CARD8 polymorphisms among bacterial meningitis patients in North-West Ethiopia.

BACKGROUND: The severity of infectious disease outcomes is dependent on the virulence factors of the pathogen and the host immune response. CARD8 is a major regulator of the innate immune proinflammatory response and has been suggested to modulate the host response to common inflammatory diseases. In the present study, the C10X genetic polymorphism in the CARD8 gene was investigated in relation to bacterial meningitis. METHODS: A total of 400 clinically suspected meningitis patients hospitalized at the University of Gondar Hospital were enrolled in the study. Cerebrospinal fluid (CSF) and blood samples were collected for laboratory investigations. The collected CSF was cultured, and all the results obtained from the culture were confirmed using direct RT‒PCR. Genotyping of whole-blood samples was performed using a TaqMan assay. The results were compared with apparently healthy controls and with PCR-negative meningitis suspected patients. RESULTS: Of the included patients, 57% were men and the most common clinical signs and symptoms were fever (81%), headache (80%), neck stiffness (76%), nausea (68%), and vomiting (67%). Microbiology culture identified 7 patients with bacterial meningitis caused by Neisseria meningitidis (n = 4) and Streptococcus pneumoniae (n = 3). The RT-PCR revealed 39 positive samples for N. meningitidis (n = 10) and S. pneumoniae (n = 29). A total of 332 whole-blood samples were genotyped with the following results: 151 (45.5%) C10X heterozygotes, 59 (17.7%) C10X homozygotes and 122 (36.7%) wild genotypes. The polymorphic gene carriers among laboratory confirmed, clinically diagnosed meningitis and healthy controls were 23(46%), 246(40%), and 1526(39%), respectively with OR = 1.27 (0.7-2.3) and OR = 1.34 (0.76-2.4). The presence of the C10X polymorphism in the CARD8 gene was more prevalent in suspected meningitis patients than in healthy controls (OR 1.2; 1.00-1.5). Homozygote C10X polymorphic gene carriers were more susceptible to infectious disease. The presence of viable or active bacterial infection was found to be associated with the presence of heterozygous C10X carriers. CONCLUSIONS: A greater proportion of C10X in the CARD8 gene in confirmed bacterial meningitis patients and clinically diagnosed meningitis patients than in healthy controls. Homozygote C10X polymorphic gene carriers were more susceptible to infectious disease than heterozygote gene carriers and healthy controls.

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 hydrophobicity of the CARD8 N-terminus tunes inflammasome activation.

Mounting evidence indicates that proteotoxic stress is a primary activator of the CARD8 inflammasome, but the complete array of signals that control this inflammasome have not yet been established. Notably, we recently discovered that several hydrophobic radical-trapping antioxidants (RTAs), including JSH-23, potentiate CARD8 inflammasome activation through an unknown mechanism. Here, we report that these RTAs directly alkylate several cysteine residues in the N-terminal disordered region of CARD8. These hydrophobic modifications destabilize the repressive CARD8 N-terminal fragment and accelerate its proteasome-mediated degradation, thereby releasing the inflammatory CARD8 C-terminal fragment from autoinhibition. Consistently, we also found that unrelated (non-RTA) hydrophobic electrophiles as well as genetic mutation of the CARD8 cysteine residues to isoleucines similarly potentiate inflammasome activation. Overall, our results not only provide further evidence that protein folding stress is a key CARD8 inflammasome-activating signal, but also indicate that the N-terminal cysteines can play key roles in tuning the response to this stress.

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.

Case Report: Identification of a CARD8 variant in all three patients with PFAPA syndrome complicated with Kawasaki disease.

