AB045. E2F6 promotes temozolomide resistance in glioblastoma by enrichment in CARD6 and POSTN promoter region.

BACKGROUND: Glioblastoma (GBM) is the most aggressive primary malignant brain tumor. Temozolomide (TMZ) is the most used first-line chemotherapeutic agent for GBM after surgery, but acquired resistance to TMZ frequently leads to treatment failure and is a major challenge in the clinical treatment of GBM. Increasing evidence suggests that E2F transcription factor 6 (E2F6) is associated with a variety of tumor malignant biological behaviors and drug resistance, but its biological function and underlying molecular mechanisms in GBM are unknown. METHODS: The study investigated the levels of E2F6 in both TMZ-sensitive and TMZ-resistant GBM cells and tissues using Western blotting and immunofluorescence assays. In vitro experiments were conducted to explore the impact of E2F6 on TMZ resistance and glioma stem cell stemness. These experiments included Western blotting, colony formation assay, flow cytometry assay, and TdT-mediated dUTP nick-end labeling (TUNEL) assay. Bioinformatic analyses were conducted to investigate the mechanism behind the high expression of E2F6 in TMZ-resistant cells and its correlation with caspase recruitment domain 6 (CARD6) and disulfide-linked cell adhesion protein (POSTN). The study employed bioinformatic analyses, messenger RNA (mRNA) sequencing, chromatin immunoprecipitation sequencing assay, immunofluorescence, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blotting. To examine the function of E2F6, an intracranial xenograft tumor mouse model was used for in vivo experiments. RESULTS: It was found that CARD6 and POSTN were significantly associated with TMZ resistance and survival of GBM patients. E2F6 was up-regulated in TMZ-resistant cells and tissues. Knockdown of E2F6 down-regulated the expression of CARD6, promoted TMZ-induced apoptosis, and enhanced chemo-sensitivity, whereas its overexpression significantly increased TMZ resistance in vitro and in vivo. In addition, E2F6 can promote TMZ resistance through stem-like properties acquisition. We identified a signaling pathway related to E2F6 and POSTN, which maintains the self-renewal of GBM stem cells (GSCs). E2F6 concentrates in the promoter region of POSTN, thereby regulating the expression of GSCs-related genes cluster of differentiation 133 (CD133), Nestin, and sex-determining region Y-box 2 (SOX2), which may be involved in tumor metabolism and drug resistance processes. Down-regulation of E2F6 down-regulated the expression of POSTN and inhibited tumor growth in nude mice. CONCLUSIONS: These results suggest that the E2F6-CARD6/POSTN signaling axis regulates the malignant biological behaviors of GBM and TMZ resistance. These findings are expected to provide promising therapeutic targets for CARD6 overcoming GBM TMZ resistance.

Pseudorabies Virus UL4 protein promotes the ASC-dependent inflammasome activation and pyroptosis to exacerbate inflammation.

Pseudorabies virus (PRV) infection causes systemic inflammatory responses and inflammatory damages in infected animals, which are associated with the activation of inflammasome and pyroptosis in infected tissues. Here, we identified a critical function of PRV non-structural protein UL4 that enhanced ASC-dependent inflammasome activation to promote pyroptosis. Whereas, the deficiency of viral UL4 was able to reduce ASC-dependent inflammasome activation and the occurrences of pyroptosis. Mechanistically, the 132-145 aa of UL4 permitted its translocation from the nucleus to the cytoplasm to interact with cytoplasmic ASC to promote the activation of NLRP3 and AIM2 inflammasome. Further research showed that UL4 promoted the phosphorylation levels of SYK and JNK to enhance the ASC phosphorylation, which led to the increase of ASC oligomerization, thus promoting the activation of NLRP3 and AIM2 inflammasome and enhanced GSDMD-mediated pyroptosis. In vivo experiments further showed that PRV UL4 (132DVAADAAAEAAAAE145) mutated strain (PRV-UL4mut) infection did not lead to a significant decrease in viral titers at 12 h. p. i, but it induced lower levels of IL-1ß, IL-18, and GSDMD-NT, which led to an alleviated inflammatory infiltration and pathological damage in the lungs and brains, and a lower death rate compared with wild-type PRV strain infection. Taken together, our findings unravel that UL4 is an important viral regulator to manipulate the inflammasome signaling and pyroptosis of host cells to promote the pathogenicity of PRV, which might be further exploited as a new target for live attenuated vaccines or therapeutic strategies against pseudorabies in the future.

