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.

Regulatory Roles of Histone Deacetylation in Metabolic Stress-Induced Expression of Caspase Recruitment Domain-Containing Protein 9 (CARD9) in Pancreatic ß-Cells.

CARD9, a scaffolding protein, has been implicated in the pathogenesis of metabolic diseases, including obesity and diabetes. We recently reported novel roles for CARD9 in islet ß-cell dysregulation under duress of gluco (HG)- and glucolipotoxic (GLT) stress. CARD9 expression was also increased in ß-cells following exposure to HG and GLT stress. The current study is aimed at understanding the putative roles of histone deacetylation in HG- and GLT-induced expression of CARD9. Using two structurally distinct inhibitors of histone deacetylases (HDACs), namely trichostatin (TSA) and suberoylanilide hydroxamic acid (SAHA), we provide the first evidence to suggest that the increased expression of CARD9 seen under duress of HG and GLT stress is under the regulatory control of histone deacetylation. Interestingly, the expression of protein kinase Cδ (PKCδ), a known upstream regulator of CARD9 activation, is also increased under conditions of metabolic stress. However, it is resistant to TSA and SAHA, suggesting that it is not regulated via histone deacetylation. Based on these data, we propose that targeting the appropriate HDACs, which mediate the expression (and function) of CARD9, might be the next step to further enhance our current understanding of the roles of CARD9 in islet dysfunction under metabolic stress and diabetes.

Eribulin is an immune potentiator in breast cancer that upregulates human leukocyte antigen class I expression via the induction of NOD-like receptor family CARD domain-containing 5.

Eribulin inhibits microtubule polymerization and improves the overall survival of patients with recurrent metastatic breast cancer. A subgroup analysis revealed a low neutrophil to lymphocyte ratio (NLR) (<3) to be a prognostic factor of eribulin treatment. We thus hypothesized that eribulin might be related to the immune response for breast cancer cells and we analyzed the effects of eribulin on the immune system. Immunohistochemical staining revealed that human leukocyte antigen (HLA) class I expression was increased in clinical samples after eribulin treatment. In vitro assays revealed that eribulin treatment increased HLA class I expression in breast cancer line cells. RNA-sequencing demonstrated that eribulin treatment increased the expression of the NOD-like family CARD domain-containing 5 (NLRC5), a master regulator of HLA class I expression. Eribulin treatment increased the NY-ESO-1-specific T-cell receptor (TCR) transduced T (TCR-T) cell response for New York oesophageal squamous cell carcinoma 1 (NY-ESO-1) overexpressed breast cancer cells. The eribulin and TCR-T combined therapy model revealed that eribulin and immunotherapy using TCR-T cells has a synergistic effect. In summary, eribulin increases the expression of HLA class 1 via HLA class 1 transactivatior NLRC5 and eribulin combination with immunotherapy can be effective for the treatment of breast cancer.

Corilagin alleviates LPS-induced sepsis through inhibiting pyroptosis via targeting TIR domain of MyD88 and binding CARD of ASC in macrophages.

Sepsis is a dysregulated systemic inflammatory response caused by infection that leads to multiple organ injury and high mortality without effective treatment. Corilagin, a natural polyphenol extracted from traditional Chinese herbs, exhibits strong anti-inflammatory properties. However, the role for Corilagin in lipopolysaccharide (LPS)-induced sepsis and the molecular mechanisms underlying this process have not been completely explored. Here we determine the effect of Corilagin on LPS-treated mice and use a screening approach integrating surface plasmon resonance with liquid chromatography-tandem mass spectrometry (SPR-LC-MS/MS) to further explore the therapeutic target of Corilagin. We discovered that Corilagin significantly prolonged the survival time of septic mice, attenuated the multi-organ injury and the expression of pyroptosis-related proteins in tissues of LPS-treated mice. In vitro studies revealed that Corilagin inhibited pyroptosis and NLRP3 inflammasome activation in LPS-treated macrophages followed with ATP stimulation, as reflected by decreased levels of GSDMD-NT and activated caspase-1, and reduced ASC specks formation. Mechanistically, Corilagin alleviated the formation of ASC specks and blocked the interaction of ASC and pro-caspase1 by competitively binding with the caspase recruitment domain (CARD) of ASC. Additionally, Corilagin interrupted the TLR4-MyD88 interaction through targeting TIR domain of MyD88, leading to the inhibition of NF-κB activation and NLRP3 production. In addition, Corilagin downregulated genes associated with several inflammatory responses and inflammasome-related signaling pathways in LPS-stimulated macrophages. Overall, our results indicate that the inhibitory effect of Corilagin on pyroptosis through targeting TIR domain of MyD88 and binding the CARD domain of ASC in macrophages plays an essential role in protection against LPS-induced sepsis.