Background: Periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA syndrome), and Kawasaki disease (KD) are both considered to be disorders of the innate immune system, and the potential role of inflammasome activation in the immunopathogenesis of both diseases has been previously described. Case presentation: Herein, we report the clinical courses of three patients who presented a rare combination of PFAPA syndrome and KD. Two patients who presented KD later developed the PFAPA syndrome, of whom one developed recurrent KD 2 years after the initial diagnosis. The third patient developed KD one year after the onset of PFAPA syndrome. The presence of both of these conditions within individual patients, combined with the knowledge that inflammasome activation is involved in both PFAPA syndrome and KD, suggests a shared background of inflammatory dysregulation. To elucidate the mechanism underlying shared inflammatory dysregulation, we investigated the roles of Nod-like receptors (NLRs) and their downstream inflammasome-related genes. All the patients had a frameshift variant in CARD8 (CARD8-FS). A previous study demonstrated a higher frequency of CARD8-FS, whose product loses CARD8 activity and activates the NLRP3 inflammasome, in patients with the PFAPA syndrome. Additionally, the NLRP3 inflammasome is known to be activated in patients with KD. Together, these results suggest that the CARD8-FS variant may also be essential in KD pathogenesis. As such, we analyzed the CARD8 variants among patients with KD. However, we found no difference in the variant frequency between patients with KD and the general Japanese population. Conclusions: We report the clinical courses of three patients with a rare combination of PFAPA syndrome and KD. All the patients had the CARD8-FS variant. However, we could not find a difference in the variant frequency between patients with KD and the general Japanese population. As the frequency of KD is much higher than that of PFAPA among Japanese patients, and the cause of KD is multifactorial, it is possible that only a small portion of patients with KD harbor CARD8-FS as a causative gene.

The CARD8 inflammasome dictates HIV/SIV pathogenesis and disease progression.

While CD4+ T cell depletion is key to disease progression in people living with HIV and SIV-infected macaques, the mechanisms underlying this depletion remain incompletely understood, with most cell death involving uninfected cells. In contrast, SIV infection of "natural" hosts such as sooty mangabeys does not cause CD4+ depletion and AIDS despite high-level viremia. Here, we report that the CARD8 inflammasome is activated immediately after HIV entry by the viral protease encapsulated in incoming virions. Sensing of HIV protease activity by CARD8 leads to rapid pyroptosis of quiescent cells without productive infection, while T cell activation abolishes CARD8 function and increases permissiveness to infection. In humanized mice reconstituted with CARD8-deficient cells, CD4+ depletion is delayed despite high viremia. Finally, we discovered loss-of-function mutations in CARD8 from "natural hosts," which may explain the peculiarly non-pathogenic nature of these infections. Our study suggests that CARD8 drives CD4+ T cell depletion during pathogenic HIV/SIV infections.

Association between C10X polymorphism in the CARD8 gene and inflammatory markers in young healthy individuals in the LBA study.

BACKGROUND: The Caspase activation and recruitment domain 8 (CARD8) protein is a component of innate immunity as a negative regulator of NF- ĸB, and has been associated with regulation of proteins involved in inflammation. Expression of CARD8 mRNA and protein has been identified in human atherosclerotic lesions, and the truncated T30A variant (rs2043211) of CARD8 has been associated with lower C-reactive (CRP) and MCP-1 levels in myocardial infarction patients. The present study examines the role of a genetic variation in the CARD8 gene in relation to a selection of markers of inflammation. METHODS: In a cross-sectional study of young healthy individuals (18.0-25.9 yrs, n = 744) the association between the rs2043211 variant in the CARD8 gene and protein markers of inflammation was assessed. Genotyping of the CARD8 C10X (rs2043211) polymorphism was performed with TaqMan real time PCR on DNA from blood samples. Protein levels were studied via Olink inflammation panel ( https://olink.com/ ). Using linear models, we analyzed men and two groups of women with and without estrogen containing contraceptives separately, due to previous findings indicating differences between estrogen users and non-estrogen using women. Genotypes were analyzed by additive, recessive and dominant models. RESULTS: The minor (A) allele of the rs2043211 polymorphism in the CARD8 gene was associated with lower levels of CCL20 and IL-6 in men (CCL20, Additive model: p = 0.023; Dominant model: p = 0.016. IL-6, Additive model: p = 0.042; Dominant model: p = 0.039). The associations remained significant also after adjustment for age and potential intermediate variables. CONCLUSIONS: Our data indicate that CARD8 may be involved in the regulation of CCL20 and IL-6 in men. No such association was observed in women. These findings strengthen and support previous in vitro data on IL-6 and CCL20 and highlight the importance of CARD8 as a factor in the regulation of inflammatory proteins. The reason to the difference between sexes is however not clear, and the influence of estrogen as a possible factor important for the inflammatory response needs to be further explored.

Innate Immune Gene Polymorphisms and COVID-19 Prognosis.

