DOP21 Role of P2X7 in intestinal fibrosis and inflammasome activation: Relevance in Crohn′s Disease

Abstract Background Crohn’s disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract whose etiology is unknown. CD is associated with complications such as fibrosis or fistula, which cannot be pharmacologically reversed, requiring repeated surgery. Although a profibrotic effect of the P2X7 receptor has been described in some scenarios such as lung, heart and liver, its role in intestinal fibrosis has not been analysed yet. Given the crosstalk between fibrosis and inflammasome, we aim to analyze the relevance of P2X7 in intestinal fibrosis and inflammasome activation. Methods Surgical intestinal resections of CD patients and healthy ileum of carcinoma patients were obtained. Murine chronic colitis was induced by 4 cycles of DSS in wild-type (WT) or P2X7-/- (KO) mice. HT29 cells were treated 24 hours with an inflammasome activator cocktail (LPS, TNF-α and IFN-γ) and the P2X7 antagonist A-80. Gene and protein expression of P2X7, inflammasome markers (NLRP3, ASC, CASPASE1, IL1β and IL18) and alternative inflammasome pathways (APIs) (NLRP1, NLRC4 and AIM2) were analysed by qPCR and Western Blot. The collagen layer was analysed by Sirius Red Staining. Results are expressed by mean±SEM. Statistical analysis was performed with one-way ANOVA and correlations were analysed with Spearman coefficient. Results In CD patients, the expression of P2X7 (2.97±0.50), Nlrp3 (2.53±0.41), Asc (5.61±0.76), Caspase1 (6.90±1.41), IL18 (4.17±0.89) and APIs Nlrp1 (3.07±0.40), Nlrc4 (6.99±1.19) is significantly increased vs non-IBD patients. Moreover, P2X7 expression positively and significantly correlates with the expression of the inflammasome markers NLRP3 (r=0.51), ASC (r=0.38), CASPASE1 (r=0.46), IL18 (r=0.36) and API such as NLRP1 (r=0.73), NRLC4 (r=0.67) and AIM2 (r=0.51) in CD patients (n≥45). The chronic murine model of DSS revealed that: a) KO-DSS showed more aggravated colitis with lower survival and greater weight loss compared with WT-DSS; b) the expression of NLRP3, IL18, IL1β and NLRP1 were significantly increased in KO-DSS (101.00±16.33, 3.28±1.49, 327.50±113.90, 4.92±1.00 respectively) vs WT-DSS; c) the thickness of the collagen layer in KO-DSS was increased vs WT-DSS. As expected, HT29 cells treated with the inflammasome cocktail increased protein expression of caspase-1 and the treatment with the P2X7 antagonist A-80 impaired the inflammasome activation since it significantly reduced the protein expression of caspase-1. Conclusion An increased expression of P2X7 receptor, the inflammasome and its APIs is detected in CD patients. Lack of P2X7 worsens chronic colitis associated with an increased activation of the inflammasome. Additional studies are needed in order to elucidate this dual role of P2X7 in intestinal fibrosis.

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miR-29a-5p Alleviates Traumatic Brain Injury (TBI)-Induced Permeability Disruption Via Regulating NLRP3 Pathway

10.21203/rs.3.rs-334899/v1 ◽

2021 ◽

Author(s):

Aijun Zhang ◽

Youming Lu ◽

Lei Yuan ◽

Pengqi Zhang ◽

Dongdong Zou ◽

...

Keyword(s):

Gene Expression ◽

Traumatic Brain Injury ◽

Endothelial Cell ◽

Brain Injury ◽

Inflammasome Activation ◽

Caspase 1 ◽

Promising Strategy ◽

Pathway Activation ◽

Nlrp3 Expression

Abstract Blood-brain barrier (BBB) dysfunction is presented during traumatic brain injury (TBI) and is dependent upon the activation of the NLRP3/Caspase-1 inflammasome pathway. MicroRNA (miRNA) was proved to inhibit signaling pathway activation by targeting gene expression and we predicated in the database that miR-29a targets to NLRP3. Herein, this study aims to define the regulating role of miR-29a in NLRP3 expression and NLRP3/Caspase-1 inflammasome activation in TBI-induced BBB dysfunction. Our results indicated that miR-29a-5p alleviates TBI-induced the increased permeability of endothelial cell and BBB via suppressing NLRP3 expression and NLRP3/Caspase-1 inflammasome activation, providing a promising strategy for relieving TBI via inhibiting NLRP3/Caspase-1 inflammasome activation.

