Caspase-1 Research Articles

CASP1 is a target for combination therapy in pancreatic cancer

Gemcitabine (GEM) is commonly used as the first-line chemotherapeutic agent for treating pancreatic cancer (PC) patients. However, drug resistance is a major hurdle in GEM-based chemotherapy for PC. Recent studies have shown that pyroptosis, a type of programmed death, plays a significant regulatory role in cancer development and therapy. In this study, we observed an increase in the expression of Caspase-1(CASP1)/Gasdermin-D (GSDMD) in PC and found that high expression of CASP1 and GSDMD was associated with poor overall survival (OS) and progression-free survival (PFS) of PC patients. Knockdown of either CASP1 or GSDMD resulted in the inhibition of cell viability and migration in PC cells. More importantly, the knockdown of CASP1 or GSDMD enhanced GEM-induced cell death in PC cells. Interestingly, subsequent investigations demonstrated that enzymatically active CASP1 promoted GEM-induced cell death in PC cells. The activation of CASP1 by the DPP8/DPP9 inhibitor (Val-boroPro, VbP) increased GEM-induced cell death by inducing pyroptosis. These findings suggest that inhibiting CASP1 to suppress its oncogenic effects or activating it to promote cell pyroptosis both enhance the sensitivity of PC cells to GEM therapy.

Qiji Shujiang granules alleviates dopaminergic neuronal injury of parkinson's disease by inhibiting NLRP3/Caspase-1 pathway mediated pyroptosis

BackgroundThe Qiji Shujiang granule (QJG) is a traditional Chinese drug widely used in treating PD patients. However, the potential mechanism of QJG in PD therapy is still unclear. PurposeThis study aims to examine the neuroprotective effects of QJG and the specific mechanism by which QJG alleviates MPTP/Probenecid-induced pyroptosis and offers an alternative for PD treatment. Study Design and MethodsWe first employed network pharmacology along with molecular docking to identify potential molecular targets and pathways. Subsequently, we validated our findings of RNA-sequencing (RNA-seq) analysis and experiments in vivo and vitro. Lentiviral systems and inhibitors were used for experiments. ResultsThe protein-protein interactions (PPI) core genes network consists of NLRP3, CASP1 (caspase-1), TP53, and MAPK8. Pathway enrichment analysis revealed that inflammatory responses related to pyroptosis were significantly enriched. The molecular docking findings showed the highest degree of centrality regarding the top three bioactive compounds following the online database. RNA-seq analysis identified that NLRP3 inflammasome was significantly downregulated in the QJG group while it was significantly upregulated in the model group. Our findings revealed that QJG dose-dependently increased the total traveled distances, enhanced the dopaminergic neurons, and accelerated the restoration of the TH protein level, showing a good antioxidant capacity through increasing the SOD levels and decreasing MDA levels. QJG significantly reduced the expression levels of NLRP3, GSDMD-N, IL-1β, and caspase-1 in striatum tissue. Furthermore, the group treated with OE-NLRP3 decreased cell viability, increased ROS and MDA levels, and promoted NLRP3, GSDMD-N, and caspase-1, in addition to IL-1β expression levels. Furthermore, OE-NLRP3+QJG treatment significantly reversed the effect. In vivo experiments, QJG dose-dependently alleviated motor impairment by increasing the total traveled distances, rescued dopaminergic neurons, inhibited oxidative stress through increasing the SOD levels and decreasing MDA levels and suppressed NLRP3-mediated pyroptosis by reducing the expression levels of NLRP3, GSDMD-N, IL-1β, and caspase-1 in MPTP induced PD Mice. Moreover, in vitro experiments, the OE-NLRP3 treated group decreased cell viability, increased ROS and MDA levels, and promoted NLRP3, GSDMD-N, caspase-1, in addition to IL-1β expression levels. Furthermore, OE-NLRP3+QJG treatment significantly reversed the effect. ConclusionsThis study provides pharmacological support for the use of QJG in the treatment of PD. Herein, we concluded that QJG induced the alleviation of pyroptosis by inhibiting the NLRP3/caspase-1 pathway to exert a neuroprotective effect.

CGRP-loaded Porous Microspheres Protect BMSCs for Alveolar Bone Regeneration in The Periodontitis Microenvironment.