COVID-19 is characterized by a heterogeneous clinical presentation and prognosis. Risk factors contributing to the development of severe disease include old age and the presence of comorbidities. However, the genetic background of the host has also been recognized as an important determinant of disease prognosis. Considering the pivotal role of innate immunity in the control of SARS-CoV-2 infection, we analyzed the possible contribution of several innate immune gene polymorphisms (including TLR2-rs5743708, TLR4-rs4986790, TLR4-rs4986791, CD14-rs2569190, CARD8-rs1834481, IL18-rs2043211, and CD40-rs1883832) in disease severity and prognosis. A total of 249 individuals were enrolled and further divided into five (5) groups, according to the clinical progression scale provided by the World Health Organization (WHO) (asymptomatic, mild, moderate, severe, and critical). We identified that elderly patients with obesity and/or diabetes mellitus were more susceptible to developing pneumonia and respiratory distress syndrome after SARS-CoV-2 infection, while the IL18-rs1834481 polymorphism was an independent risk factor for developing pneumonia. Moreover, individuals carrying either the TLR2-rs5743708 or the TLR4-rs4986791 polymorphisms exhibited a 3.6- and 2.5-fold increased probability for developing pneumonia and a more severe disease, respectively. Our data support the notion that the host's genetic background can significantly affect COVID-19 clinical phenotype, also suggesting that the IL18-rs1834481, TLR2-rs5743708, and TLR4-rs4986791 polymorphisms may be used as molecular predictors of COVID-19 clinical phenotype.

Hemophagocytic lymphohistiocytosis-like hyperinflammation due to a de novo mutation in DPP9.

BACKGROUND: Genetic defects in components of inflammasomes can cause autoinflammation. Biallelic loss-of-function mutations in dipeptidyl peptidase 9 (DPP9), a negative regulator of the NLRP1 and CARD8 inflammasomes, have recently been shown to cause an inborn error of immunity characterized by pancytopenia, skin manifestations, and increased susceptibility to infections. OBJECTIVE: We sought to study the molecular basis of autoinflammation in a patient with severe infancy-onset hyperinflammation associated with signs of fulminant hemophagocytic lymphohistiocytosis. METHODS: Using heterologous cell models as well as patient cells, we performed genetic, immunologic, and molecular investigations to identify the genetic cause and to assess the impact of the identified mutation on inflammasome activation. RESULTS: The patient exhibited pancytopenia with decreased neutrophils and T, B, and natural killer cells, and markedly elevated levels of lactate dehydrogenase, ferritin, soluble IL-2 receptor, and triglycerides. In addition, serum levels of IL-1ß and IL-18 were massively increased, consistent with inflammasome activation. Genetic analysis revealed a previously undescribed de novo mutation in DPP9 (c.755G>C, p.Arg252Pro) affecting a highly conserved amino acid residue. The mutation led to destabilization of the DPP9 protein as shown in transiently transfected HEK293T cells and in patient-derived induced pluripotent stem cells. Using functional inflammasome assays in HEK293T cells, we demonstrated that mutant DPP9 failed to restrain the NLRP1 and CARD8 inflammasomes, resulting in constitutive inflammasome activation. These findings suggest that the Arg252Pro DPP9 mutation acts in a dominant-negative manner. CONCLUSIONS: A de novo mutation in DPP9 leads to severe infancy-onset autoinflammation because of unleashed inflammasome activation.

A human-specific motif facilitates CARD8 inflammasome activation after HIV-1 infection.

Inflammasomes are cytosolic innate immune complexes that assemble upon detection of diverse pathogen-associated cues and play a critical role in host defense and inflammatory pathogenesis. Here, we find that the human inflammasome-forming sensor CARD8 senses HIV-1 infection via site-specific cleavage of the CARD8 N-terminus by the HIV protease (HIV-1PR). HIV-1PR cleavage of CARD8 induces pyroptotic cell death and the release of pro-inflammatory cytokines from infected cells, processes regulated by Toll-like receptor stimulation prior to viral infection. In acutely infected cells, CARD8 senses the activity of both de novo translated HIV-1PR and packaged HIV-1PR that is released from the incoming virion. Moreover, our evolutionary analyses reveal that the HIV-1PR cleavage site in human CARD8 arose after the divergence of chimpanzees and humans. Although chimpanzee CARD8 does not recognize proteases from HIV or simian immunodeficiency viruses from chimpanzees (SIVcpz), SIVcpz does cleave human CARD8, suggesting that SIVcpz was poised to activate the human CARD8 inflammasome prior to its cross-species transmission into humans. Our findings suggest a unique role for CARD8 inflammasome activation in response to lentiviral infection of humans.