CARD14 signalosome formation is associated with its endosomal relocation and mTORC1-induced keratinocyte proliferation.

Rare mutations in CARD14 promote psoriasis by inducing CARD14-BCL10-MALT1 complexes that activate NF-κB and MAP kinases. Here, the downstream signalling mechanism of the highly penetrant CARD14E138A alteration is described. In addition to BCL10 and MALT1, CARD14E138A associated with several proteins important in innate immune signalling. Interactions with M1-specific ubiquitin E3 ligase HOIP, and K63-specific ubiquitin E3 ligase TRAF6 promoted BCL10 ubiquitination and were essential for NF-κB and MAP kinase activation. In contrast, the ubiquitin binding proteins A20 and ABIN1, both genetically associated with psoriasis development, negatively regulated signalling by inducing CARD14E138A turnover. CARD14E138A localized to early endosomes and was associated with the AP2 adaptor complex. AP2 function was required for CARD14E138A activation of mTOR complex 1 (mTORC1), which stimulated keratinocyte metabolism, but not for NF-κB nor MAP kinase activation. Furthermore, rapamycin ameliorated CARD14E138A-induced keratinocyte proliferation and epidermal acanthosis in mice, suggesting that blocking mTORC1 may be therapeutically beneficial in CARD14-dependent psoriasis.

Regulation of MYC by CARD14 in human epithelium is a determinant of epidermal homeostasis and disease.

Caspase recruitment domain family member 14 (CARD14) and its variants are associated with both atopic dermatitis (AD) and psoriasis, but their mechanistic impact on skin barrier homeostasis is largely unknown. CARD14 is known to signal via NF-κB; however, CARD14-NF-κB signaling does not fully explain the heterogeneity of CARD14-driven disease. Here, we describe a direct interaction between CARD14 and MYC and show that CARD14 signals through MYC in keratinocytes to coordinate skin barrier homeostasis. CARD14 directly binds MYC and influences barrier formation in an MYC-dependent fashion, and this mechanism is undermined by disease-associated CARD14 variants. These studies establish a paradigm that CARD14 activation regulates skin barrier function by two distinct mechanisms, including activating NF-κB to bolster the antimicrobial (chemical) barrier and stimulating MYC to bolster the physical barrier. Finally, we show that CARD14-dependent MYC signaling occurs in other epithelia, expanding the impact of our findings beyond the skin.

Macrophage OTUD1-CARD9 axis drives isoproterenol-induced inflammatory heart remodelling.

BACKGROUND: Chronic inflammation contributes to the progression of isoproterenol (ISO)-induced heart failure (HF). Caspase-associated recruitment domain (CARD) families are crucial proteins for initiation of inflammation in innate immunity. Nonetheless, the relevance of CARDs in ISO-driven cardiac remodelling is little explored. METHODS: This study utilized Card9-/- mice and reconstituted C57BL/6 mice with either Card9-/- or Otud1-/- marrow-derived cells. Mechanistic studies were conducted in primary macrophages, cardiomyocytes, fibroblasts and HEK-293T cells. RESULTS: Here, we demonstrated that CARD9 was substantially upregulated in murine hearts infused with ISO. Either whole-body CARD9 knockout or myeloid-specific CARD9 deletion inhibited ISO-driven murine cardiac inflammation, remodelling and dysfunction. CARD9 deficiency in macrophages prevented ISO-induced inflammation and alleviated remodelling changes in cardiomyocytes and fibroblasts. Mechanistically, we found that ISO enhances the activity of CARD9 by upregulating ovarian tumour deubiquitinase 1 (OTUD1) in macrophages. We further demonstrated that OTUD1 directly binds to the CARD9 and then removes the K33-linked ubiquitin from CARD9 to promote the assembly of the CARD9-BCL10-MALT1 (CBM) complex, without affecting CARD9 stability. The ISO-activated CBM complex results in NF-κB activation and macrophage-based inflammatory gene overproduction, which then enhances cardiomyocyte hypertrophy and fibroblast fibrosis, respectively. Myeloid-specific OTUD1 deletion also attenuated ISO-induced murine cardiac inflammation and remodelling. CONCLUSIONS: These results suggested that the OTUD1-CARD9 axis is a new pro-inflammatory signal in ISO-challenged macrophages and targeting this axis has a protective effect against ISO-induced HF. KEY POINTS: Macrophage CARD9 was elevated in heart tissues of mice under chronic ISO administration. Either whole-body CARD9 knockout or myeloid-specific CARD9 deficiency protected mice from ISO-induced inflammatory heart remodeling. ISO promoted the assembly of CBM complex and then activated NF-κB signaling in macrophages through OTUD1-mediated deubiquitinating modification. OTUD1 deletion in myeloid cells protected hearts from ISO-induced injuries in mice.