Periodontitis is a prevalent dental disease marked by progressive destruction of tooth-supporting tissues, and the recovery of bone defects after periodontitis remains challenging. Although stem cell-based therapy is a promising treatment for periodontal tissue regeneration, the function of mesenchymal stem cells is constantly impaired by the inflammatory microenvironment, leading to compromised treatment outcomes. Herein, calcitonin gene-related peptide (CGRP)-loaded porous microspheres (PMs) are prepared to protect bone marrow mesenchymal stem cells (BMSCs) against inflammatory mediators in periodontitis. The released CGRP could effectively ameliorate the inflammation-induced dysfunction of BMSCs, which may involve suppressing the ROS (reactive oxygen species)/NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3)/Caspase-1 pathway. Moreover, the porous architecture of PMs provides effective cell-carrying capacity and physical protection for BMSCs during transplantation. In vivo experiments demonstrate that CGRP/BMSC-loaded PMs could effectively inhibit inflammation and improve osteogenic activity, resulting in better periodontal bone regeneration. This study focuses on the protection of stem cell function in the inflammatory microenvironment, which is important for stem cell-mediated tissue regeneration and repair under inflammatory conditions. This article is protected by copyright. All rights reserved.

Effect of caspase-1 (CASP1) combined with multimodal ultrasound features on the prognosis of breast cancer patients.

Breast cancer (BRCA) is the malignant tumor with the highest incidence rate among women in the world, and its mortality rate ranks second. The purpose of our study is to explore the correlation between caspase-1 (CASP1) and the prognosis of BRCA patients and the potential mechanism of action, and to analyze the clinical value of CASP1 combined with multimodal ultrasound features in early screening and prognosis of BRCA. We analyzed The Cancer Genome Atlas (TCGA) database to confirm that CASP1 was expressed in BRCA patients and determine whether its expression was correlated with patient prognosis. The relationship between CASP1 expression and survival was measured by the clinicopathological parameters. Multivariate analysis was performed using Cox regression, and a nomogram was developed using these results for quality assurance purposes. The correlations between CASP1 and immune cells were investigated using the Tumor Immune Estimation Resource (TIMER) and TCGA databases. Next, we performed gene set enrichment analysis (GSEA) to determine the potential mechanism of action. Finally, to analyze the effect of CASP1 combined with multimodal ultrasonography characteristics on the prognosis of BRCA patients was studied by analyzing the clinical data of patients. CASP1 expression was lower in BRCA tumor tissues than in the surrounding tissues. Patients with high CASP1 expression had better overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI) than those with low CASP1 expression. GSEA suggested that CASP1 may affect the cell cycle, immune environment, inflammation, apoptosis, the HIPPOMERLIN pathway, Natural killer (NK) cell regulation of cytotoxicity, p53 expression, the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, the mitogen-activated protein kinase (MAPK) pathway, extracellular matrix, etc., thereby influencing the biological events in BRCA. Among conventional ultrasound features and contrast-enhanced ultrasound (CEUS) features, mass margin status and blood flow grade were associated with the expression of CASP1. Meanwhile, patients with poor ultrasound features tended to have low CASP1 expression. CASP1 may be a novel predictive marker for BRCA patients. CASP1 combined with multimodal ultrasound features has good clinical value in the early screening and prognostic prediction of BRCA.

Cardiac and noncardiac biomarkers in patients undergoing anthracycline chemotherapy – a prospective analysis

BackgroundBiomarkers represent a potential tool to identify individuals at risk for anthracycline-induced cardiotoxicity (AICT) prior to symptom onset or left ventricular dysfunction.MethodsThis study examined the levels of cardiac and noncardiac biomarkers before, after the last dose of, and 3–6 months after completion of doxorubicin chemotherapy. Cardiac biomarkers included 5th generation high-sensitivity cardiac troponin T (cTnT), N-terminal pro-brain natriuretic peptide, growth/differentiation factor-15 (GDF-15), and soluble suppression of tumorigenesis-2 (sST2). Noncardiac biomarkers included activated caspase-1 (CASP-1), activated caspase-3, C-reactive protein, tumor necrosis factor-α, myeloperoxidase (MPO), galectin-3, and 8-hydroxy-2’-deoxyguanosine. Echocardiographic data (LVEF and LVGLS) were obtained at pre- and post-chemotherapy. Subanalysis examined interval changes in biomarkers among high (cumulative doxorubicin dose ≥ 250 mg/m2) and low exposure groups.ResultsThe cardiac biomarkers cTnT, GDF-15, and sST2 and the noncardiac biomarkers CASP-1 and MPO demonstrated significant changes over time. cTnT and GDF-15 levels increased after anthracycline exposure, while CASP-1 and MPO decreased significantly. Subanalysis by cumulative dose did not demonstrate a larger increase in any biomarker in the high-dose group.ConclusionsThe results identify biomarkers with significant interval changes in response to anthracycline therapy. Further research is needed to understand the clinical utility of these novel biomarkers.

