Vaccinia virus F1L blocks the ribotoxic stress response to subvert ZAKα-dependent NLRP1 inflammasome activation.

Inflammasomes are essential for host defense, recognizing foreign or stress signals to trigger immune responses, including maturation of IL-1 family cytokines and pyroptosis. Here, NLRP1 is emerging as an important sensor of viral infection in barrier tissues. NLRP1 is activated by various stimuli, including viral double-stranded (ds) RNA, ribotoxic stress, and inhibition of dipeptidyl peptidases 8 and 9 (DPP8/9). However, certain viruses, most notably the vaccinia virus, have evolved strategies to subvert inflammasome activation or effector functions. Using the modified vaccinia virus Ankara (MVA) as a model, we investigated how the vaccinia virus inhibits inflammasome activation. We confirmed that the early gene F1L plays a critical role in inhibiting NLRP1 inflammasome activation. Interestingly, it blocks dsRNA and ribotoxic stress-dependent NLRP1 activation without affecting its DPP9-inhibition-mediated activation. Complementation and loss-of-function experiments demonstrated the sufficiency and necessity of F1L in blocking NLRP1 activation. Furthermore, we found that F1L-deficient, but not wild-type MVA, induced ZAKα activation. Indeed, an F1L-deficient virus was found to disrupt protein translation more prominently than an unmodified virus, suggesting that F1L acts in part upstream of ZAKα. These findings underscore the inhibitory role of F1L on NLRP1 inflammasome activation and provide insight into viral evasion of host defenses and the intricate mechanisms of inflammasome activation.

Unexplained fever with consumptive syndrome in the elderly: two cases of VEXAS syndrome with inflammasome dysregulation.

The aim of this study is to investigate the inflammasome dysregulation in peripheral blood leukocytes of VEXAS patients. The constitutive and in vitro triggered activation of inflammasome in PBMC and neutrophils was analysed in two Brazilian patients with typical UBA1 mutations, and compared with heathy donors. Our findings highlight the constitutive activation of caspase-1 in VEXAS leukocytes, accompanied by increased plasma levels of IL-18. Furthermore, upon stimulation of isolated peripheral blood mononuclear cells (PBMC) and neutrophils, we observed not only the exhaustion of NLRP3 and NLRP1/CARD8 pathways in VEXAS PBMC but also a significant increase in NLRP3-mediated NETs release in VEXAS neutrophils. These findings support previous studies on the contribution of the inflammasome to VEXAS pathogenesis, identifying at least two profoundly affected pathways (NLRP3 and NLRP1/CARD8) in VEXAS peripheral blood.

The Serum NLRP1 Level and Coronary Artery Calcification: From Association to Development of a Risk-Prediction Nomogram.

Background: To investigate the correlation between inflammasomes and coronary artery calcification (CAC), and develop and validating a nomogram for predicting the risk of CAC in patients with coronary artery disease (CAD). Methods: A total of 626 patients with CAD at the Affiliated Hospital of Xuzhou Medical University were enrolled in this study. The patients were divided into the calcification group and the non-calcification group based on the assessment of coronary calcification. We constructed a training set and a validation set through random assignment. The least absolute shrinkage and selection operator (LASSO) regression and multivariate analysis were performed to identify independent risk factors of CAC in patients with CAD. Based on these independent predictors, we developed a web-based dynamic nomogram prediction model. The area under the receiver operating characteristic curve (AUC-ROC), calibration curves, and decision curve analysis (DCA) were used to evaluate this nomogram. Results: Age, smoking, diabetes mellitus (DM), hyperlipidemia, the serum level of nucleotide-binding oligomerization domain (NOD)-like receptor protein 1 (NLRP1), alkaline phosphatase (ALP) and triglycerides (TG) were identified as independent risk factors of CAC. The AUC-ROC of the nomogram is 0.881 (95% confidence interval (CI): 0.850-0.912) in the training set and 0.825 (95% CI: 0.760-0.876) in the validation set, implying high discriminative ability. Satisfactory performance of this model was confirmed using calibration curves and DCA. Conclusions: The serum NLRP1 level is an independent predictor of CAC. We established a web-based dynamic nomogram, providing a more accurate estimation and comprehensive perspective for predicting the risk of CAC in patients with CAD.