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Macrophage Uptake of Necrotic Cell DNA Activates the AIM2 Inflammasome to Regulate a Proinflammatory Phenotype in CKD

Journal of the American Society of Nephrology ◽

10.1681/asn.2017080863 ◽

2018 ◽

Vol 29 (4) ◽

pp. 1165-1181 ◽

Cited By ~ 32

Author(s):

Takanori Komada ◽

Hyunjae Chung ◽

Arthur Lau ◽

Jaye M. Platnich ◽

Paul L. Beck ◽

...

Keyword(s):

Bone Marrow ◽

Inflammasome Activation ◽

Dna Uptake ◽

Chronic Injury ◽

Caspase 1 ◽

Double Stranded Dna ◽

Molecular Pattern ◽

Macrophage Uptake

Nonmicrobial inflammation contributes to CKD progression and fibrosis. Absent in melanoma 2 (AIM2) is an inflammasome-forming receptor for double-stranded DNA. AIM2 is expressed in the kidney and activated mainly by macrophages. We investigated the potential pathogenic role of the AIM2 inflammasome in kidney disease. In kidneys from patients with diabetic or nondiabetic CKD, immunofluorescence showed AIM2 expression in glomeruli, tubules, and infiltrating leukocytes. In a mouse model of unilateral ureteral obstruction (UUO), Aim2 deficiency attenuated the renal injury, fibrosis, and inflammation observed in wild-type (WT) littermates. In bone marrow chimera studies, UUO induced substantially more tubular injury and IL-1β cleavage in Aim2−/− or WT mice that received WT bone marrow than in WT mice that received Aim2−/− bone marrow. Intravital microscopy of the kidney in LysM(gfp/gfp) mice 5–6 days after UUO demonstrated the significant recruitment of GFP+ proinflammatory macrophages that crawled along injured tubules, engulfed DNA from necrotic cells, and expressed active caspase-1. DNA uptake occurred in large vacuolar structures within recruited macrophages but not resident CX3CR1+ renal phagocytes. In vitro, macrophages that engulfed necrotic debris showed AIM2-dependent activation of caspase-1 and IL-1β, as well as the formation of AIM2+ ASC specks. ASC specks are a hallmark of inflammasome activation. Cotreatment with DNaseI attenuated the increase in IL-1β levels, confirming that DNA was the principal damage-associated molecular pattern in this process. Therefore, the activation of the AIM2 inflammasome by DNA from necrotic cells drives a proinflammatory phenotype that contributes to chronic injury in the kidney.

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The signaling adaptor BCAP inhibits NLRP3 and NLRC4 inflammasome activation in macrophages through interactions with Flightless-1

Science Signaling ◽

10.1126/scisignal.aau0615 ◽

2019 ◽

Vol 12 (581) ◽

pp. eaau0615 ◽

Cited By ~ 6

Author(s):

Samuel J. Carpentier ◽

Minjian Ni ◽

Jeffrey M. Duggan ◽

Richard G. James ◽

Brad T. Cookson ◽

...

Keyword(s):

B Cell ◽

Yersinia Pseudotuberculosis ◽

Cell Receptor ◽

Pi3k Pathway ◽

Inflammasome Activation ◽

Interacting Protein ◽

Caspase 1 ◽

B Cell Receptor Signaling ◽

Phosphoinositide 3 Kinase

B cell adaptor for phosphoinositide 3-kinase (PI3K) (BCAP) is a signaling adaptor that activates the PI3K pathway downstream of B cell receptor signaling in B cells and Toll-like receptor (TLR) signaling in macrophages. BCAP binds to the regulatory p85 subunit of class I PI3K and is a large, multidomain protein. We used proteomic analysis to identify other BCAP-interacting proteins in macrophages and found that BCAP specifically associated with the caspase-1 pseudosubstrate inhibitor Flightless-1 and its binding partner leucine-rich repeat flightless-interacting protein 2. Because these proteins inhibit the NLRP3 inflammasome, we investigated the role of BCAP in inflammasome function. Independent of its effects on TLR priming, BCAP inhibited NLRP3- and NLRC4-induced caspase-1 activation, cell death, and IL-1β release from macrophages. Accordingly, caspase-1–dependent clearance of a Yersinia pseudotuberculosis mutant was enhanced in BCAP-deficient mice. Mechanistically, BCAP delayed the recruitment and activation of pro–caspase-1 within the NLRP3/ASC preinflammasome through its association with Flightless-1. Thus, BCAP is a multifunctional signaling adaptor that inhibits key pathogen-sensing pathways in macrophages.