Pyroptosis-Mediated Damage Mechanism by Deoxynivalenol in Porcine Small Intestinal Epithelial Cells

Deoxynivalenol (DON) is known as a vomitoxin, which frequently contaminates feedstuffs, such as corn, wheat, and barley. Intake of DON-contaminated feed has been known to cause undesirable effects, including diarrhea, emesis, reduced feed intake, nutrient malabsorption, weight loss, and delay in growth, in livestock. However, the molecular mechanism of DON-induced damage of the intestinal epithelium requires further investigation. Treatment with DON triggered ROS in IPEC-J2 cells and increased the mRNA and protein expression levels of thioredoxin interacting protein (TXNIP). To investigate the activation of the inflammasome, we confirmed the mRNA and protein expression levels of the NLR family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and caspase-1 (CASP-1). Moreover, we confirmed that caspase mediates the mature form of interleukin-18, and the cleaved form of Gasdermin D (GSDMD) was increased. Based on these results, our study suggests that DON can induce damage through oxidative stress and pyroptosis in the epithelial cells of the porcine small intestine via NLRP3 inflammasome.

Shigella IpaH9.8 limits GBP1-dependent LPS release from intracytosolic bacteria to suppress caspase-4 activation.

Pyroptosis is an inflammatory form of cell death induced upon recognition of invading microbes. During an infection, pyroptosis is enhanced in interferon-gamma-exposed cells via the actions of members of the guanylate-binding protein (GBP) family. GBPs promote caspase-4 (CASP4) activation by enhancing its interactions with lipopolysaccharide (LPS), a component of the outer envelope of Gram-negative bacteria. Once activated, CASP4 promotes the formation of noncanonical inflammasomes, signaling platforms that mediate pyroptosis. To establish an infection, intracellular bacterial pathogens, like Shigella species, inhibit pyroptosis. The pathogenesis of Shigella is dependent on its type III secretion system, which injects ~30 effector proteins into host cells. Upon entry into host cells, Shigella are encapsulated by GBP1, followed by GBP2, GBP3, GBP4, and in some cases, CASP4. It has been proposed that the recruitment of CASP4 to bacteria leads to its activation. Here, we demonstrate that two Shigella effectors, OspC3 and IpaH9.8, cooperate to inhibit CASP4-mediated pyroptosis. We show that in the absence of OspC3, an inhibitor of CASP4, IpaH9.8 inhibits pyroptosis via its known degradation of GBPs. We find that, while some LPS is present within the host cell cytosol of epithelial cells infected with wild-type Shigella, in the absence of IpaH9.8, increased amounts are shed in a GBP1-dependent manner. Furthermore, we find that additional IpaH9.8 targets, likely GBPs, promote CASP4 activation, even in the absence of GBP1. These observations suggest that by boosting LPS release, GBP1 provides CASP4-enhanced access to cytosolic LPS, thus promoting host cell death via pyroptosis.

Dual Role of Mitogen-Activated Protein Kinase 8 Interacting Protein-1 in Inflammasome and Pancreatic β-Cell Function

Inflammasomes have been implicated in the pathogenesis of type 2 diabetes (T2D). However, their expression and functional importance in pancreatic β-cells remain largely unknown. Mitogen-activated protein kinase 8 interacting protein-1 (MAPK8IP1) is a scaffold protein that regulates JNK signaling and is involved in various cellular processes. The precise role of MAPK8IP1 in inflammasome activation in β-cells has not been defined. To address this gap in knowledge, we performed a set of bioinformatics, molecular, and functional experiments in human islets and INS-1 (832/13) cells. Using RNA-seq expression data, we mapped the expression pattern of proinflammatory and inflammasome-related genes (IRGs) in human pancreatic islets. Expression of MAPK8IP1 in human islets was found to correlate positively with key IRGs, including the NOD-like receptor (NLR) family pyrin domain containing 3 (NLRP3), Gasdermin D (GSDMD) and Apoptosis-associated speck-like protein containing a CARD (ASC), but correlate inversely with Nuclear factor kappa β1 (NF-κβ1), Caspase-1 (CASP-1), Interleukin-18 (IL-18), Interleukin-1β (IL-1β) and Interleukin 6 (IL-6). Ablation of Mapk8ip1 by siRNA in INS-1 cells down-regulated the basal expression levels of Nlrp3, NLR family CARD domain containing 4 (Nlrc4), NLR family CARD domain containing 1 (Nlrp1), Casp1, Gsdmd, Il-1β, Il-18, Il-6, Asc, and Nf-κβ1 at the mRNA and/or protein level and decreased palmitic acid (PA)-induced inflammasome activation. Furthermore, Mapk8ip1-silened cells substantially reduced reactive oxygen species (ROS) generation and apoptosis in palmitic acid-stressed INS-1 cells. Nonetheless, silencing of Mapk8ip1 failed to preserve β-cell function against inflammasome response. Taken together, these findings suggest that MAPK8IP1 is involved in regulating β-cells by multiple pathways.