HMGB1/TLR4 axis promotes pyroptosis after ICH by activating the NLRP3 inflammasome.

BACKGROUND: We previously reported that the HMGB1/TLR4 axis promoted inflammation during the acute phase of intracerebral hemorrhage. Given that this phase is known to involve neuronal pyroptosis and neuroinflammation, here we explore whether HMGB1/TLR signaling activate inflammasome and pyroptosis after intracerebral hemorrhage. METHODS: Autologous blood was injected into Sprague-Dawley rats to induce intracerebral hemorrhage. Neurological deficits were assessed using a modified neurological severity score. These expression and localization of NLRP1 and NLRP3 inflammasomes, as well as the levels of pyroptosis and pyroptosis-associated proteins were assessed using Western blot or immunocytochemistry. These experiments were repeated in animals that received treatment with short interfering RNAs against NLRP1 or NLRP3, with HMGB1 inhibitor ethyl pyruvate or TLR4 inhibitor TAK-242. RESULTS: Intracerebral hemorrhage upregulated NLRP1 and NLRP3 in the ipsilateral striatum and increased the proportions of these cells that were pyroptosis-positive. Additionally, the levels of caspase protein family (e.g., pro-caspase-1 and caspase-1), apoptosis-associated speck-like protein (ASC), pro-interleukin-1ß (IL-1ß), and IL-1ß were also elevated. These effects on pyroptosis and associated neurological deficit, were partially reversed by knockdown of NLRP1 or NLRP3, or by inhibition of HMGB1 or TLR4. Inhibition of HMGB1 or TLR4 resulted in the downregulation NLRP3 but not NLRP1. CONCLUSIONS: The HMGB1/TLR4 signaling may activate the NLRP3 inflammasome during the acute phase of intracerebral hemorrhage, resulting in the inflammatory process known as pyroptosis. These insights suggest potential therapeutic targets for the mitigation tissue injury and associated neurological deficits following hemorrhagic stroke.

Celecoxib inhibits NLRP1 inflammasome pathway in MDA-MB-231 Cells.

NLRP1 is predominantly overexpressed in breast cancer tissue, and the evaluated activation of NLRP1 inflammasome is associated with tumor growth, angiogenesis, and metastasis. Therefore, targeting NLRP1 activation could be a crucial strategy in anticancer therapy. In this study, we investigated the hypothesis that NLRP1 pathway may contribute to the cytotoxic effects of celecoxib and nimesulide in MDA-MB-231 cells. First of all, IC50 values and inhibitory effects on the colony-forming ability of drugs were evaluated in cells. Then, the alterations in the expression levels of NLRP1 inflammasome components induced by drugs were investigated. Subsequently, the release of inflammatory cytokine IL-1ß and the activity of caspase-1 in drug-treated cells were measured. According to our results, celecoxib and nimesulide selectively inhibited the viability of MDA-MB-231 cells. These drugs remarkably inhibited the colony-forming ability of cells. The expression levels of NLRP1 inflammasome components decreased in celecoxib-treated cells, accompanied by decreased caspase-1 activity and IL-1ß release. In contrast, nimesulide treatment led to the upregulation of the related protein expressions with unchanged caspase-1 activity and increased IL-1ß secretion. Our results indicated that the NLRP1 inflammasome pathway might contribute to the antiproliferative effects of celecoxib in MDA-MB-231 cells but is not a crucial mechanism for nimesulide.

