NLRP1B allele 2 does not respond to Val-boro-Pro (VbP) in intestinal epithelial cells.

The intestinal mucosa must balance tolerance to commensal microbes and luminal antigens with rapid detection of enteric pathogens in order to maintain homeostasis. This balance is facilitated through the regulation of epithelial layer integrity by innate immune receptors. Certain NOD-like receptors (NLRs) expressed in intestinal epithelial cells, including NLRC4 and NLRP9B, form inflammasomes that protect against pathogens by activating caspase-1 to cause extrusion of infected cells. NLRP1B is a murine NLR encoded by five alleles of a highly polymorphic gene homologous to human NLRP1. NLRP1B forms inflammasomes in response to a variety of pathogens that cause intestinal infections, but it has almost exclusively been studied in immune cells and has not been characterized in cells of the intestinal epithelium. Here, we show that Nlrp1b allele 2 is expressed in ileal and colonic organoids derived for C57BL/6J mice, while the related gene Nlrp1a was not expressed. Nlrp1b was upregulated by interleukin-13 in organoids and by the protozoan Tritrichomonas muris in vivo, suggesting that NLRP1B may be involved in defense against enteric parasites. Surprisingly, while Val-boro-Pro (VbP) activated C57BL/6J-derived bone marrow-derived macrophages, which expressed both Nlrp1a and Nlrp1b, it did not activate intestinal organoids of the same genotype. We furthermore did not detect Nlrp1b in organoids derived from Balb/cJ mice, which express a different allele than the one expressed in C57BL/6J mice. Together, our results suggest that NLRP1B may have an allele-dependent function in murine IECs whose regulation is distinct from that of macrophages, and that the response to VbP might be exclusively driven by NLRP1A in C57BL/6J mice.

Methods to Activate the NLRP1 Inflammasome.

In addition to being the first NLR protein proposed to form inflammasome, NLRP1s have attracted much attention in their activation mechanism by post-translational auto-proteolysis to generate C-terminal CARD containing fragment to form inflammasome. Among NLRP1, mouse NLRP1B but not human NLRP1 is well studied for its activation by lethal toxin. As dissecting the cellular components involved in NLRP1-associated diseases is highly dependent on NLRP1 inflammasome activation, experiments that can lead to NLRP1 activation is of pivotal importance to elucidate the biological role and the activation mechanism of NLRP1 especially in human. In this chapter we describe methods commonly used for mouse NLRP1B inflammasome activation as well as activation of human NLRP1 inflammasome visualized by ASC speck formation in our laboratory.

Epigenetic combined with transcriptomic analysis of the m6A methylome after spared nerve injury-induced neuropathic pain in mice.

Epigenetic changes in the spinal cord play a key role in the initiation and maintenance of nerve injury-induced neuropathic pain. N6-methyladenosine (m6A) is one of the most abundant internal RNA modifications and plays an essential function in gene regulation in many diseases. However, the global m6A modification status of mRNA in the spinal cord at different stages after neuropathic pain is unknown. In this study, we established a neuropathic pain model in mice by preserving the complete sural nerve and only damaging the common peroneal nerve. High-throughput methylated RNA immunoprecipitation sequencing results showed that after spared nerve injury, there were 55 m6A methylated and differentially expressed genes in the spinal cord. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway results showed that m6A modification triggered inflammatory responses and apoptotic processes in the early stages after spared nerve injury. Over time, the differential gene function at postoperative day 7 was enriched in "positive regulation of neurogenesis" and "positive regulation of neural precursor cell proliferation." These functions suggested that altered synaptic morphological plasticity was a turning point in neuropathic pain formation and maintenance. Results at postoperative day 14 suggested that the persistence of neuropathic pain might be from lipid metabolic processes, such as "very-low-density lipoprotein particle clearance," "negative regulation of cholesterol transport" and "membrane lipid catabolic process." We detected the expression of m6A enzymes and found elevated mRNA expression of Ythdf2 and Ythdf3 after spared nerve injury modeling. We speculate that m6A reader enzymes also have an important role in neuropathic pain. These results provide a global landscape of mRNA m6A modifications in the spinal cord in the spared nerve injury model at different stages after injury.