TRIM29 (Tripartite Motif Containing 29) Alleviates NLRC4 (NLR Family CARD Domain Containing Protein 4) Inflammasome Related Cerebral Injury via Promoting Proteasomal Degradation of NLRC4 in Ischemic Stroke.

BACKGROUND: Neuroinflammation plays extremely crucial roles in the neurological damage mediated by ischemic stroke. TRIM29 (tripartite motif containing 29) has previously been proposed to contribute to the regulation of innate immunity, however, the effect of TRIM29 on ischemic stroke induced neurodegenerative processes and neuroinflammation still largely unexplored. In the current article, we aimed to investigate the function and the precise mechanisms of TRIM29 in ischemic stroke. METHODS: Middle cerebral artery occlusion mice model and oxygen-glucose deprivation cell model were established as in vivo and in vitro models of ischemic stroke. Quantitative real-time polymerase chain reaction (PCR), Western blot, and ELSIA were used to detect the expression levels of TRIM29, cytokines, and marker proteins. Immunofluorescence assay was performed to examine the extent of cell death. Different truncations were generated, and coimmunoprecipitation assays were used to confirm the protein interaction. Ubiquitination assay was performed to detect the ubiquitination levels. RESULTS: We found that the cerebral ischemia-reperfusion induced injury was aggravated in TRIM29 knockout mice after middle cerebral artery occlusion operation as well as the increased neurological deficits score. TRIM29 expression was also found to be up-regulated upon middle cerebral artery occlusion or OGD administration, and loss of TRIM29 promoted the apoptosis and pyroptosis of neurons and microglial cells induced by middle cerebral artery occlusion or OGD, consistent with the enhanced proinflammatory mediators production and activation of NLRC4 (NLR [NOD-like receptor] family CARD [caspase recruitment domain] domain containing protein 4) inflammasome. Furthermore, we observed that TRIM29 interacted with NLRC4 directly and promoted the K48-linked polyubiquitination of NLRC4, lead to the proteasomal degradation of NLRC4. CONCLUSIONS: In conclusion, for the first time, we revealed the role of TRIM29 in ischemic stroke and illustrated the direct relationship between TRIM29 and NLRC4.

[Effects of caspase recruitment domain protein 9 gene knockout on airway inflammation in C57BL/6 mice with steroid resistant asthma].

Objective: To investigate the role of caspase recruitment domain protein 9 (CARD9) in airway injury and inflammation of steroid resistant asthma in C57BL/6 mice. Methods: C57BL/6 mice were divided into A group (control group), B group (model group) and C group (dexamethasone treatment group), with 6 mouse in each group using random number table. The mouse asthma model was established in B and C group by subcutaneous injection of ovalbumin (OVA)/complete Freund adjuvant (CFA) in the abdomen and OVA aerosol challenge, the pathological change and cell count in broncho alveolar lavage fluid (BALF) were detected in order to confirm the model as steroid resistant asthma, and the lung tissue inflammatory infiltration was scored. Western blot was used to detect the changes of CARD9 protein between the group A and B; then wild-type and CARD9 knockout mice were divided into D group (wild-type control group), E group (wild-type model group), F group (CARD9 knockout control group) and G group (CARD9 knockout model group), the following indicators were observed and compared after establishing steroid resistant asthma model separately: HE staining was used to observe the pathological changes of lung tissue, ELISA was used to detect the protein levels of interleukin-4 (IL-4), interleukin-5 (IL-5) and interleukin-17(IL-17) in BALF, and RT-PCR was used to detect the mRNA levels of CXC motif chemokine ligand-10 (CXCL-10) and IL-17 in lung. Results: The inflammatory score (3.33±0.82 vs 0.67±0.52) and BALF total cell count [(10.13±4.83) ×105/ml vs (3.76±0.84) ×105/ml] in B group were higher than those in the A group with statistical significance (P<0.05). There was no significant difference between group C and group B in inflammatory infiltration score (2.83±0.75 vs 3.33±0.82) and BALF total cell count [(9.80±3.19) ×105/ml vs (10.13±4.83) ×105/ml] (P>0.05). Moreover the protein level of CARD9 was increased in the B group than A group (0.245±0.090 vs 0.047±0.014, P=0.004). Compared to E group and F group, more obviously inflammatory cells, neutrophils, eosinophils infiltration and tissue injury were observed in G group (P<0.05), so did the expression of IL-4 (P<0.05), IL-5 and IL-17. Meanwhile the mRNA expression levels of IL-17 and CXCL-10 also increased in lung tissue (P<0.05) of G group. Conclusion: CARD9 gene deletion may aggravate the steroid resistant of asthma by increasing neutrophil chemokines, such as IL-17 and CXCL-10, therefore increasing infiltration of neutrophils in C57BL/6 mice asthma model.