Thioredoxin-1 Protects Neurons Through Inhibiting NLRP1-Mediated Neuronal Pyroptosis in Models of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common neurodegenerative disease all over the world. In the last decade, accumulating proofs have evidenced that neuroinflammation is intimately implicated in the pathogenesis of AD and activation of NOD-like receptor family pyrin domain-containing 1 (NLRP1) inflammasome can induce neuronal pyroptosis and in turn lead to neuronal loss in AD. Thioredoxin-1 (Trx-1), a multifunctional molecule with anti-inflammation in human tissues, displays crucial neuroprotective roles in AD. Our previous research preliminarily found that Trx-1 inhibition enhanced the expression of NLRP1, caspase-1, and gasdermin D (GSDMD) in Aß25-35-treated PC12 cells. However, it is largely unknown if Trx-1 can inhibit NLRP1-mediated neuronal pyroptosis in AD neurons. In this study, it was verified that the protein levels of NLRP1, caspase-1, and GSDMD were significantly increased in Aß25-35-treated mouse HT22 and primary hippocampal neurons. Suppression of Trx-1 with PX-12, a selective inhibitor of Trx-1, or Trx-1 knockdown further activated NLRP1-mediated neuronal pyroptosis. On the contrary, lentivirus infection-mediated Trx-1 overexpression in differentiated PC12 cells dramatically reversed expression of NLRP1, caspase-1, and GSDMD. Furthermore, Trx-1 overexpression mediated by adeno-associated virus in the hippocampal tissues of APP/PS1 mice likewise attenuated the activation of NLRP1-mediated neuronal pyroptosis, as well as reduced the hippocampal deposition of Aß and ameliorated the cognitive function of APP/PS1 mice. In conclusion, this article predicates a novel molecular mechanism by which Trx-1 exploits neuroprotection through attenuating NLRP1-mediated neuronal pyroptosis in AD models, suggesting that Trx-1 may be a promising therapeutic target for AD.

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.

NLRP1 inflammasome promotes senescence and senescence-associated secretory phenotype.

BACKGROUND: Senescence is a cellular aging-related process triggered by different stresses and characterized by the secretion of various inflammatory factors referred to as senescence-associated secretory phenotype (SASP), some of which are produced by the NLRP3 inflammasome. Here, we present evidence that the NLRP1 inflammasome is a DNA damage sensor and a key mediator of senescence. METHODS: Senescence was induced in fibroblasts in vitro and in mice. Cellular senescence was assessed by Western blot analysis of several proteins, including p16, p21, p53, and SASP factors, released in the culture media or serum. Inflammasome components, including NLRP1, NLRP3 and GSDMD were knocked out or silenced using siRNAs. RESULTS: In vitro and in vivo results suggest that the NLRP1 inflammasome promotes senescence by regulating the expression of p16, p21, p53, and SASP factors in a Gasdermin D (GSDMD)-dependent manner. Mechanistically, the NLRP1 inflammasome is activated in response to genomic damage detected by the cytosolic DNA sensor cGMP-AMP (cGAMP) synthase (cGAS). CONCLUSION: Our findings show that NLRP1 is a cGAS-dependent DNA damage sensor during senescence and a mediator of SASP release through GSDMD. This study advances the knowledge on the biology of the NLRP1 inflammasome and highlights this pathway as a potential pharmcological target to modulate senescence.

CARD8: A Novel Inflammasome Sensor with Well-Known Anti-Inflammatory and Anti-Apoptotic Activity.

Inflammasomes comprise a group of protein complexes with fundamental roles in the induction of inflammation. Upon sensing stress factors, their assembly induces the activation and release of the pro-inflammatory cytokines interleukin (IL)-1ß and -18 and a lytic type of cell death, termed pyroptosis. Recently, CARD8 has joined the group of inflammasome sensors. The carboxy-terminal part of CARD8, consisting of a function-to-find-domain (FIIND) and a caspase activation and recruitment domain (CARD), resembles that of NLR family pyrin domain containing 1 (NLRP1), which is recognized as the main inflammasome sensor in human keratinocytes. The interaction with dipeptidyl peptidases 8 and 9 (DPP8/9) represents an activation checkpoint for both sensors. CARD8 and NLRP1 are activated by viral protease activity targeting their amino-terminal region. However, CARD8 also has some unique features compared to the established inflammasome sensors. Activation of CARD8 occurs independently of the inflammasome adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), leading mainly to pyroptosis rather than the activation and secretion of pro-inflammatory cytokines. CARD8 was also shown to have anti-inflammatory and anti-apoptotic activity. It interacts with, and inhibits, several proteins involved in inflammation and cell death, such as the inflammasome sensor NLRP3, CARD-containing proteins caspase-1 and -9, nucleotide-binding oligomerization domain containing 2 (NOD2), or nuclear factor kappa B (NF-κB). Single nucleotide polymorphisms (SNPs) of CARD8, some of them occurring at high frequencies, are associated with various inflammatory diseases. The molecular mechanisms underlying the different pro- and anti-inflammatory activities of CARD8 are incompletely understood. Alternative splicing leads to the generation of multiple CARD8 protein isoforms. Although the functional properties of these isoforms are poorly characterized, there is evidence that suggests isoform-specific roles. The characterization of the functions of these isoforms, together with their cell- and disease-specific expression, might be the key to a better understanding of CARD8's different roles in inflammation and inflammatory diseases.