UVB-Induced Skin Autoinflammation Due to Nlrp1b Mutation and Its Inhibition by Anti-IL-1ß Antibody.

NLRP1 (NACHT and leucine-rich repeat-containing protein family, pyrin domain-containing protein 1) is an innate immune sensor that is involved in the formation of inflammasome complexes. NLRP1 hyperactivity has been reported to cause inherited autoinflammatory diseases including familial keratosis lichenoides chronica and NLRP1-associated autoinflammation with arthritis and dyskeratosis. We generated Nlrp1b (the mouse homologue of human NLRP1) gain-of-function knock-in (Nlrp1b KI) mice with UVB irradiation-induced autoinflammatory skin lesions. We demonstrated that UVB irradiation induces IL-1ß upregulation and IL-1ß-dependent inflammation via caspase-1 activation in these Nlrp1b KI mice. RNA sequencing revealed the upregulation of inflammasome pathway-related genes, keratinocyte stress marker genes, and keratinocyte differentiation marker genes in the Nlrp1b KI mice after UVB irradiation. The skin inflammation and hyperkeratosis from UVB irradiation in the Nlrp1b KI mice were inhibited by both intraperitoneal and subcutaneous administration of anti-IL-1ß antibodies before UVB irradiation. UVB irradiation and the IL-1ß pathway are important in the pathogenesis of NLRP1-associated autoinflammatory skin lesions.

Role of pyroptosis in spinal cord injury and its therapeutic implications.

BACKGROUND: Currently, spinal cord injury (SCI) is a pathological incident that triggers several neuropathological conditions, leading to the initiation of neuronal damage with several pro-inflammatory mediators' release. However, pyroptosis is recognized as a new programmed cell death mechanism regulated by the stimulation of caspase-1 and/or caspase-11/-4/-5 signaling pathways with a series of inflammatory responses. AIM: Our current review concisely summarizes the potential role of pyroptosis-regulated programmed cell death in SCI, according to several molecular and pathophysiological mechanisms. This review also highlights the targeting of pyroptosis signaling pathways and inflammasome components and its therapeutic implications for the treatment of SCI. KEY SCIENTIFIC CONCEPTS: Multiple pieces of evidence have illustrated that pyroptosis plays significant roles in cell swelling, plasma membrane lysis, chromatin fragmentation and intracellular pro-inflammatory factors including IL-18 and IL-1ß release. In addition, pyroptosis is directly mediated by the recently discovered family of pore-forming protein known as GSDMD. Current investigations have documented that pyroptosis-regulated cell death plays a critical role in the pathogenesis of multiple neurological disorders as well as SCI. Our narrative article suggests that inhibiting the pyroptosis-regulated cell death and inflammasome components could be a promising therapeutic approach for the treatment of SCI in the near future.

BPTES inhibits anthrax lethal toxin-induced inflammatory response.

Bacillus anthracis is a lethal agent of anthrax disease and the toxins are required in anthrax pathogenesis. The anthrax lethal toxin can trigger NLRP1b inflammasome activation and pyroptosis. Although the underlying mechanism is well understood, the medications targeting the NLRP1b inflammasome are not available in the clinic. Herein, we describe that BPTES, a known Glutaminase (GLS) inhibitor, is an effective NLRP1b inflammasome inhibitor. BPTES could effectively and specifically suppress NLRP1b inflammasome activation in macrophages but have no effects on NLRP3, NLRC4 and AIM2 inflammasome activation. Mechanistically, BPTES alleviated the UBR2 mediated proteasomal degradation pathway of the NLRP1b N terminus, thus blocking the release of the CARD domain for subsequent caspase-1 processing. Furthermore, BPTES could prevent disease progression in mice challenged with the anthrax lethal toxin. Taken together, our studies indicate that BPTES can be a promising pharmacological inhibitor to treat anthrax lethal toxin-related inflammatory diseases.

Angiostrongylus cantonensis activates inflammasomes in meningoencephalitic BALB/c mice.