Lyssavirus matrix protein inhibits NLRP3 inflammasome assembly by binding to NLRP3.

Lyssavirus is a kind of neurotropic pathogen that needs to evade peripheral host immunity to enter the central nervous system to accomplish infection. NLRP3 inflammasome activation is essential for the host to defend against pathogen invasion. This study demonstrates that the matrix protein (M) of lyssavirus can inhibit both the priming step and the activation step of NLRP3 inflammasome activation. Specifically, M of lyssavirus can compete with NEK7 for binding to NLRP3, which restricts downstream apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization. The serine amino acid at the 158th site of M among lyssavirus is critical for restricting ASC oligomerization. Moreover, recombinant lab-attenuated lyssavirus rabies (rabies lyssavirus [RABV]) with G158S mutation at M decreases interleukin-1ß (IL-1ß) production in bone-marrow-derived dendritic cells (BMDCs) to facilitate lyssavirus invasion into the brain thereby elevating pathogenicity in mice. Taken together, this study reveals a common mechanism by which lyssavirus inhibits NLRP3 inflammasome activation to evade host defenses.

Targeting IL-1 controls refractory pityriasis rubra pilaris.

Pityriasis rubra pilaris (PRP) is a rare inflammatory skin disease with a poorly understood pathogenesis. Through a molecularly driven precision medicine approach and an extensive mechanistic pathway analysis in PRP skin samples, compared to psoriasis, atopic dermatitis, healed PRP, and healthy controls, we identified IL-1ß as a key mediator, orchestrating an NF-κB-mediated IL-1ß-CCL20 axis, including activation of CARD14 and NOD2. Treatment of three patients with the IL-1 antagonists anakinra and canakinumab resulted in rapid clinical improvement and reversal of the PRP-associated molecular signature with a 50% improvement in skin lesions after 2 to 3 weeks. This transcriptional signature was consistent with in vitro stimulation of keratinocytes with IL-1ß. With the central role of IL-1ß underscoring its potential as a therapeutic target, our findings propose a redefinition of PRP as an autoinflammatory keratinization disorder. Further clinical trials are needed to validate the efficacy of IL-1ß antagonists in PRP.

Extracellular Vesicle ASC: A Novel Mediator for Lung-Brain Axis in Preterm Brain Injury.