Molecular Characterization, Expression, and Regulatory Signal Pathway Analysis of Inflammasome Component Apoptosis-Associated Speck-like Protein Containing a CARD Domain (ASC) in Large Yellow Croaker (Larimichthys crocea).

ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD)) is the only adaptor involved in the formation of multiple types of inflammasomes. Accumulating evidence demonstrates that ASC plays a critical role in the protection of the host against pathogen infection. In this study, we identified an ASC gene in the large yellow croaker (Larimichthys crocea), namely LcASC, and then investigated the expression characteristics and related signal pathways. On one hand, LcASC has several conserved protein modules, i.e., an N-terminal PYD region, a C-terminal CARD region, and twelve α-helix structures. On the other hand, it has a high variable linker between PYD and CARD domains. Moreover, LcASC has varying degrees of expression in different tissues, among which the highest expression is observed in the spleen followed by the gills and skin. It also shows induced expressions in the head kidney, liver, and spleen following immune stimulation, especially Vibrio Parahaemolyticus infection. Further subcellular localization analysis showed that LcASC formed a clear aggregated speck in the cytoplasm close to the nucleus. In addition, we found 46 DEGs in a comparative transcriptome analysis between the LcASC overexpression group and the control vector group. Notedly, the up-regulated gene Fos and down-regulated gene DOK3 in LcASC overexpressed cells play important roles in the immune system. How ASC contacts these two genes needs to be clarified in upcoming studies. These findings collectively provide new insights into finfish ASC and its potential regulatory signaling pathway as well.

A double-edged sword: role of apoptosis repressor with caspase recruitment domain (ARC) in tumorigenesis and ischaemia/reperfusion (I/R) injury.

Apoptosis repressor with caspase recruitment domain (ARC) acts as a potent and multifunctional inhibitor of apoptosis, which is mainly expressed in postmitotic cells, including cardiomyocytes. ARC is special for its N-terminal caspase recruitment domain and caspase recruitment domain. Due to the powerful inhibition of apoptosis, ARC is mainly reported to act as a cardioprotective factor during ischaemia‒reperfusion (I/R) injury, preventing cardiomyocytes from being devastated by various catastrophes, including oxidative stress, calcium overload, and mitochondrial dysfunction in the circulatory system. However, recent studies have found that ARC also plays a potential regulatory role in tumorigenesis especially in colorectal cancer and renal cell carcinomas, through multiple apoptosis-associated pathways, which remains to be explored in further studies. Therefore, ARC regulates the body and maintains the balance of physiological activities with its interesting duplex. This review summarizes the current research progress of ARC in the field of tumorigenesis and ischaemia/reperfusion injury, to provide overall research status and new possibilities for researchers.

Mechanism of action of IC 100, a humanized IgG4 monoclonal antibody targeting apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC).