Angiostrongylus cantonensis is a metastrongyloid nematode that causes eosinophilic meningoencephalitis in humans. A high infestation of A. cantonensis can cause permanent brain damage or even death. The inflammasome is an oligomeric molecular platform that can detect microbial pathogens and activate inflammatory cytokines. The recognition of larval surface antigens by pattern recognition receptors (PRRs) can cause oligomerization of the NOD-like receptor (NLR) or absent in melanoma 2 (AIM2) with the adaptor apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) to form a caspase-1-activating scaffold. Activated caspase-1 converts pro-inflammatory cytokines into their mature, active forms. Helminths infection has been shown to activate NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasomes. In this study, we aimed to investigate the mechanism of inflammasome activation upon A. cantonensis infection in a mouse model. This study provides evidence that A. cantonensis infection can activate NLRP1B and NLRC4 inflammasomes and promote pyroptosis to cause meningoencephalitis.

The N-end rule ubiquitin ligase UBR2 mediates NLRP1B inflammasome activation by anthrax lethal toxin.

Anthrax lethal toxin (LT) is known to induce NLRP1B inflammasome activation and pyroptotic cell death in macrophages from certain mouse strains in its metalloprotease activity-dependent manner, but the underlying mechanism is unknown. Here, we establish a simple but robust cell system bearing dual-fluorescence reporters for LT-induced ASC specks formation and pyroptotic lysis. A genome-wide siRNA screen and a CRISPR-Cas9 knockout screen were applied to this system for identifying genes involved in LT-induced inflammasome activation. UBR2, an E3 ubiquitin ligase of the N-end rule degradation pathway, was found to be required for LT-induced NLRP1B inflammasome activation. LT is known to cleave NLRP1B after Lys44. The cleaved NLRP1B, bearing an N-terminal leucine, was targeted by UBR2-mediated ubiquitination and degradation. UBR2 partnered with an E2 ubiquitin-conjugating enzyme UBE2O in this process. NLRP1B underwent constitutive autocleavage before the C-terminal CARD domain. UBR2-mediated degradation of LT-cleaved NLRP1B thus triggered release of the noncovalent-bound CARD domain for subsequent caspase-1 activation. Our study illustrates a unique mode of inflammasome activation in cytosolic defense against bacterial insults.

Inhibition of Dpp8/9 Activates the Nlrp1b Inflammasome.

Val-boroPro (PT-100, Talabostat) induces powerful anti-tumor immune responses in syngeneic cancer models, but its mechanism of action has not yet been established. Val-boroPro is a non-selective inhibitor of post-proline-cleaving serine proteases, and the inhibition of the highly related cytosolic serine proteases Dpp8 and Dpp9 (Dpp8/9) by Val-boroPro was recently demonstrated to trigger an immunostimulatory form of programmed cell death known as pyroptosis selectively in monocytes and macrophages. Here we show that Dpp8/9 inhibition activates the inflammasome sensor protein Nlrp1b, which in turn activates pro-caspase-1 to mediate pyroptosis. This work reveals a previously unrecognized mechanism for activating an innate immune pattern recognition receptor and suggests that Dpp8/9 serve as an intracellular checkpoint to restrain Nlrp1b and the innate immune system.

Listeria monocytogenes and Shigella flexneri Activate the NLRP1B Inflammasome.

Activation of the innate immune receptor NLRP1B leads to the formation of an inflammasome, which induces autoproteolytic processing of pro-caspase-1, and ultimately to the release of inflammatory cytokines and to the execution of pyroptosis. One of the signals to which NLRP1B responds is metabolic stress that occurs in cells deprived of glucose or treated with metabolic inhibitors. NLRP1B might therefore sense microbial infection, as intracellular pathogens such as Listeria monocytogenes and Shigella flexneri cause metabolic stress as a result of nutrient scavenging and host cell damage. Here we addressed whether these pathogens activate the NLRP1B inflammasome. We found that Listeria infection activated the NLRP1B inflammasome in a reconstituted fibroblast model. Activation of NLRP1B by Listeria was diminished in an NLRP1B mutant shown previously to be defective at detecting energy stress and was dependent on the expression of listeriolysin O (LLO), a protein required for vacuolar escape. Infections of either Listeria or Shigella activated NLRP1B in the RAW264.7 murine macrophage line, which expresses endogenous NLRP1B. We conclude that NLRP1B senses cellular infection by distinct invasive pathogens.