Bronchopulmonary dysplasia (BPD) and neurodevelopmental impairment are among the most common morbidities affecting preterm infants. Although BPD is a predictor of poor neurodevelopmental outcomes, it is currently uncertain how BPD contributes to brain injury in preterm infants. Extracellular vesicles (EVs) are involved in interorgan communication in diverse pathological processes. ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) is pivotal in inflammasome assembly and activation of inflammatory response. We assessed expression profiles of the alveolar macrophage (AM) markers CD11b, CD11c, and CD206 as well as ASC in EVs isolated from the plasma of preterm infants at risk for BPD at 1 week of age. We found that infants on higher fraction of inspired oxygen therapy (HO2⩾30%) had increased concentrations of AM-derived EV-ASC compared with infants on lower fraction of inspired oxygen (LO2<30%). To assess the function of these EVs, we performed adoptive transfer experiments by injecting them into the circulation of newborn mice. We discovered that mice that received EVs from infants on HO2 had increased lung inflammation, decreased alveolarization, and disrupted vascular development, the hallmarks of BPD. Importantly, these EVs crossed the blood-brain barrier, and the EVs from infants on HO2 caused inflammation, reduced cell survival, and increased cell death, with features of pyroptosis and necroptosis in the hippocampus. These results highlight a novel role for AM-derived EV-ASC in mediating the lung-to-brain cross-talk that is critical in the pathogenesis of BPD and brain injury and identify potential novel targets for preventing and treating BPD and brain injury in preterm infants.

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.

The ASC inflammasome adapter governs SAA-derived protein aggregation in inflammatory amyloidosis.

Extracellularly released molecular inflammasome assemblies -ASC specks- cross-seed Aß amyloid in Alzheimer's disease. Here we show that ASC governs the extent of inflammation-induced amyloid A (AA) amyloidosis, a systemic disease caused by the aggregation and peripheral deposition of the acute-phase reactant serum amyloid A (SAA) in chronic inflammatory conditions. Using super-resolution microscopy, we found that ASC colocalized tightly with SAA in human AA amyloidosis. Recombinant ASC specks accelerated SAA fibril formation and mass spectrometry after limited proteolysis showed that ASC interacts with SAA via its pyrin domain (PYD). In a murine model of inflammatory AA amyloidosis, splenic amyloid load was conspicuously decreased in Pycard-/- mice which lack ASC. Treatment with anti-ASCPYD antibodies decreased amyloid loads in wild-type mice suffering from AA amyloidosis. The prevalence of natural anti-ASC IgG (-logEC50 ≥ 2) in 19,334 hospital patients was <0.01%, suggesting that anti-ASC antibody treatment modalities would not be confounded by natural autoimmunity. These findings expand the role played by ASC and IL-1 independent inflammasome employments to extraneural proteinopathies and suggest that anti-ASC immunotherapy may contribute to resolving such diseases.

Dexmedetomidine's protective mechanism against hyperoxic injury in neonatal rats.

Bronchopulmonary dysplasia (BPD) is a common serious complication of premature babies. No effective means control it. Hyperoxia damage is one of the important mechanisms of BPD. The reaserach confirmed pyroptosis existed in BPD. Dexmedetomidine is a new, high-specific α2 receptor agonist. Previous research foundation found that dexmedetomidine has a protective effect on BPD. To investigate how dexmedetomidine improves hyperoxic lung injury in neonatal mice by regulating pyroptosis. Neonatal rats were randomly divided into four groups: normal control group, hyperoxic injury group, air plus dexmedetomidine group, and hyperoxia plus dexmedetomidine group. After seven days the lungs of rats in each group were extracted, and the wet-to-dry weight ratio of the lung was measured. The lung injury in rats was observed using hematoxylin-eosin staining. Additionally, the expression and localization of nucleotide-binding oligomerization domain-like receptor thermal protein domain associated protein 3 (NLRP3), apoptosis-associated speck-like protein (ASC), and gasdermin D (GSDMD) proteins were examined in the lungs of rats using immunofluorescence staining. The mRNA levels of NLRP3, ASC, caspase-1, and interleukin 18 (IL-18) in the lungs of rats were determined using real-time PCR. Moreover, the protein levels of NLRP3, ASC, caspase-1/cleaved caspase-1, interleukin 1beta (IL-1ß), IL-18, and tunor necrosis factor alpha (TNF-α) were detected in lungs of rats using Western blot. The extent of mitochondrial damage in lung tissues of each group was observed by transmission electron microscopy. The lung tissue injury of the neonatal rats was significantly improved in the hyperoxia plus dexmedetomidine group compared to the hyperoxic injury group. Furthermore, the expressions of pyroptosis-related proteins such as NLRP3, ASC, cleaved-caspase-1, and GSDMD were significantly decreased, along with the expressions of inflammatory factors in lung tissues. By inhibiting the NLRP3/caspase-1/GSDMD pyroptosis pathway, dexmedetomidine reduces the activation and release of inflammatory factors and provides a protective effect against hyperoxic lung injury in neonatal mice.