Combined therapy with IL-1 and JAK inhibitors in a patient with the NLRP1 gene mutation and a complex inflammatory phenotype.

A patient presented with overlapping clinical and laboratory features of 2 rare autoinflammatory diseases, NLRP1-associated autoinflammation with arthritis and dyskeratosis and familial multiple self-healing palmoplantar carcinoma. Her severe inflammatory attack was treated with the IL-1 receptor-α inhibitor anakinra along with the Janus kinase inhibitor ruxolitinib. Three years into the treatment, the patient's inflammatory symptoms are completely in remission.

A Prospective Cohort Study of Elevated Serum NLRP1 Levels to Prognosticate Neurological Outcome After Acute Intracerebral Hemorrhage at a Single Academic Institution.

Background: Nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 1 (NLRP1) participates in neuroinflammation. This study aimed to identify serum NLRP as a potential prognostic biomarker of acute intracerebral hemorrhage (ICH). Methods: This prospective cohort study enrolled 145 patients with supratentorial ICH and 51 healthy controls. Serum NLRP1 levels were quantified on admission of all 145 patients, on days 1, 3, 5, 7, and 10 after stroke in 51 of 145 patients and at entry into the study of controls. Poststroke 6-month modified Rankin Scale (mRS) scores of 3-6 signified a poor prognosis. Results: Compared to controls, patients had prominently increased serum NLRP1 levels until day 10 after ICH, with the highest levels at days 1 and 3. Serum NLRP1 levels were independently correlated with National Institutes of Health Stroke Scale (NIHSS) scores, hematoma volume and six-month mRS scores, and independently predicted six-month bad prognosis. A linear relationship was observed between serum NLRP1 levels and the risk of poor prognosis in a restricted cubic spline. Under the receiver operating characteristic (ROC) curve, serum NLRP levels efficiently discriminated poor prognosis. Serum NLRP1, NIHSS, and hematoma volume were merged into a prognosis prediction model, which was portrayed using a nomogram. Good performance of the model was verified using calibration curve, decision curve, and ROC curve. Conclusion: Serum NLRP1 levels are elevated during the early period following ICH and are independently related to hemorrhagic severity and poor prognosis, suggesting that serum NLRP1 may represent a promising prognostic biomarker of ICH.

Inflammasomes in epithelial innate immunity: front line warriors.

Our epithelium represents a battle ground against a variety of insults including pathogens and danger signals. It encodes multiple sensors that detect and respond to such insults, playing an essential role in maintaining and defending tissue homeostasis. One key set of defense mechanisms is our inflammasomes which drive innate immune responses including, sensing and responding to pathogen attack, through the secretion of pro-inflammatory cytokines and cell death. Identification of physiologically relevant triggers for inflammasomes has greatly influenced our ability to decipher the mechanisms behind inflammasome activation. Furthermore, identification of patient mutations within inflammasome components implicates their involvement in a range of epithelial diseases. This review will focus on exploring the roles of inflammasomes in epithelial immunity and cover: the diversity and differential expression of inflammasome sensors amongst our epithelial barriers, their ability to sense local infection and damage and the contribution of the inflammasomes to epithelial homeostasis and disease.

Increased NLRP1 mRNA and Protein Expression Suggests Inflammasome Activation in the Dorsolateral Prefrontal and Medial Orbitofrontal Cortex in Schizophrenia.