Associations of genetic variations in pyroptosis related genes with acute adverse events in postoperative rectal cancer patients receiving concurrent chemoradiotherapy

Objective: This study aims to investigate the associations between genetic variations of pyroptosis pathway related key genes and adverse events (AEs) of postoperative chemoradiotherapy (CRT) in patients with rectal cancer. Methods: DNA was extracted from the peripheral blood which was collected from 347 patients before CRT. Sequenom MassARRAY was used to detect the genotypes of 43 haplotype-tagging single nucleotide polymorphisms (htSNPs) in eight pyroptosis genes, including absent in melanoma 2 (AIM2), caspase-1 (CASP1), caspase-4(CASP4), caspase-5 (CASP5), caspase-11 (CASP11), gasdermin D (GSDMD), gasdermin E (GSDME) and NLR family pyrin domain containing 3 (NLRP3). The associations between 43 htSNPs and AEs were evaluated by the odd ratios (ORs) and 95% confidence intervals (CIs) by unconditional logistic regression models, adjusted for sex, age, clinical stage, tumor grade, Karnofsky performance status (KPS), surgical procedure, and tumor location. Results: Among the 347 patients with rectal cancer underwent concurrent CRT with capecitabine after surgery, a total of 101(29.1%) occurred grade ≥ 2 leukopenia. rs11226565 (OR=0.41, 95% CI: 0.21-0.79, P=0.008), rs579408(OR=1.54, 95% CI: 1.03-2.29, P=0.034) and rs543923 (OR=0.63, 95% CI: 0.41-0.98, P=0.040) were significantly associated with the occurrence of grade ≥ 2 leukopenia. One hundred and fifty-six (45.0%) had grade ≥ 2 diarrhea, two SNPs were significantly associated with the occurrence of grade ≥ diarrhea, including CASP11 rs10880868 (OR=0.55, 95% CI: 0.33-0.91, P=0.020) and GSDME rs2954558 (OR=1.52, 95% CI: 1.01-2.31, P=0.050). In addition, sixty-six cases (19.0%) developed grade ≥2 dermatitis, three SNPs that significantly associated with the risk of grade ≥2 dermatitis included GSDME rs2237314 (OR=0.36, 95% CI: 0.16-0.83, P=0.017), GSDME rs12540919 (OR=0.52, 95% CI: 0.27-0.99, P=0.045) and NLRP3 rs3806268 (OR=1.51, 95% CI: 1.03-2.22, P=0.037). There was no significant difference in the association between other genetic variations and AEs of rectal cancer patients (all P>0.05). Surgical procedure and tumor location had great impacts on the occurrence of grade ≥2 diarrhea and dermatitis (all P<0.01). Conclusion: The genetic variants of CASP4, CASP11, GSDME and NLRP3 are associated with the occurrence of AEs in patients with rectal cancer who received postoperative CRT, suggesting they may be potential genetic markers in predicting the grade of AEs to achieve individualized treatment of rectal cancer.

Overexpression of CASP1 triggers acute promyelocytic leukemia cell pyroptosis and differentiation

Caspase-1 (CASP1)-mediated classical pyroptosis plays a key role in cancer development and management, however, the role of CASP1 and its regulation has not yet been documented for acute promyelocytic leukemia (APL). Here, we found that CASP1/GSDMD had lower expression in patients with APL and most other subtypes of primary de novo acute myeloid leukemia (AML) and was increased in all-trans-retinoic acid (ATRA)-treated APL cells. We showed that ATRA increases and activates CASP1 to trigger the pyroptosis and differentiation of APL cells. Mechanistically, ATRA could induce CASP1 expression via the IFNγ/STAT1 pathway in APL cells. In conclusion, ATRA-induced activation of CASP1 may serve as a suppressor in APL progression, as it triggers pyroptotic cell death and differentiation.