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.

NCF4 attenuates colorectal cancer progression by modulating inflammasome activation and immune surveillance.

The spatiotemporal regulation of inflammasome activation remains unclear. To examine the mechanism underlying the assembly and regulation of the inflammasome response, here we perform an immunoprecipitation-mass spectrometry analysis of apoptosis-associated speck-like protein containing a CARD (ASC) and identify NCF4/1/2 as ASC-binding proteins. Reduced NCF4 expression is associated with colorectal cancer development and decreased five-year survival rate in patients with colorectal cancer. NCF4 cooperates with NCF1 and NCF2 to promote NLRP3 and AIM2 inflammasome activation. Mechanistically, NCF4 phosphorylation and puncta distribution switches from the NADPH complex to the perinuclear region, mediating ASC oligomerization, speck formation and inflammasome activation. NCF4 functions as a sensor of ROS levels, to establish a balance between ROS production and inflammasome activation. NCF4 deficiency causes severe colorectal cancer in mice, increases transit-amplifying and precancerous cells, reduces the frequency and activation of CD8+ T and NK cells, and impairs the inflammasome-IL-18-IFN-γ axis during the early phase of colorectal tumorigenesis. Our study implicates NCF4 in determining the spatial positioning of inflammasome assembly and contributing to inflammasome-mediated anti-tumor responses.

Tryptanthrin suppresses multiple inflammasome activation to regulate NASH progression by targeting ASC protein.

BACKGROUND: The adaptor protein apoptosis-associated speck-like protein (ASC) containing a caspase recruitment domain (CARD) can be activated through pyrin domain (PYD) interactions between sensors and ASC, and through CARD interactions between caspase-1 and ASC. Although the majority of ternary inflammasome complexes depend on ASC, drugs targeting ASC protein remain scarce. After screening natural compounds from Isatidis Radixin, we found that tryptanthrin (TPR) could inhibit NLRP3-induced IL-1ß and caspase-1 production, but the underlying anti-inflammatory mechanisms remain to be elucidated. PURPOSE: The purpose of this study was to determine the impact of TPR on the NLRP3, NLRC4, and AIM2 inflammasomes and the underlying mechanisms. Additionally, the efficacy of TPR was analysed in the further course of methionine- and choline-deficient (MCD)-induced NASH and lipopolysaccharide (LPS)-induced sepsis models of mice. METHODS: In vitro studies used bone marrow-derived macrophages to assess the anti-inflammatory activity of TPR, and the techniques included western blot, testing of intracellular K+ and Ca2+, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), co-immunoprecipitation, ASC oligomerization assay, surface plasmon resonance (SPR), and molecular docking. We used LPS-induced sepsis models and MCD-induced NASH models in vivo to evaluate the effectiveness of TPR in inhibiting inflammatory diseases. RESULTS: Our observations suggested that TPR could inhibit NLRP3, NLRC4, and AIM2 inflammasome activation. As shown in a mouse model of inflammatory diseases caused by MCD-induced NASH and LPS-induced sepsis, TPR significantly alleviated the progression of diseases. TPR interrupted the interactions between ASC and NLRP3/NLRC4/AIM2 in the co-immunoprecipitation experiment, and stable binding of TPR to ASC was also evident in SPR experiments. The underlying mechanisms of anti-inflammatory activities of TPR might be associated with targeting ASC, in particular, PYD domain of ASC. CONCLUSION: In general, the requirement for ASC in multiple inflammasome complexes makes TPR, as a novel broad-spectrum inflammasome inhibitor, potentially useful for treating a wide range of multifactorial inflammasome-related diseases.

The ROS/TXNIP/NLRP3 pathway mediates LPS-induced microglial inflammatory response.