Schizophrenia is a complex mental condition, with key symptoms marked for diagnosis including delusions, hallucinations, disorganized thinking, reduced emotional expression, and social dysfunction. In the context of major developmental hypotheses of schizophrenia, notably those concerning maternal immune activation and neuroinflammation, we studied NLRP1 expression and content in the postmortem brain tissue of 10 schizophrenia and 10 control subjects. In the medial orbitofrontal cortex (Brodmann's area 11/12) and dorsolateral prefrontal cortex (area 46) from both hemispheres of six schizophrenia subjects, the NLRP1 mRNA expression was significantly higher than in six control brains (p < 0.05). As the expression difference was highest for the medial orbitofrontal cortex in the right hemisphere, we assessed NLRP1-immunoreactive pyramidal neurons in layers III, V, and VI in the medial orbitofrontal cortex in the right hemisphere of seven schizophrenia and five control brains. Compared to controls, we quantified a significantly higher number of NLRP1-positive pyramidal neurons in the schizophrenia brains (p < 0.01), suggesting NLRP1 inflammasome activation in schizophrenia subjects. Layer III pyramidal neuron dysfunction aligns with working memory deficits, while impairments of pyramidal neurons in layers V and VI likely disrupt predictive processing. We propose NLRP1 inflammasome as a potential biomarker and therapeutic target in schizophrenia.

Does NLRP1 Inflammasome Activation in Immune Cells in Kidney Transplantation Relate with Donor Organ Age?

Inflammation causes a wide range of health disorders. In this process, the formation of inflammasome complexes plays a key role. Although inflammasomes have been extensively studied during kidney disease, their role in kidney transplantation has not been fully elucidated. In this study, we evaluate the gene and protein expression of several components of the inflammasome pathway before and at several time points after kidney transplantation in a cohort of patients of different ages and receiving an organ from older or younger donors. Our findings indicate the activation of the NLRP1 inflammasome in several immune cell population, monocytes and CD4+ and CD8+ cells mainly, in renal transplant, and its level increases gradually in patients who receive an older organ, whereas it has the opposite effect on older patients who receive a younger organ. Despite treatment with immunosuppressants, inflammation persists in some patients. These results lead to the hypothesis that the donor's age is a critical factor in post-transplant inflammasome activation and that specific NLRP1 inflammasome inhibitors should be considered to increase the success of kidney transplantation long-term.

Host E3 ubiquitin ligase ITCH mediates Toxoplasma gondii effector GRA35-triggered NLRP1 inflammasome activation and cell-autonomous immunity.

Toxoplasma gondii is an intracellular parasite that can activate the NLRP1 inflammasome leading to macrophage pyroptosis in Lewis rats, but the underlying mechanism is not well understood. In this study, we performed a genome-wide CRISPR screen and identified the dense granule proteins GRA35, GRA42, and GRA43 as the Toxoplasma effectors mediating cell death in Lewis rat macrophages. GRA35 localizes on the parasitophorous vacuole membrane, where it interacts with the host E3 ubiquitin ligase ITCH. Inhibition of proteasome activity or ITCH knockout prevented pyroptosis in Toxoplasma-infected Lewis rat macrophages, consistent with the "NLRP1 functional degradation model." However, there was no evidence that ITCH directly ubiquitinates or interacts with rat NLRP1. We also found that GRA35-ITCH interaction affected Toxoplasma fitness in IFNγ-activated human fibroblasts, likely due to ITCH's role in recruiting ubiquitin and the parasite-restriction factor RNF213 to the parasitophorous vacuole membrane. These findings identify a new role of host E3 ubiquitin ligase ITCH in mediating effector-triggered immunity, a critical concept that involves recognizing intracellular pathogens and initiating host innate immune responses.IMPORTANCEEffector-triggered immunity represents an innate immune defense mechanism that plays a crucial role in sensing and controlling intracellular pathogen infection. The NLRP1 inflammasome in the Lewis rats can detect Toxoplasma infection, which triggers proptosis in infected macrophages and eliminates the parasite's replication niche. The work reported here revealed that host E3 ubiquitin ligase ITCH is able to recognize and interact with Toxoplasma effector protein GRA35 localized on the parasite-host interface, leading to NLRP1 inflammasome activation in Lewis rat macrophages. Furthermore, ITCH-GRA35 interaction contributes to the restriction of Toxoplasma in human fibroblasts stimulated by IFNγ. Thus, this research provides valuable insights into understanding pathogen recognition and restriction mediated by host E3 ubiquitin ligase.