Inflammasomes are multiprotein complexes of the innate immune response that recognize a diverse range of intracellular sensors of infection or cell damage and recruit the adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) into an inflammasome signaling complex. The recruitment, polymerization and cross-linking of ASC is upstream of caspase-1 activation and interleukin-1ß release. Here we provide evidence that IC 100, a humanized IgG4κ monoclonal antibody against ASC, is internalized into the cell and localizes with endosomes, while another part is recycled and redistributed out of the cell. IC 100 binds intracellular ASC and blocks interleukin-1ß release in a human whole blood cell inflammasome assay. In vitro studies demonstrate that IC 100 interferes with ASC polymerization and assembly of ASC specks. In vivo bioluminescence imaging showed that IC 100 has broad tissue distribution, crosses the blood brain barrier, and readily penetrates the brain and spinal cord parenchyma. Confocal microscopy of fluorescent-labeled IC 100 revealed that IC 100 is rapidly taken up by macrophages via a mechanism utilizing the Fc region of IC 100. Coimmunoprecipitation experiments and confocal immunohistochemistry showed that IC 100 binds to ASC and to the atypical antibody receptor Tripartite motif-containing protein-21 (TRIM21). In A549 WT and TRIM21 KO cells treated with either IC 100 or IgG4κ isotype control, the levels of intracellular IC 100 were higher than in the IgG4κ-treated controls at 2 hours, 1 day and 3 days after administration, indicating that IC 100 escapes degradation by the proteasome. Lastly, electron microscopy studies demonstrate that IC 100 binds to ASC filaments and alters the architecture of ASC filaments. Thus, IC 100 readily penetrates a variety of cell types, and it binds to intracellular ASC, but it is not degraded by the TRIM21 antibody-dependent intracellular neutralization pathway.

NLRC5 overexpression in ovarian tumors remodels the tumor microenvironment and increases T-cell reactivity toward autologous tumor-associated antigens.

Introduction: Epithelial ovarian cancer (OC) stands as one of the deadliest gynecologic malignancies, urgently necessitating novel therapeutic strategies. Approximately 60% of ovarian tumors exhibit reduced expression of major histocompatibility complex class I (MHC I), intensifying immune evasion mechanisms and rendering immunotherapies ineffective. NOD-like receptor CARD domain containing 5 (NLRC5) transcriptionally regulates MHC I genes and many antigen presentation machinery components. We therefore explored the therapeutic potential of NLRC5 in OC. Methods: We generated OC cells overexpressing NLRC5 to rescue MHC I expression and antigen presentation and then assessed their capability to respond to PD-L1 blockade and an infected cell vaccine. Results: Analysis of microarray datasets revealed a correlation between elevated NLRC5 expression and extended survival in OC patients; however, NLRC5 was scarcely detected in the OC tumor microenvironment. OC cells overexpressing NLRC5 exhibited slower tumor growth and resulted in higher recruitment of leukocytes in the TME with lower CD4/CD8 T-cell ratios and increased activation of T cells. Immune cells from peripheral blood, spleen, and ascites from these mice displayed heightened activation and interferon-gamma production when exposed to autologous tumor-associated antigens. Finally, as a proof of concept, NLRC5 overexpression within an infected cell vaccine platform enhanced responses and prolonged survival in comparison with control groups when challenged with parental tumors. Discussion: These findings provide a compelling rationale for utilizing NLRC5 overexpression in "cold" tumor models to enhance tumor susceptibility to T-cell recognition and elimination by boosting the presentation of endogenous tumor antigens. This approach holds promise for improving antitumoral immune responses in OC.

BMI1 induces ubiquitination and protein degradation of Nod-like receptor family CARD domain containing 5 and suppresses human leukocyte antigen class I expression to induce immune escape in non-small cell lung cancer.

The Nod-like receptor (NLR) family CARD domain containing 5 (NLRC5) has been reported as an activator of human leukocyte antigen (HLA) class I that is responsible for immune activity in cancer treatment. This work focuses on the role of BMI1 proto-oncogene (BMI1) in the NLRC5-HLA class I axis and in immune escape in non-small cell lung cancer (NSCLC). First, immunoblot analysis and/or reverse transcription-quantitative polymerase chain reaction were performed, which identified decreased NLRC5 and HLA class I levels in NSCLC tissues and cell lines. NSCLCs were co-cultured with activated CD8+ T cells. Overexpression of NLRC5 in NSCLC cells elevated the expression of HLA class I and increased the activity of T cells and IL-2 production, and it reduced the PD-1/PD-L1 levels. The ubiquitination and immunoprecipitation assays confirmed that BMI1 bound to NLRC5 to induce is ubiquitination and protein degradation. Downregulation of BMI1 in NSCLC cells elevated NLRC5 and HLA class I levels, and consequently promoted T cell activation and decreased PD-1/PD-L1 levels in the co-culture system. However, overexpression of BMI1 in cells led to inverse trends. In summary, this study demonstrates that BMI1 induces ubiquitination and protein degradation of NLRC5 and suppresses HLA class I expression, which potentially helps immune escape in NSCLC.