BACKGROUND: Sepsis-associated encephalopathy (SAE) is a diffuse brain dysfunction activated by microglia. The potential pathological changes of SAE are complex, and the cellular pathophysiological characteristics remains unclear. This study aims to explore the ROS/TXNIP/NLRP3 pathway mediated lipopolysaccharide (LPS)-induced inflammatory response in microglia. METHODS: BV-2 cells were pre-incubated with 10 µM N-acetyl-L-cysteine (NAC) for 2 h, which were then reacted with 1 µg/mL LPS for 24 h. Western blot assay examined the protein levels of IBA1, CD68, TXNIP, NLRP3, ASC, and Cleaved Caspase-1 in BV-2 cells. The contents of inflammatory factor were detected by ELISA assay. The co-immunoprecipitation assay examined the interaction between TXNIP and NLRP3. RESULTS: LPS was confirmed to promote the positive expressions of IBA1 and CD68 in BV-2 cells. The further experiments indicated that LPS enhanced ROS production and NLRP3 inflammasome activation in BV-2 cells. Moreover, we also found that NAC partially reversed the facilitation of LPS on the levels of ROS, IL-1ß, IL-18, TXNIP, NLRP3, ASC, and Cleaved Caspase-1 in BV-2 cells. NAC treatment also notably alleviated the interaction between TXNIP and NLRP3 in BV-2 cells. CONCLUSION: ROS inhibition mediated NLRP3 signaling inactivation by decreasing TXNIP expression.

Correlation of LLT-1 and NLRC4 inflammasome and its effect on glioblastoma prognosis.

PURPOSE: LLT-1 is a well-known ligand for the natural killer (NK) cell inhibitory receptor NKRP1A. Here, we examined NLRC4 inflammasome components and LLT-1 expression in glioblastoma (GBM) tissues to elucidate potential associations and interactions between these factors. METHODS: GBM tissues were collected for RNA sequencing (RNA-seq) and Immunofluorescent experiments. Colocalization of LLT-1 and other proteins was assessed by immunofluorescence. Computational analyses utilized RNA-seq data from 296 to 52 patients from the Chinese Glioma Genome Atlas and CHA medical records, respectively. These data were subjected to survival, non-negative matrix factorization clustering, Gene Ontology enrichment, and protein-protein interaction analyses. Receptor-ligand interactions between tumor and immune cells were confirmed by single-cell RNA-seq analysis. RESULTS: In GBM tissues, LLT-1 was predominantly colocalized with glial fibrillary acidic protein (GFAP)-expressing astrocytes, but not with microglial markers like Iba-1. Additionally, LLT-1 and activated NLRC4 inflammasomes were mainly co-expressed in intratumoral astrocytes, suggesting an association between LLT-1, NLRC4, and glioma malignancy. High LLT-1 expression correlates with poor prognosis, particularly in the mesenchymal subtype, and is associated with TNF and NOD-like receptor signaling pathway enrichment, indicating a potential role in tumor inflammation and progression. At the single-cell level, mesenchymal-like malignant cells showed high NF, NLR, and IL-1 signaling pathway enrichment compared to other malignant cell types. CONCLUSION: We revealed an association between NLRC4 inflammasome activity and LLT-1 expression, suggesting a novel regulatory pathway involving TNF, inflammasomes, and IL-1, potentially offering new NK-cell-mediated anti-glioma approaches.

MALT1 substrate cleavage: what is it good for?