NLRP3 inflammasome activation By 17β-estradiol is a potential therapeutic target in hepatocellular carcinoma treatment

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and it mostly arises as a consequence of persistent chronic inflammation. Recently, NLRP3 inflammasome has caught the attention of many research groups due to its involvement in different types of cancer. However, its direct role in HCC remains elusive. Our study aimed to evaluate the role of NLRP3 inflammasome and pyroptosis in HCC and to clarify the potential mechanism by which 17β-estradiol (E2) can be used as a protective factor against HCC. NLRP3, caspase-1 (CASP1) as well as gasdermin-D (GSDMD) mRNA expression levels were assessed in human HCC tissues and adjacent non-cancerous liver tissues. Also, HepG2 HCC cells were cultured and treated with E2, followed by detection of the mRNA levels of these three genes. Our results revealed that NLRP3, CASP1, and GSDMD mRNA expressions were significantly lower in HCC tissues than in controls, and this under-expression was closely correlated with advanced HCC stages and grades. In contrast, HepG2 HCC cells displayed significantly higher expression levels of NLRP3 inflammasome components and GSDMD in the two E2-treated groups compared to the untreated group. Also, NLRP3, CASP1, and GSDMD mRNA expression levels were positively correlated with each other. This study confirmed that lack of NLRP3 inflammasome is involved in HCC progression and 17β-estradiol-induced activation of NLRP3 inflammasome may be effective in HCC treatment as it inhibited tumor cell growth and proliferation by triggering CASP1-dependent pyroptosis in HCC cells.

Circ_0013613 Promotes the recovery of spinal cord injury by regulating mir-370-3p/caspase1 to regulate neuronal pyroptosis.

Spinal cord injury (SCI) is a common disease of the central nervous system. Circular RNAs (circRNAs) play an essential role in neuron disorders. This study aimed to investigate the role and regulatory mechanism of circ_0013613 in promoting functional recovery of spinal cord injury (SCI). The mRNA and miRNA expressions were calculated by RT-QPCR. The characteristics of circ_0013613 in nerve cells was determined by actinomycin D and RNase R assay. Protein expression was detected using Western blot. Lactate dehydrogenase (LDH) activity assay was performed to determine cytotoxicity. ELISA was conducted to detect the release of cytokines. MTT assay and flow cytometry were applied to measure cellular functions. In SCI patients and lipopolysaccharide (LPS)-induced primary spinal cord neurons circ_0013613 was increased. Knockdown of circ_0013613 suppressed LPS-stimulated cytotoxicity, inflammation and inflammation-induced pyroptosis. Additionally, circ_0013613 negatively regulated miR-370-3p, downregulation of which degraded cellular functions of spinal cord neurons. Moreover, miR-370-3p targeted Caspase1 (CASP1), overexpression of which promoted the inflammation and pyroptosis of spinal cord neurons. circ_0013613 can promote the recovery of SCI by regulating miR-370-3p/CASP1 to mediate neuronal pyroptosis.

Association of caspase-1 gene polymorphisms with rheumatoid arthritis risk in a Chinese Han population.

The goals of present research are to investigate if the genetic polymorphisms in the caspase-1 (CASP1) gene are associated with the risk of rheumatoid arthritis (RA) and the clinical characteristics of the illness in Han patients from China. Our team studied the CASP1 rs2409062 A/G polymorphisms in 1095 healthy controls and 805 RA patients, while the genotype was identified via a custom-by-design 48-Plex single nucleotide polymorphism (SNP) scan™ Kit. The mRNA expression levels of the CASP1 in 40 RA cases and 40 healthy controls were detected by qRT-PCR, while blood plasma levels of the CASP1 in 40 RA cases and 40 paired controls measured via ELISA. Our research showed that the CASP1 rs2409062 A/G polymorphisms were related to an elevated risk for RA. By stratified analysis, our team discovered a remarkably elevated RA risk in females sufferers, age≥55, CRP-positive, or DAS28<3.20. In contrast to the control group, the mean level of CASP1 protein in the plasma of RA cases rised significantly. Moreover, RA cases displayed significantly greater levels of CASP1 mRNA versus the control group (P<0.05). Those outcomes reveal that the CASP1 rs2409062 A/G gene polymorphisms are associated with an elevated risk for RA in a Chinese Han population.