Gene Delivery of a Caspase Activation and Recruitment Domain Improves Retinal Pigment Epithelial Function and Modulates Inflammation in a Mouse Model with Features of Dry Age-Related Macular Degeneration.

Purpose: The NLRP3 inflammasome, a cytoplasmic signal transduction complex that regulates inflammation, has been implicated in the pathogenesis of age-related macular degeneration (AMD), the leading cause of visual impairment in industrialized countries. We tested the therapeutic effect of anti-inflammatory gene therapy, delivered preventively, in Liver-X-Receptor alpha knockout (LXRα-/-) mice, which exhibit features of dry AMD. Methods:LXRα-/- mice were treated with an adeno-associated virus (AAV) vector that delivers a secretable and cell-penetrating form of the caspase activation and recruitment domain (CARD). A sGFP-FCS-TatCARD-AAV or sGFP-FCS (control) vector was delivered intravitreally to 3-5 month-old, LXRα-/- mice, who were then aged to 15-18 months (12-13 month treatment). Retinal function and morphology were assessed pre- and post-treatment. Results: TatCARD treated LXRα-/- mice did not show improvement in rod and cone photoreceptor function, measured by dark adapted a- and b-wave amplitudes, and rod-saturated b-wave amplitudes. We found a sex-dependent, significant therapeutic effect in c-wave amplitudes in the TatCARD treated mice, which exhibited maintenance of amplitudes in comparison to the significant decline recorded in the control treated group, indicating a therapeutic effect mediated in part through retinal pigment epithelial (RPE) cells. Additionally, the retinas of the TatCARD treated mice exhibited a significant decline in the concentration of interleukin-1 beta (IL-1ß) concomitant with modulation of several inflammatory cytokines in the retina and RPE-choroid tissues, as measured by ELISA and cytokine array, respectively. Conclusion: Collectively, these results support that anti-inflammatory gene constructs such as AAV-TatCARD may be considered for the treatment of inflammation in AMD and other ocular diseases of the posterior pole in which inflammation may play a role. Furthermore, our findings emphasize the need to carefully consider potential sex-different responses when assessing potential therapies in pre-clinical models.

Inflammasome regulation by adaptor isoforms, ASC and ASCb, via differential self-assembly.

ASC is an essential adaptor of the inflammasome, a micrometer-size multiprotein complex that processes proinflammatory cytokines. Inflammasome formation depends on ASC self-association into large assemblies via homotypic interactions of its two death domains, PYD and CARD. ASCb, an alternative splicing isoform, activates the inflammasome to a lesser extent compared with ASC. Thus, it has been postulated that adaptor isoforms differentially regulate inflammasome function. At the amino acid level, ASC and ASCb differ only in the length of the linker connecting the two death domains. To understand inflammasome regulation at the molecular level, we investigated the self-association properties of ASC and ASCb using real-time NMR, dynamic light scattering (DLS), size-exclusion chromatography, and transmission electron microscopy (TEM). The NMR data indicate that ASC self-association is faster than that of ASCb; a kinetic model for this oligomerization results in differing values for both the reaction order and the rate constants. Furthermore, DLS analysis indicates that ASC self-associates into more compact macrostructures compared with ASCb. Finally, TEM data show that ASCb has a reduced tendency to form densely packed filaments relative to ASC. Overall, these differences can only be explained by an effect of the linker length, as the NMR results show structural equivalence of the PYD and CARD in both proteins. The effect of linker length was corroborated by molecular docking with the procaspase-1 CARD domain. Altogether, our results indicate that ASC's faster and less polydisperse polymerization is more efficient, plausibly explaining inflammasome activation differences by ASC isoforms at the molecular level.