CARD-BCL10-MALT1 (CBM) signalosomes connect distal signaling of innate and adaptive immune receptors to proximal signaling pathways and immune activation. Four CARD scaffold proteins (CARD9, 10, 11, 14) can form seeds that nucleate the assembly of BCL10-MALT1 filaments in a cell- and stimulus-specific manner. MALT1 (also known as PCASP1) serves a dual function within the assembled CBM complexes. By recruiting TRAF6, MALT1 acts as a molecular scaffold that initiates IκB kinase (IKK)/NF-κB and c-Jun N-terminal kinase (JNK)/AP-1 signaling. In parallel, proximity-induced dimerization of the paracaspase domain activates the MALT1 protease which exerts its function by cleaving a set of specific substrates. While complete MALT1 ablation leads to immune deficiency, selective destruction of either scaffolding or protease function provokes autoimmune inflammation. Thus, balanced MALT1-TRAF6 recruitment and MALT1 substrate cleavage are critical to maintain immune homeostasis and to promote optimal immune activation. Further, MALT1 protease activity drives the survival of aggressive lymphomas and other non-hematologic solid cancers. However, little is known about the relevance of the cleavage of individual substrates for the pathophysiological functions of MALT1. Unbiased serendipity, screening and computational predictions have identified and validated ~20 substrates, indicating that MALT1 targets a quite distinct set of proteins. Known substrates are involved in CBM auto-regulation (MALT1, BCL10 and CARD10), regulation of signaling and adhesion (A20, CYLD, HOIL-1 and Tensin-3), or transcription (RelB) and mRNA stability/translation (Regnase-1, Roquin-1/2 and N4BP1), indicating that MALT1 often targets multiple proteins involved in similar cellular processes. Here, we will summarize what is known about the fate and functions of individual MALT1 substrates and how their cleavage contributes to the biological functions of the MALT1 protease. We will outline what is needed to better connect critical pathophysiological roles of the MALT1 protease with the cleavage of distinct substrates.

Cutaneous inflammasome driving ASC / gasdermin-D activation and IL-1ß-secreting macrophages in severe atopic dermatitis.

Atopic dermatitis (AD) is an inflammatory skin disease with intense pruritus, and chronic skin colonization by Staphylococcus aureus. To understand the inflammatory status in AD, we investigated the inflammasome complex, that activates ASC (Apoptosis-associated speck-like protein containing a CARD), caspase-1 and GSDMD (gasdermin-D), and production of IL-1ß and IL-18. We aimed to evaluate the expression of the inflammasome pathway in the skin of adults with AD. Thirty patients with moderate to severe AD and 20 healthy controls were enrolled in the study. We performed the analysis of the inflammasome components NLRP1, NLRP3, AIM-2, IL-1ß, IL-18, Caspase-1, ASC, GSDMD, and CD68 expression (macrophage marker) by immunohistochemistry and immunofluorescence. The main findings included increased expression of NLRP3, NLRP1 and AIM-2 at dermal level of severe AD; augmented IL-18 and IL-1ß expression at epidermis of moderate and severe patients, and in the dermis of severe AD; augmented expression of ASC, caspase-1 and GSDMD in both epidermis and dermis of moderate and severe AD. We detected positive correlation between caspase-1, GSDMD and IL-1ß (epidermis) and caspase-1 (dermis) and AD severity; NLRP3, AIM-2 and IL-1ß, and NLRP3 with IL-18 in the epidermis; ASC, GSDMD and IL-1ß, and NLRP3, AIM-2, caspase-1, and IL-18 in the dermis. We also evidenced the presence of CD68+ macrophages secreting GSDMD, ASC and IL-1ß in moderate and severe AD. Cutaneous macrophages, early detected in moderate AD, have its role in the disease inflammatory mechanisms. Our study indicates a canonical activation pathway of inflammasomes, reinforced by the chronic status of inflammation in AD. The analysis of the inflammasome complex evidenced an imbalance in its regulation, with increased expression of the evaluated components, which is remarkably in severe AD, emphasizing its relevance as potential disease biomarkers and targets for immunomodulatory interventions.

IC100, a humanized therapeutic monoclonal anti-ASC antibody alleviates oxygen-induced retinopathy in mice.