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Rab GTPase Mediating Regulation of NALP3 in Colorectal Cancer

Molecules ◽

10.3390/molecules25204834 ◽

2020 ◽

Vol 25 (20) ◽

pp. 4834

Author(s):

Gülçin Tezcan ◽

Ekaterina E. Garanina ◽

Margarita N. Zhuravleva ◽

Shaimaa Hamza ◽

Albert A. Rizvanov ◽

...

Keyword(s):

Colorectal Cancer ◽

Vesicle Trafficking ◽

Magnetic Bead ◽

Dominant Negative ◽

Enzyme Linked Immunosorbent Assay ◽

Inflammasome Activation ◽

Rab Gtpase ◽

Rab Proteins ◽

Caspase 1

The NALP3 inflammasome signaling contributes to inflammation within tumor tissues. This inflammation may be promoted by the vesicle trafficking of inflammasome components and cytokines. Rab5, Rab7 and Rab11 regulate vesicle trafficking. However, the role of these proteins in the regulation of inflammasomes remains largely unknown. To elucidate the role of these Rab proteins in inflammasome regulation, HCT-116, a colorectal cancer (CRC) cell line expressing pDsRed-Rab5 wild type (WT), pDsRed-Rab5 dominant-negative (DN), pDsRed-Rab7 WT, pDsRed-Rab7 DN, pDsRed-Rab11 WT and pDsRed-Rab11 DN were treated with lipopolysaccharide (LPS)/nigericin. Inflammasome activation was analyzed by measuring the mRNA expression of NLRP3, Pro-CASP1, RAB39A and Pro-IL-1β, conducting immunofluorescence imaging and western blotting of caspase-1 and analysing the secretion levels of IL-1β using enzyme-linked immunosorbent assay (ELISA). The effects of Rabs on cytokine release were evaluated using MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel-Premixed 41 Plex. The findings showed that LPS/nigericin-treated cells expressing Rab5-WT indicated increased NALP3 expression and secretion of the IL-1β as compared to Rab5-DN cells. Caspase-1 was localized in the nucleus and cytosol of Rab5-WT cells but was localized in the cytosol in Rab5-DN cells. There were no any effects of Rab7 and Rab11 expression on the regulation of inflammasomes. Our results suggest that Rab5 may be a potential target for the regulation of NALP3 in the treatment of the CRC inflammation.

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Epigenetic regulation of somatostatin and somatostatin receptors in neuroendocrine tumors and other types of cancer

Reviews in Endocrine and Metabolic Disorders ◽

10.1007/s11154-020-09607-z ◽

2020 ◽

Author(s):

M.J. Klomp ◽

S.U. Dalm ◽

M. de Jong ◽

R.A. Feelders ◽

J. Hofland ◽

...

Keyword(s):

Protein Expression ◽

Neuroendocrine Tumors ◽

Epigenetic Regulation ◽

Somatostatin Receptors ◽

Secretory Activity ◽

Expression Levels ◽

Epigenetic Machinery ◽

Gene And Protein Expression

Abstract Both somatostatin (SST) and somatostatin receptors (SSTRs) are proteins with important functions in both physiological tissue and in tumors, particularly in neuroendocrine tumors (NETs). NETs are frequently characterized by high SSTRs expression levels. SST analogues (SSAs) that bind and activate SSTR have anti-proliferative and anti-secretory activity, thereby reducing both the growth as well as the hormonal symptoms of NETs. Moreover, the high expression levels of SSTR type-2 (SSTR2) in NETs is a powerful target for therapy with radiolabeled SSAs. Due to the important role of both SST and SSTRs, it is of great importance to elucidate the mechanisms involved in regulating their expression in NETs, as well as in other types of tumors. The field of epigenetics recently gained interest in NET research, highlighting the importance of this process in regulating the expression of gene and protein expression. In this review we will discuss the role of the epigenetic machinery in controlling the expression of both SSTRs and the neuropeptide SST. Particular attention will be given to the epigenetic regulation of these proteins in NETs, whereas the involvement of the epigenetic machinery in other types of cancer will be discussed as well. In addition, we will discuss the possibility to target enzymes involved in the epigenetic machinery to modify the expression of the SST-system, thereby possibly improving therapeutic options.