CASP4 can be a diagnostic biomarker and correlated with immune infiltrates in gliomas.

Gliomas are the most common and invasive malignant tumors that originate in the central nervous system. Currently, the primary treatment modality for gliomas is maximum surgical resection, supplemented by radiotherapy and chemotherapy. However, the long-term survival rate has not signifificantly increased. Pyroptosis is a new form of programmed lytic death that has been recently discovered. Caspase 4 (CASP4) plays a key role in pyroptosis. Many studies have shown that pyroptosis is not only related to inflflammation but is also closely related to the occurrence and development of most tumors. This study aimed to prove that CASP4 has a key role in the mechanism of gliomas. We used expression data from The Cancer Genome Atlas and the Chinese Glioma Genome Atlas to explore the relationship between CASP4 expression and glioma prognosis. The differential expression of CASP4 in gliomas and normal tissues was fifirst tested, and then the connection between CASP4 and tumor prognosis was explored. The relationship between CASP4 expression and immune cell infifiltration was also investigated. Finally, the possible pathways were analyzed using Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Gene Set Enrichment Analysis. CASP4 was highly expressed and associated with a signifificantly lower survival rate in patients with glioma. It could also inflfluence immune cell infifiltration by releasing cytokines. CASP4 can be a diagnostic biomarker and is a promising therapeutic target for gliomas.

Puerarin protects against human retinal endothelial cells injury induced by high glucose via regulating miR-16-5p/CASP1 axis.

We aimed to investigate the effects of puerarin on high glucose (HG)-induced human retinal endothelial cells (HRECs) injury. Cells were exposed to puerarin in the presence or absence of HG challenge. Cell viability was determined using a CCK-8 assay. Then, the levels of the oxidative stress-related factors were evaluated using corresponding kits. Pyroptosis was assessed by measurement of gasdermin-N domain (GSDMD-N) and caspase-1 (CASP1) expression. Subsequently, miR-16-5p expression was detected using RT-qPCR. The levels of oxidative stress and pyroptosis were examined after miR-16-5p silencing. The Starbase database predicted that CASP1 is a potential target of miR-16-5p, which was verified through a luciferase reporter assay. Moreover, CASP1 expression was determined after miR-16-5p silencing in HG-stimulated HRECs with puerarin exposure. Results revealed that puerarin significantly enhanced cell viability and inhibited oxidative stress. Furthermore, puerarin markedly downregulated GSDMD-N and CASP1 expression, and miR-16-5p level was notably inhibited in HG-stimulated HRECs, which was reversed after puerarin intervention. Following transfection with miR-16-5p inhibitor, the effects of puerarin on cell viability, oxidative stress and pyroptosis were attenuated in HG-induced HRECs. CASP1 was confirmed as a direct target gene of miR-16-5p. Taken together, puerarin alleviates oxidative stress and pyroptosis in HG-stimulated HRECs through regulating the miR- 16-5p/CASP1 axis.

Disulfiram with Cu2+ alleviates dextran sulfate sodium-induced ulcerative colitis in mice.

Background: Disulfiram (DSF), a Food and Drug Administration (FDA)-approved drug for chronic alcohol addiction, has anti-inflammatory effects that help prevent various cancers, and Cu2+ can enhance the effects of DSF. Inflammatory bowel diseases (IBD) are characterized by chronic or recurrent relapsing gastrointestinal inflammation. Many drugs targeting the immune responses of IBD have been developed, but their application has many problems, including side effects and high costs. Therefore, there is an urgent need for new drugs. In this study, we investigated the preventive effects of DSF+Cu2+ on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in mice. Methods: The anti-inflammatory effects were investigated using the DSS-induced colitis mouse model and lipopolysaccharide (LPS)-induced macrophages. DSS-induced TCRβ-/- mice were used to demonstrate the effect of DSF in conjunction with Cu2+ on CD4+ T cell-secreted interleukin 17 (IL-17). In addition, the effect of DSF+Cu2+ on intestinal flora was studied by 16S rRNA microflora sequencing. Results: DSF and Cu2+ could significantly reverse the symptom of DSS-induced UC in mice, such as weight loss, disease activity index score, colon length shortening, and reversal of colon pathological changes. DSF and Cu2+ could inhibit colonic macrophage activation by blocking the nuclear factor kappa B (NF-κB) pathway, reducing nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 (NLRP3)-inflammasome-derived interleukin 1 beta (IL-1β) secretion and caspase-1 (CASP1) activation, and decreasing IL-17 secretion by CD4+ T cells. Moreover, the treatment of DSF and Cu2+ could protect the intestinal barrier by reversing the expression of tight junction proteins, zonula occluden-1 (ZO-1), occludin, and mucoprotein-2 (MUC2). Additionally, DSF+Cu2+ could reduce the abundance of harmful bacteria and increase beneficial bacteria in the intestinal tract of mice, effectively improving intestinal microecology. Conclusion: Our study evaluated the effect of DSF+Cu2+ on the immune system and gut microbiota in colonic inflammation and highlighted its potential to treat UC in the clinic.