Mechanistic basis for potassium efflux-driven activation of the human NLRP1 inflammasome.

Nigericin, an ionophore derived from Streptomyces hygroscopicus, is arguably the most commonly used tool compound to study the NLRP3 inflammasome. Recent findings, however, showed that nigericin also activates the NLRP1 inflammasome in human keratinocytes. In this study, we resolve the mechanistic basis of nigericin-driven NLRP1 inflammasome activation. In multiple nonhematopoietic cell types, nigericin rapidly and specifically inhibits the elongation stage of the ribosome cycle by depleting cytosolic potassium ions. This activates the ribotoxic stress response (RSR) sensor kinase ZAKα, p38, and JNK, as well as the hyperphosphorylation of the NLRP1 linker domain. As a result, nigericin-induced pyroptosis in human keratinocytes is blocked by extracellular potassium supplementation, ZAKα knockout, or pharmacologic inhibitors of ZAKα and p38 kinase activities. By surveying a panel of ionophores, we show that electroneutrality of ion movement is essential to activate ZAKα-driven RSR and a greater extent of K+ depletion is necessary to activate ZAKα-NLRP1 than NLRP3. These findings resolve the mechanism by which nigericin activates NLRP1 in nonhematopoietic cell types and demonstrate an unexpected connection between RSR, perturbations of potassium ion flux, and innate immunity.

Pathogen recognition pathway gene variants and inflammasome sensors gene expression in tuberculosis patients under treatment.

BACKGROUND: Several epidemiological studies have suggested that genetic variations in encoding pattern recognition receptors (PRRs) genes such as Toll Like Receptors (TLRs) and their signaling products, may influence the susceptibility, severity and outcome of tuberculosis (TB). After sensing a pathogen, the cell responds producing an inflammatory response, to restrain the pathogen's successful course of infection. Herein we assessed single nucleotide polymorphisms (SNP) and gene expression from pathogen recognition and inflammasome pathways in Brazilian TB patients. METHODS AND RESULTS: For genetic association analysis we included MYD88 and TLR4, PRRs sensing proteins. Allele distribution for MYD88 rs6853 (A > G) and TLR4 rs7873784 (C > G) presented conserved among the tested samples with statistically differential distribution in TB patients versus controls. However, when testing according to sample ethnicity (African or Caucasian-derived individuals) we identified that the rs6853 G/G genotype was associated with a lower susceptibility to TB in Caucasian population. Meanwhile, the rs7873784 G/G genotype was associated with a higher TB susceptibility in Afro-descendant ethnicity individuals. We also aimed to verify MYD88 and the inflammasome genes NLRP1 and NLRC4 expression in order to connect to active TB and/or clinical aspects. CONCLUSIONS: We identified that inflammasome gene expression in TB patients under treatment display a similar pattern as in healthy controls, indicating that TB treatment impairs NLRP1 inflammasome activation.

The interaction between NLRP1 and oxidized TRX1 involves a transient disulfide bond.

NLRP1 is an innate immune receptor that detects pathogen-associated signals, assembles into a multiprotein structure called an inflammasome, and triggers a proinflammatory form of cell death called pyroptosis. We previously discovered that the oxidized, but not the reduced, form of thioredoxin-1 directly binds to NLRP1 and represses inflammasome formation. However, the molecular basis for NLRP1's selective association with only the oxidized form of TRX1 has not yet been established. Here, we leveraged AlphaFold-Multimer, site-directed mutagenesis, thiol-trapping experiments, and mass spectrometry to reveal that a specific cysteine residue (C427 in humans) on NLRP1 forms a transient disulfide bond with oxidized TRX1. Overall, this work demonstrates how NLRP1 monitors the cellular redox state, further illuminating an unexpected connection between the intracellular redox potential and the innate immune system.

Autophagy dysfunction contributes to NLRP1 inflammasome-linked depressive-like behaviors in mice.