NLR family CARD domain containing 5 promotes hypoxia-induced cancer progress and carboplatin resistance by activating PI3K/AKT via carcinoembryonic antigen related cell adhesion molecule 1 in non-small cell lung cancer.

It is well known that non-small cell lung cancer (NSCLC) is a malignant tumor with high incidence in the world. We aimed to clarify a possible target and identify its precise molecular biological mechanism in NSCLC. NLR family CARD domain containing 5 (NLRC5) is widely expressed in tissues and exerts a vital role in anti-tumor immunity. We determined NLRC5 expression by RT-qPCR and western blot assay. The role of NLRC5 in the development of NSCLC was assessed by a loss-of-function assay. CCK-8, Annexin-V-FITC/PI Apoptosis Detection Kit, Transwell, and wound healing assays were used to determine the cell functions. Drug resistance-related proteins were analyzed by western blot assay. Furthermore, the modulation of NLRC5 on carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) expression and subsequent PI3K/AKT signaling was assessed. In this study, a hyper-expression of NLRC5 was found in NSCLC tissues and cell lines. Knockdown of NLRC5 suppressed cell viability, invasion, and migration, and furthermore promoted cell apoptosis in NSCLC cells. Moreover, under normoxia or hypoxia treatment, the upregulation of NLRC5 was related to carboplatin resistance. NLRC5 silencing increased carboplatin-resistant cell chemosensitivity, as evidenced by the increase in the cell inhibition rate and decrease in drug resistance-related protein expression. Mechanistically, NLRC5 knockdown inhibited the expression of CEACAM1 and subsequently blocked the PI3K/AKT signaling pathway. In conclusion, NLRC5 promotes the malignant biological behaviors of NSCLC cells by activating the PI3K/AKT signaling pathway via the regulation of CEACAM1 expression under normoxia and hypoxia.

Effect of luteolin on the gene level expression of apoptosis-associated speck-like protein containing a caspase recruitment domain of NLRP3 inflammasome and NF-κB in rats subjected to experimental pancreatitis - influence of HSP70.

OBJECTIVES: Nod-like receptor pyrin domain containing 3 (NLRP3) is one of the well characterized inflammasome that controls the maturation of pro-inflammatory cytokines and thereby the inflammation in pancreas which could be a promising target for anti-inflammatory drugs. The present study is aimed to explore whether luteolin can target the NLRP3 inflammasome and modulate its activity through the signaling protein, HSP70 in the ethanol-cerulein model of experimental pancreatitis. METHODS: Male albino Wistar rats were divided into four groups. Groups 1 and 2 rats received normal diet. Groups 3 and 4 rats received isocalorically adjusted diet containing ethanol for 5 weeks and cerulein (20 µg/kg body weight i.p., thrice weekly for the last 3 weeks of the experimental period). Additionally, group 2 and 4 rats received 2 mg/kg body weight of luteolin orally from third week. RESULTS: Luteolin co-administration decreased the serum levels of HSP70, oxidative stress markers, myeloperoxidase, GSH/GSSG and GST with concomitant downregulation in the mRNA expression of HSP70, caspase-1, ASC-NLRP3 and NF-κB. Spearman's rank correlation test showed that serum HSP70 has positive correlation with the expression of ASC-NLRP3, caspase-1, NF-κB and 4-hydroxynonenal and negative correlation with GSH:GSSG ratio. CONCLUSIONS: The modulating effect of luteolin on the expression of HSP70, NF-κB and thereby on ASC-NLRP3 complex may be claimed for its pancreato-protective activity.

Dual function of a turbot inflammatory caspase in mediating both canonical and non-canonical inflammasome activation.