NLRP3-Independent Inflammasome Activation By Proteasome Inhibitors

Introduction Proteasome inhibitors (PIs) such as Bortezomib (BTZ) have revolutionized multiple myeloma (MM) treatment. However, their efficacy is limited by the emergence of drug resistance and treatment-limiting side effects, including peripheral neuropathy and lymphopenia. Treatment resistance in MM has been demonstrated to be promoted by an NLRP3 inflammasome-mediated innate immune response to MM-associated proteins. Thus, we set out to investigate the impact of PI treatment on innate immune cytokine production. Materials and Methods The PIs BTZ, marizomib, epoxomicin and onx-0914 were applied to a variety of inflammasome-competent human immortalized and primary cells, including the cell line THP-1 (monocytic), primary human peripheral blood mononuclear cells (PBMC), M-CSF or ascites-derived macrophages, keratinocytes, and epithelial cells. Inflammasome activation was characterized via Interleukin-1b (IL-1β) release in cellular supernatants (ELISA, all cell types) and ASC-speck formation using an ASC-GFP reporter cell line (THP-1). To characterize the contribution of Nod-like receptor pyrin domain-containing protein 3 (NLRP3) and caspase-1 (CASP-1) to inflammasome formation, NLRP3 and CASP-1-deficient THP1 cells were generated using CRISPR/Cas9-mediated genome engineering, and NLRP3 and caspase-1 were pharmacologically inhibited using MCC-950 (Invitrogen) and VX-765 (Invivogen). Results We report PI-mediated activation of the inflammasome in all cells tested. Treatment with PI induced secretion of IL-1b by THP1 cells and PBMC with an EC50 that was similar to the IC50 in BTZ-sensitive, but not BTZ-resistant, KMS-11 MM cells. We observed hallmarks of canonical inflammasome activation such as the formation of ASC specks in PI-exposed cells. Moreover, pharmacological inhibition of caspase-1 with VX-765 or genetic caspase-1 deficiency abrogated IL-1b. A variety of inflammatory stimuli, including soluble proteins secreted by MM cells, induce inflammasome formation via activation of NLRP3, the only inflammasome receptor currently targetable by a small molecule and investigated in clinical trials. Surprisingly, neither genetic ablation of NLRP3 in THP-1 cells nor MCC-950 treatment of PBMC inhibited IL-1b signaling following BTZ exposure. Conclusion Here, we identify PIs as activators of the inflammasome. In contrast to previously identified myeloma-intrinsic mechanisms leading to inflammasome assembly, PI-mediated IL-1b secretion was independent of NLRP3. Further investigation will include the identification of the receptor mediating BTZ-driven inflammasome assembly in order to identify mechanisms of action that may be amenable to therapeutic intervention.

Significance of elevated serum and hepatic NOD-like receptor pyrin domain containing 3 (NLRP3) in hepatitis C virus-related liver disease

NOD-like receptor pyrin domain containing 3 (NLRP3) is a microbial and danger signal sensor that acts as a regulator of inflammation via activation of Caspase-1 (CASP1) and has been identified as a major contributor to human liver diseases. The present study was conducted to investigate the association between NLRP3 and the progression of hepatitis C virus (HCV)-related liver disease. Serum NLRP3 levels were analyzed in 49 patients with chronic HCV infection and 18 healthy controls and liver tissues from 34 patients were examined to assess the protein expression of NLRP3 and its activation marker CASP1 using immunohistochemical staining. The results showed that the median serum NLRP3 levels was significantly higher in HCV-infected patients compared with healthy controls (1040 pg/ml vs 695 pg/ml respectively, P < 0.001) and were positively correlated with hepatic NLRP3 and CASP1 expression (r = 0.749, P < 0.001 and r = 0.557, P = 0.001 respectively). The NLRP3 levels in serum and the liver significantly increased with worsening liver pathology and showed positive correlations with serum aminotransferases levels, HCV viremia, and albumin-bilirubin score (P < 0.05). The receiver operating characteristic curve analysis revealed a high diagnostic performance of serum NLRP3 in determining the extent of liver necroinflammation, fibrosis, and steatosis (area under the curve = 0.951, 0.971, and 0.917 respectively, P < 0.001). In conclusion, NLRP3 plays an important role in liver disease progression during HCV infection via CASP1 activation and might be a promising therapeutic target. Serum NLRP3 could be an additional biomarker for liver inflammation and fibrosis.

