has impeded our understanding of this important human disease, but why mice are resistant is unknown. Here we show that in human cells, but not in mice, Shigella evades detection by the NAIP-NLRC4 inflammasome, an immune sensor present in intestinal epithelial cells (IECs). We find that NAIP-NLRC4-deficient mice are highly susceptible to oral Shigella infection and recapitulate the clinical features of human shigellosis, including bacterial replication in IECs and neutrophilic inflammation of the colon. Confirming a role for bacterial replication in IECs in our new model, a Shigella mutant lacking IcsA, a factor required for cell-to-cell spread among IECs, is attenuated in otherwise susceptible NAIP-NLRC4-deficient mice. Although inflammasome-mediated cell death is widely held to promote Shigella infection and pathogenesis, we instead demonstrate that IEC-specific NAIP-NLRC4-induced cell death is sufficient to protect the host from shigellosis. Thus, NAIP-NLRC4-deficient mice are a physiologically relevant and experimentally tractable model for shigellosis. More broadly, our results suggest that the lack of an inflammasome response in IECs may help explain the extreme susceptibility of humans to shigellosis.

microbiology↗

bioRxiv2018

Functional degradation: a mechanism of NLRP1 inflammasome activation by diverse pathogen enzymes

Inflammasomes are multi-protein platforms that initiate innate immunity by recruitment and activation of Caspase-1. The NLRP1B inflammasome is activated upon direct cleavage by the anthrax lethal toxin protease. However, the mechanism by which cleavage results in NLRP1B activation is unknown. Here we find that cleavage results in proteasome-mediated degradation of the N-terminal domains of NLRP1B, liberating a C-terminal fragment that is a potent Caspase-1 activator. Proteasome-mediated degradation of NLRP1B is both necessary and sufficient for NLRP1B activation. Consistent with our new functional degradation model, we identify IpaH7.8, a Shigella flexneri ubiquitin ligase secreted effector, as an enzyme that induces NLRP1B degradation and activation. Our results provide a unified mechanism for NLRP1B activation by diverse pathogen-encoded enzymatic activities.\n\nOne Sentence SummaryTwo distinct pathogen enzymes activate an innate immune sensor called NLRP1B by a mechanism that requires proteasome-mediated degradation of NLRP1B.

immunology↗