BACKGROUND: Retinopathy of prematurity (ROP), which often presents with bronchopulmonary dysplasia (BPD), is among the most common morbidities affecting extremely premature infants and is a leading cause of severe vision impairment in children worldwide. Activations of the inflammasome cascade and microglia have been implicated in playing a role in the development of both ROP and BPD. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is pivotal in inflammasome assembly. Utilizing mouse models of both oxygen-induced retinopathy (OIR) and BPD, this study was designed to test the hypothesis that hyperoxia induces ASC speck formation, which leads to microglial activation and retinopathy, and that inhibition of ASC speck formation by a humanized monoclonal antibody, IC100, directed against ASC, will ameliorate microglial activation and abnormal retinal vascular formation. METHODS: We first tested ASC speck formation in the retina of ASC-citrine reporter mice expressing ASC fusion protein with a C-terminal citrine (fluorescent GFP isoform) using a BPD model that causes both lung and eye injury by exposing newborn mice to room air (RA) or 85% O2 from postnatal day (P) 1 to P14. The retinas were dissected on P14 and retinal flat mounts were used to detect vascular endothelium with AF-594-conjugated isolectin B4 (IB4) and citrine-tagged ASC specks. To assess the effects of IC100 on an OIR model, newborn ASC citrine reporter mice and wildtype mice (C57BL/6 J) were exposed to RA from P1 to P6, then 75% O2 from P7 to P11, and then to RA from P12 to P18. At P12 mice were randomized to the following groups: RA with placebo PBS (RA-PBS), O2 with PBS (O2-PBS), O2 + IC100 intravitreal injection (O2-IC100-IVT), and O2 + IC100 intraperitoneal injection (O2-IC100-IP). Retinal vascularization was evaluated by flat mount staining with IB4. Microglial activation was detected by immunofluorescence staining for allograft inflammatory factor 1 (AIF-1) and CD206. Retinal structure was analyzed on H&E-stained sections, and function was analyzed by pattern electroretinography (PERG). RNA-sequencing (RNA-seq) of the retinas was performed to determine the transcriptional effects of IC100 treatment in OIR. RESULTS: ASC specks were significantly increased in the retinas by hyperoxia exposure and colocalized with the abnormal vasculature in both BPD and OIR models, and this was associated with increased microglial activation. Treatment with IC100-IVT or IC100-IP significantly reduced vaso-obliteration and intravitreal neovascularization. IC100-IVT treatment also reduced retinal microglial activation, restored retinal structure, and improved retinal function. RNA-seq showed that IC100 treatment corrected the induction of genes associated with angiogenesis, leukocyte migration, and VEGF signaling caused by O2. IC100 also corrected the suppression of genes associated with cell junction assembly, neuron projection, and neuron recognition caused by O2. CONCLUSION: These data demonstrate the crucial role of ASC in the pathogenesis of OIR and the efficacy of a humanized therapeutic anti-ASC antibody in treating OIR mice. Thus, this anti-ASC antibody may potentially be considered in diseases associated with oxygen stresses and retinopathy, such as ROP.

Sirtuin 6 Deacetylates Apoptosis-Associated Speck-Like Protein (ASC) to Inhibit Endothelial Cell Pyroptosis in Atherosclerosis.

Endothelial cell dysfunction is the main pathology of atherosclerosis (AS). Sirtuin 6 (SIRT6), a deacetylase, is involved in AS progression. This study aimed to investigate the impacts of SIRT6 on the pyroptosis of endothelial cells and its underlying mechanisms. ApoE-/- mice were fed a high-fat diet (HFD) to establish the AS mouse model, atherosclerotic lesions were evaluated using oil red O staining, and blood lipids and inflammatory factors were measured using corresponding kits. Human umbilical vein endothelial cells (HUVECs) were treated with oxidized low-density lipoprotein (ox-LDL) to establish the cell model, and pyroptosis was evaluated by flow cytometry, ELISA, and western blot. Immunoprecipitation (IP), co-IP, western blot, and immunofluorescence were used to detect the molecular mechanisms. The results showed that SIRT6 expression was downregulated in the blood of HFD-induced mice and ox-LDL-induced HUVECs. Overexpression of SIRT6 reduced atherosclerotic lesions, blood lipids, and inflammation in vivo and suppressed pyroptosis of HUVECs in vitro. Moreover, SIRT6 interacted with ASC to inhibit the acetylation of ASC, thus, reducing the interaction between ASC and NLRP3. Moreover, SIRT6 inhibits endothelial cell pyroptosis in the aortic roots of mice by deacetylating ASC. In conclusion, SIRT6 deacetylated ASC to inhibit its interaction with NLRP3 and then suppressed pyroptosis of endothelial cells, thus, decelerating the progression of AS. The findings provide new insights into the function of SIRT6 in AS.