Transcriptome analysis provides novel insights into the immune mechanisms of Macrobrachium nipponense during molting

Molting is a basic physiological behavior of the Oriental river prawn (Macrobrachium nipponense), however, the gene expression patterns and immune mechanisms during the molting process of Oriental river prawn are unclear. In the current study, the gene expression levels of the hepatopancreas of the Oriental river prawn at different molting stages (pre-molting, Prm; mid-molting, Mm; and post-molting, Pom) were detected by mRNA sequencing. A total of 1721, 551, and 1054 differentially expressed genes (DEGs) were identified between the Prm hepatopancreas (PrmHe) and Mm hepatopancreas (MmHe), MmHe and Pom hepatopancreas (PomHe) and PrmHe and PomHe, respectively. The results showed that a total of 1151 DEGs were annotated into 316 signaling pathways, and the significantly enriched immune-related pathways were "Lysosome", "Hippo signaling pathway", "Apoptosis", "Autophagy-animal", and "Endocytosis". The qRT-PCR verification results of 30 randomly selected DEGs were consistent with RNA-seq. The expression patterns of eight immune related genes in different molting stages of the Oriental river prawn were analyzed by qRT-PCR. The function of Caspase-1 (CASP1) was further investigated by bioinformatics, qRT-PCR, and RNAi analysis. CASP1 has two identical conserved domains: histidine active site and pentapeptide motif, and the expression of CASP1 is the highest in ovary. The expression levels of triosephosphate isomerase (TPI), Cathepsin B (CTSB) and Hexokinase (HXK) were evaluated after knockdown of CASP1. This research provides a valuable basis to improve our understanding the immune mechanisms of Oriental river prawns at different molting stages. The identification of immune-related genes is of great significance for enhancing the immunity of the Oriental river prawn, or other crustaceans, by transgenic methods in the future.

Investigating the Role of the NLRP3 Inflammasome Pathway in Acute Intestinal Inflammation: Use of THP-1 Knockout Cell Lines in an Advanced Triple Culture Model.

The NLRP3 inflammasome plays an important role in intestinal homeostasis as well as inflammation. However, in vivo studies investigating the role of the NLRP3 inflammasome in inflammatory bowel disease (IBD) report contrasting results, leaving it unclear if the NLRP3 inflammasome augments or attenuates intestinal inflammation. To investigate the role of the NLRP3/caspase-1 pathway in a model of acute intestinal inflammation, we modified a previously established in vitro triple culture model of the healthy and inflamed intestine (Caco-2/HT29-MTX-E12/THP-1). Using THP-1 knockout cell lines, we analyzed how the NLRP3 inflammasome and its downstream enzyme caspase-1 (CASP1) affect inflammatory parameters including barrier integrity and cytotoxicity, as well as gene expression and secretion of pro-inflammatory cytokines and mucus. Furthermore, we investigated differences in inflammation-mediated cytotoxicity towards enterocyte-like (Caco-2) or goblet-like (HT29-MTX-E12) epithelial cells. As a complementary approach, inflammation-related cytotoxicity and gene expression of cytokines was analyzed in intestinal tissue explants from wildtype (WT) and Nlrp3-/- mice. Induction of intestinal inflammation impaired the barrier, caused cytotoxicity, and altered gene expression of pro-inflammatory cytokines and mucins in vitro, while the knockout of NLRP3 and CASP1 in THP 1 cells led to attenuation of these inflammatory parameters. The knockout of CASP1 tended to show a slightly stronger attenuating effect compared to the NLRP3 knockout model. We also found that the inflammation-mediated death of goblet-like cells is NLRP3/caspase-1 dependent. Furthermore, inflammation-related cytotoxicity and upregulation of pro-inflammatory cytokines was present in ileal tissue explants from WT, but not Nlrp3-/- mice. The here presented observations indicate a pro-inflammatory and adverse role of the NLRP3 inflammasome in macrophages during acute intestinal inflammation.