BACKGROUND: Major depressive disorder (MDD) is a common but severe psychiatric illness characterized by depressive mood and diminished interest. Both nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 1 (NLRP1) inflammasome and autophagy have been reported to implicate in the pathological processes of depression. However, the mechanistic interplay between NLRP1 inflammasome, autophagy, and depression is still poorly known. METHODS: Animal model of depression was established by chronic social defeat stress (CSDS). Depressive-like behaviors were determined by social interaction test (SIT), sucrose preference test (SPT), open field test (OFT), forced swim test (FST), and tail-suspension test (TST). The protein expression levels of NLRP1 inflammasome complexes, pro-inflammatory cytokines, phosphorylated-phosphatidylinositol 3-kinase (p-PI3K)/PI3K, phosphorylated-AKT (p-AKT)/AKT, phosphorylated-mechanistic target of rapamycin (p-mTOR)/mTOR, brain-derived neurotrophic factor (BDNF), phosphorylated-tyrosine kinase receptor B (p-TrkB)/TrkB, Bcl-2-associated X protein (Bax)/B-cell lymphoma-2 (Bcl2) and cleaved cysteinyl aspartate-specific proteinase-3 (caspase-3) were examined by western blotting. The mRNA expression levels of pro-inflammatory cytokines were tested by quantitative real-time PCR. The interaction between proteins was detected by immunofluorescence and coimmunoprecipitation. Neuronal injury was assessed by Nissl staining. The autophagosomes were visualized by transmission electron microscopy. Nlrp1a knockdown was performed using an adeno-associated virus (AAV) vector containing Nlrp1a-shRNA-eGFP infusion. RESULTS: CSDS exposure caused a bidirectional change in hippocampal autophagy function, which was activated in the initial period but impaired at the later stage. In addition, CSDS exposure increased the expression levels of hippocampal NLRP1 inflammasome complexes, pro-inflammatory cytokines, p-PI3K, p-AKT and p-mTOR in a time-dependent manner. Interestingly, NLRP1 is immunoprecipitated with mTOR but not PI3K/AKT and CSDS exposure facilitated the immunoprecipitation between them. Hippocampal Nlrp1a knockdown inhibited the activity of PI3K/AKT/mTOR signaling, rescued the impaired autophagy and ameliorated depressive-like behavior induced by CSDS. In addition, rapamycin, an autophagy inducer, abolished NLRP1 inflammasome-driven inflammatory reactions, alleviated depressive-like behavior and exerted a neuroprotective effect. CONCLUSIONS: Autophagy dysfunction contributes to NLRP1 inflammasome-linked depressive-like behavior in mice and the regulation of autophagy could be a valuable therapeutic strategy for the management of depression.

SREBP1 deficiency diminishes glutamate-mediated HT22 cell damage and hippocampal neuronal pyroptosis induced by status epilepticus.

Status epilepticus (SE) is a life-threatening disorder that can result in death or severe brain damage, and there is a substantial body of evidence suggesting a strong association between pyroptosis and SE. Sterol regulatory element binding protein 1 (SREBP1) is a significant transcription factor participating in both lipid homeostasis and glucose metabolism. However, the function of SREBP1 in pyroptosis during SE remains unknown. In this study, we established a SE rat model by intraperitoneal injection of lithium chloride and pilocarpine in vivo. Additionally, we treated HT22 hippocampal cells with glutamate to create neuronal injury models in vitro. Our results demonstrated a significant induction of SREBP1, inflammasomes, and pyroptosis in the hippocampus of SE rats and glutamate-treated HT22 cells. Moreover, we found that SREBP1 is regulated by the mTOR signaling pathway, and inhibiting mTOR signaling contributed to the amelioration of SE-induced hippocampal neuron pyroptosis, accompanied by a reduction in SREBP1 expression. Furthermore, we conducted siRNA-mediated knockdown of SREBP1 in HT22 cells and observed a significant reversal of glutamate-induced cell death, activation of inflammasomes, and pyroptosis. Importantly, our confocal immunofluorescence analysis revealed the co-localization of SREBP1 and NLRP1. In conclusion, our findings suggest that deficiency of SREBP1 attenuates glutamate-induced HT22 cell injury and hippocampal neuronal pyroptosis in rats following SE. Targeting SREBP1 may hold promise as a therapeutic strategy for SE.