Effect of NLRP3 inflammasome genes polymorphism on disease susceptibility and response to TNF-α inhibitors in Iraqi patients with rheumatoid arthritis.

Background: Rheumatoid arthritis (RA) is a genetically predisposed, systemic, chronic, inflammatory disease. Immune system dysregulation and inherited susceptibility polymorphisms suggest that this type of variation is functional and may help predict disease susceptibility and develop new therapeutic strategies. Anti-TNF-alpha (TNF-α) drugs are highly effective RA treatments, but not all patients respond the same way. It's important to figure out whether RA risk alleles can identify and predict anti-TNF-α-responsiveness in RA patients. Aims of the study: Examine the function of the NLR family pyrin domain containing 3 (NLRP3) and caspase recruitment domain family member 8 (CARD8) genes polymorphisms and their morbid genotypes and alleles in RA patients and apparently healthy controls. In addition, their role in disease susceptibility, severity, and response to anti-TNF-α therapy. Also, examine how single nucleotide polymorphisms (SNPs) affect serum levels of pro-inflammatory cytokines like TNF-α and interleukin (IL)-1ß. Materials and methods: 100 RA patients (88 females, 12 males) and 100 apparently healthy people (86 females, 14 males) were examined. To measure serum TNF-α and IL-1ß, Elabscience sandwich ELISA kits were used. Iraq Biotech, Turkey DNA extraction kit was used to extract genomic DNA from whole blood. CARD8 (rs2043211) and NLRP3 (rs4612666) were genotyped using Agilent, AriaMx, USA, through Tri-Plex SYBR Green-based real-time PCR allelic discrimination assays. Geneious software, version 2019.2.2, used to design primers from published sequences (GenBank accession no. GCA 009914755.1). Primer specificity was determined by NCBI's BLAST. Results: Study found that there is association between cytokines serum level and 28-joints disease activity score (DAS-28). The level of TNF-α increases with the higher DAS-28 (r2 = 0.45, P < 0.0001). Also, IL- 1ß level increases with higher DAS-28 (r2 = 0.51, P < 0.0001). There were no statistically significant variations between patients with RA and the control group in the distribution of CARD8 SNP rs2043211 and NLRP3 SNP rs4612666 genotypes (P = 0.17 and 0.08 respectively) as well their alleles (P = 0.059 and 0.879 respectively). CARD8 (rs2043211) TT genotype was more frequent in patients with higher DAS-28 (P < 0.0001) and higher TNF-α and IL-1ß serum levels (P < 0.0001 for both). Also, NLRP3 (rs4612666) TT genotype was more frequent in patients with higher DAS-28 (P < 0.0001) and higher TNF-α and IL- 1ß serum levels (P < 0.0001 for both). Interestingly, this study revealed that CARD8 (rs2043211) and NLRP3 (rs4612666) variant genotypes are associated with lower response to anti-TNF-α drugs. Conclusions: Serum TNF-α and IL-1ß correlate with DAS-28 and disease activity. Non-responders have elevated TNF-α and IL-1ß. CARD8 rs2043211 and NLRP3 rs4612666 variant polymorphisms are associated with high serum TNF-α and IL-1ß, active disease course, poor disease outcomes, and low response to anti-TNF-α therapy.

A 360° view of the inflammasome: Mechanisms of activation, cell death, and diseases.

Inflammasomes are critical sentinels of the innate immune system that respond to threats to the host through recognition of distinct molecules, known as pathogen- or damage-associated molecular patterns (PAMPs/DAMPs), or disruptions of cellular homeostasis, referred to as homeostasis-altering molecular processes (HAMPs) or effector-triggered immunity (ETI). Several distinct proteins nucleate inflammasomes, including NLRP1, CARD8, NLRP3, NLRP6, NLRC4/NAIP, AIM2, pyrin, and caspases-4/-5/-11. This diverse array of sensors strengthens the inflammasome response through redundancy and plasticity. Here, we present an overview of these pathways, outlining the mechanisms of inflammasome formation, subcellular regulation, and pyroptosis, and discuss the wide-reaching effects of inflammasomes in human disease.

CARD8 Inflammasome Activation by HIV-1 Protease.