Host protective inflammatory caspase activity must be tightly regulated to prevent pathogens infection, however, the inflammatory caspase-engaged inflammasome activation in teleost fish remains largely unknown. In this study, we reveal a bifurcated evolutionary role of the inflammatory caspase in mediating both non-canonical and canonical inflammasome pathways in teleost fish. Through characterization of a unique inflammatory SmCaspase from the teleost Scophthalmus maximus (turbot), we found it can directly recognize cytosolic lipopolysaccharide (LPS) via its N-terminal CARD domain, resulting in caspase-5-like proteolytic enzyme activity-mediated pyroptosis in Turbot Muscle Fibroblasts. Interestingly, we also found that this inflammatory caspase can be recruited to SmNLRP3-SmASC to form the NLRP3 inflammasome complex, engaging the SmIL-1ß release in Head Kidney-derived Macrophages. Consequently, the SmCaspase activation can recognize and cleave the SmGSDMEb to release its N-terminal domain, mediating both pyroptosis and bactericidal activities. Furthermore, the SmCaspase-SmGSDMEb axis-gated pyroptosis governs the bacterial clearance and epithelial desquamation in fish gill filaments in vivo. To our knowledge, this study is the first to identify an inflammatory caspase acting as a central coordinator in NLRP3 inflammasome, as well as a cytosolic LPS receptor; thus uncovering a previously unrecognized function of inflammatory caspase in turbot innate immunity.

Apoptosis repressor with caspase recruitment domain (ARC) promotes bone regeneration of bone marrow-derived mesenchymal stem cells by activating Fgf-2/PI3K/Akt signaling.

OBJECTIVES: This study aims to investigate whether apoptosis repressor with caspase recruitment domain (ARC) could promote survival and enhance osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). MATERIALS AND METHODS: The lentivirus transfection method was used to establish ARC-overexpressing BMSCs. The CCK-8 method was used to detect cell proliferation. The BD Pharmingen™ APC Annexin V Apoptosis Detection kit was used to detect cell apoptosis. The osteogenic capacity was investigated by OCN immunofluorescence staining, ALP analysis, ARS assays, and RT-PCR analysis. Cells were seeded into calcium phosphate cement (CPC) scaffolds and then inserted subcutaneously into nude mice and the defect area of the rat calvarium. Histological analysis was conducted to evaluate the in vivo cell apoptosis and new bone formation of the ARC-overexpressing BMSCs. RNA-seq was used to detect the possible mechanism of the effect of ARC on BMSCs. RESULTS: ARC promoted BMSC proliferation and inhibited cell apoptosis. ARC enhanced BMSC osteogenic differentiation in vitro. An in vivo study revealed that ARC can inhibit BMSC apoptosis and increase new bone formation. ARC regulates BMSCs mainly by activating the Fgf-2/PI3K/Akt pathway. CONCLUSIONS: The present study suggests that ARC is a powerful agent for promoting bone regeneration of BMSCs and provides a promising method for bone tissue engineering.

Vitamin-B12-conjugated PLGA-PEG nanoparticles incorporating miR-532-3p induce mitochondrial damage by targeting apoptosis repressor with caspase recruitment domain (ARC) on CD320-overexpressed gastric cancer.

Among various methods, the use of targeting nucleic acid therapy is a promising method for inhibiting gastric cancer (GC) cells' rapid growth and metastasis abilities. In this study, vitamin B12-labeled poly (d,l-lactide-co-glycolide) and polyethylene glycol nanoparticles (PLGA-PEG-VB12 NPs) were developed for microRNAs-532-3p mimics incorporating as targeting gene delivery systems (miR-532-3p@PLGA-PEG-VB12 NPs) to fight against transcobalamin II (CD320)-overexpressed GC cells' progression. The PLGA-PEG-VB12 NPs with appropriate particle sizes and good bio-compatibility could be selectively delivered into CD320-overexpressed GC cells, and significantly decrease the expression of apoptosis repressor with caspase recruitment domain (ARC). Following that, more pro-apoptotic protein (Bax) flowed from cytoplasm into mitochondria to form Bax oligomerization, thus induced mitochondrial damage, including mitochondrial membrane potentials (MMPs) loss and excessive production of mitochondrial reactive oxygen species (mitoROS). Since that, mitochondrial permeability transition pore (mPTP) was opened, followed by induced more cytochrome c (Cyto C) releasing from mitochondria into cytosol, and finally activated caspase-depended cell apoptosis pathway. Therefore, our designed miR-532-3p@PLGA-PEG-VB12 NPs showed enhanced GC targeting ability, and could induce apoptosis through activating ARC/Bax/mitochondria-mediated apoptosis signaling pathway, finally remarkably suppressed proliferation of GC cells both in vitro and in vivo, which presented a promising treatment for GC.