The pattern recognition receptor CARD8 is an inflammasome sensor for intracellular HIV-1 protease activity. Previously, the only method for studying the CARD8 inflammasome has been through utilizing DPP8/DPP9 inhibitors including Val-boroPro (VbP) to modestly and nonspecifically activate the CARD8 inflammasome. The identification of HIV-1 protease as a target for sensing by CARD8 has opened the door for a new method of studying the underlying mechanism of CARD8 inflammasome activation. Additionally, triggering the CARD8 inflammasome offers a promising strategy for reducing HIV-1 latent reservoirs. Here we describe the methods to study CARD8 sensing of HIV-1 protease activity through non-nucleoside reverse transcriptase inhibitor (NNRTI)-mediated pyroptosis of HIV-1-infected immune cells and through an HIV and CARD8 co-transfection model.

Association between NLRP3 rs10754558 and CARD8 rs2043211 Variants and Susceptibility to Chronic Kidney Disease.

Nod-like receptor protein 3 (NLRP3) is a multi-protein complex belonging to the innate immune system, whose activation by danger stimuli promotes inflammatory cell death. Evidence supports the crucial role of NLRP3 inflammasome activation in the transition of acute kidney injury to Chronic Kidney Disease (CKD), by promoting both inflammation and fibrotic processes. Variants of NLRP3 pathway-related genes, such as NLRP3 itself and CARD8, have been associated with susceptibility to different autoimmune and inflammatory diseases. In this study, we investigated for the first time the association of functional variants of NLRP3 pathway-related genes (NLRP3-rs10754558, CARD8-rs2043211), with a susceptibility to CKD. A cohort of kidney transplant recipients, dialysis and CKD stage 3-5 patients (303 cases) and a cohort of elderly controls (85 subjects) were genotyped for the variants of interest and compared by using logistic regression analyses. Our analysis showed a significantly higher G allele frequency of the NLRP3 variant (67.3%) and T allele of the CARD8 variant (70.8%) among cases, compared with the control sample (35.9 and 31.2%, respectively). Logistic regressions showed significant associations (p < 0.001) between NLRP3 and CARD8 variants and cases. Our results suggest that the NLRP3 rs10754558 and CARD8 rs2043211 variants could be associated with a susceptibility to CKD.

The NLRP1 and CARD8 inflammasomes detect reductive stress.

The danger signals that activate the related nucleotide-binding domain leucine-rich repeat pyrin domain-containing 1 (NLRP1) and caspase activation and recruitment domain-containing 8 (CARD8) inflammasomes have not been fully established. We recently reported that the oxidized form of TRX1 binds to NLRP1 and represses inflammasome activation. These findings suggested that intracellular reductive stress, which would reduce oxidized TRX1 and thereby abrogate the NLRP1-TRX1 interaction, is an NLRP1 inflammasome-activating danger signal. However, no agents that induce reductive stress were known to test this premise. Here, we identify and characterize several radical-trapping antioxidants, including JSH-23, that induce reductive stress. We show that these compounds accelerate the proteasome-mediated degradation of the repressive N-terminal fragments of both NLRP1 and CARD8, releasing the inflammasome-forming C-terminal fragments from autoinhibition. Overall, this work validates chemical probes that induce reductive stress and establishes reductive stress as a danger signal sensed by both the NLRP1 and CARD8 inflammasomes.

Protein folding stress potentiates NLRP1 and CARD8 inflammasome activation.

NLRP1 and CARD8 are related pattern-recognition receptors (PRRs) that detect intracellular danger signals and form inflammasomes. Both undergo autoproteolysis, generating N-terminal (NT) and C-terminal (CT) fragments. The proteasome-mediated degradation of the NT releases the CT from autoinhibition, but the stimuli that trigger NT degradation have not been fully elucidated. Here, we show that several distinct agents that interfere with protein folding, including aminopeptidase inhibitors, chaperone inhibitors, and inducers of the unfolded protein response, accelerate NT degradation. However, these agents alone do not trigger inflammasome formation because the released CT fragments are physically sequestered by the serine dipeptidase DPP9. We show that DPP9-binding ligands must also be present to disrupt these complexes and allow the CT fragments to oligomerize into inflammasomes. Overall, these results indicate that NLRP1 and CARD8 detect a specific perturbation that induces both protein folding stress and DPP9 ligand accumulation.

The CARD8 T60 variant associates with NLRP1 and negatively regulates its activation.

The NLRP1 inflammasome functions as canonical cytosolic sensor in response to intracellular infections and is implicated in auto-inflammatory diseases. But the regulation and signal transduction mechanisms of NLRP1 are incompletely understood. Here, we show that the T60 variant of CARD8, but not the canonical T48 isoform, negatively regulates the NLRP1 inflammasome activation by directly interacting with the receptor molecule NLRP1 and inhibiting inflammasome assembly. Furthermore, our results suggest that different ASC preference in three types of inflammasomes, namely the ASC-indispensable NLRP1 inflammasome, ASC-dispensable mNLRP1b inflammasome and ASC-independent CARD8 inflammasome, is mainly caused by the CARD domain, not the UPA subdomain. Based on the systematic site-directed mutagenesis and structural analysis, we find that signal transduction of the NLRP1 inflammasome relies on multiple interaction surfaces at its CARD domain. Finally, our results partly explain how mutations in NLRP1 lead to its constitutive activation in auto-inflammatory diseases. In conclusion, our study not only reveals how CARD8 downregulates the NLRP1 inflammasome activation, but also provides insights into the assembly mechanisms of CARD-containing inflammasomes.

Identification of LncRNA CARD8-AS1 as a Potential Prognostic Biomarker Associated With Progression of Lung Adenocarcinoma.

Introduction: Long non-coding RNAs (lncRNAs) exhibit crucial roles in human tumors. However, the role of lncRNA CARD8-AS1 in lung adenocarcinoma remains elusive. This study investigated the role of CARD8-AS1 in lung adenocarcinoma. Materials and Methods: The expression of CARD8-AS1 was detected by RT-qPCR analysis and confirmed using an online database. The clinical value of CARD8-AS1 was evaluated using the Kaplan-Meier curve and multivariate Cox regression analyses. The effects of CARD8-AS1 on cancer cell proliferation, migration, and invasion potential were assessed through several cellular experiments. Western blot assay was used to measure Bcl-2 and Bax protein levels. The interaction among CARD8-AS1, miR-650, and Bax, was assessed using a dual-luciferase reporter assay. Results: The expression of CARD8-AS1 was decreased in lung adenocarcinoma tissues and cell lines (p < 0.001). Low expression of CARD8-AS1 was related to tumor size (p = 0.042), TNM stage (p = 0.021), lymph node metastasis (p = 0.025), and poor overall survival (p < 0.05). Elevated expression of CARD8-AS1 could suppress cellular viability, migration potential, and invasion ability (p < 0.05). The Bcl-2 protein levels were decreased while Bax levels were increased by overexpression of CARD8-AS1 (p < 0.001). miR-650 may thus be a direct target of CARD8-AS1 and Bax may be a direct target of miR-650. Discussion: CARD8-AS1 expression was downregulated in lung adenocarcinoma and associated with several clinical parameters. CARD8-AS1 exerted tumor-suppressive effects by targeting the miR-650 and then regulating Bax expression. CARD8-AS1/miR-650 may serve as novel prognostic biomarkers and potential therapeutic targets for the treatment of lung adenocarcinoma.

Beyond Inhibition: A Novel Strategy of Targeting HIV-1 Protease to Eliminate Viral Reservoirs.

HIV-1 protease (PR) is a viral enzyme that cleaves the Gag and Gag-Pol polyprotein precursors to convert them into their functional forms, a process which is essential to generate infectious viral particles. Due to its broad substrate specificity, HIV-1 PR can also cleave certain host cell proteins. Several studies have identified host cell substrates of HIV-1 PR and described the potential impact of their cleavage on HIV-1-infected cells. Of particular interest is the interaction between PR and the caspase recruitment domain-containing protein 8 (CARD8) inflammasome. A recent study demonstrated that CARD8 can sense HIV-1 PR activity and induce cell death. While PR typically has low levels of intracellular activity prior to viral budding, premature PR activation can be achieved using certain non-nucleoside reverse transcriptase inhibitors (NNRTIs), resulting in CARD8 cleavage and downstream pyroptosis. Used together with latency reversal agents, the induction of premature PR activation to trigger CARD8-mediated cell killing may help eliminate latent reservoirs in people living with HIV. This represents a novel strategy of utilizing PR as an antiviral target through premature activation rather than inhibition. In this review, we discuss the viral and host substrates of HIV-1 protease and highlight potential applications and advantages of targeting CARD8 sensing of HIV-1 PR.