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Role of 12-lipoxygenase in hypoxia-induced rat pulmonary artery smooth muscle cell proliferation

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00114.2005 ◽

2006 ◽

Vol 290 (2) ◽

pp. L367-L374 ◽

Cited By ~ 61

Author(s):

Ioana R. Preston ◽

Nicholas S. Hill ◽

Rod R. Warburton ◽

Barry L. Fanburg

Keyword(s):

Endothelial Cells ◽

Pulmonary Hypertension ◽

Smooth Muscle ◽

Pulmonary Artery ◽

Protein Expression ◽

Pulmonary Arteries ◽

Mek Inhibitor ◽

Gene And Protein Expression ◽

12 Lipoxygenase

The 12-lipoxygenase (12-LO) pathway of arachidonic acid metabolism stimulates cell growth and metastasis of various cancer cells and the 12-LO metabolite, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], enhances proliferation of aortic smooth muscle cells (SMCs). However, pulmonary vascular effects of 12-LO have not been previously studied. We sought evidence for a role of 12-LO and 12(S)-HETE in the development of hypoxia-induced pulmonary hypertension. We found that 12-LO gene and protein expression is elevated in lung homogenates of rats exposed to chronic hypoxia. Immunohistochemical staining with a 12-LO antibody revealed intense staining in endothelial cells of large pulmonary arteries, SMCs (and possibly endothelial cells) of medium and small-size pulmonary arteries and in alveolar walls of hypoxic lungs. 12-LO protein expression was increased in hypoxic cultured rat pulmonary artery SMCs. 12(S)-HETE at concentrations as low as 10−5 μM stimulated proliferation of pulmonary artery SMCs. 12(S)-HETE induced ERK 1/ERK 2 phosphorylation but had no effect on p38 kinase expression as assessed by Western blotting. 12(S)-HETE-stimulated SMC proliferation was blocked by the MEK inhibitor PD-98059, but not by the p38 MAPK inhibitor SB-202190. Hypoxia (3%)-stimulated pulmonary artery SMC proliferation was blocked by both U0126, a MEK inhibitor, and baicalein, an inhibitor of 12-LO. We conclude that 12-LO and its product, 12(S)-HETE, are important intermediates in hypoxia-induced pulmonary artery SMC proliferation and may participate in hypoxia-induced pulmonary hypertension.

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Inflammatory effect of Bothropstoxin-I from Bothrops jararacussu venom mediated by NLRP3 inflammasome involves ATP and P2X7 receptor

Clinical Science ◽

10.1042/cs20201419 ◽

2021 ◽

Vol 135 (5) ◽

pp. 687-701

Author(s):

Priscila Andrade Ranéia e Silva ◽

Dhêmerson Souza de Lima ◽

João Paulo Mesquita Luiz ◽

Niels Olsen Saraiva Câmara ◽

José Carlos Farias Alves-Filho ◽

...

Keyword(s):

Muscle Tissue ◽

Nlrp3 Inflammasome ◽

Tissue Damage ◽

P2x7 Receptor ◽

Tissue Injury ◽

C2c12 Myotubes ◽

Inflammasome Activation ◽

Bothrops Jararacussu ◽

Caspase 1 ◽

Inflammatory Effect

Abstract Muscle tissue damage is one of the local effects described in bothropic envenomations. Bothropstoxin-I (BthTX-I), from Bothrops jararacussu venom, is a K49-phospholipase A2 (PLA2) that induces a massive muscle tissue injury, and, consequently, local inflammatory reaction. The NLRP3 inflammasome is a sensor that triggers inflammation by activating caspase 1 and releasing interleukin (IL)-1β and/or inducing pyroptotic cell death in response to tissue damage. We, therefore, aimed to address activation of NLRP3 inflammasome by BthTX-I-associated injury and the mechanism involved in this process. Intramuscular injection of BthTX-I results in infiltration of neutrophils and macrophages in gastrocnemius muscle, which is reduced in NLRP3- and Caspase-1-deficient mice. The in vitro IL-1β production induced by BthTX-I in peritoneal macrophages (PMs) requires caspase 1/11, ASC and NLRP3 and is dependent on adenosine 5′-triphosphate (ATP)-induced K+ efflux and P2X7 receptor (P2X7R). BthTX-I induces a dramatic release of ATP from C2C12 myotubes, therefore representing the major mechanism for P2X7R-dependent inflammasome activation in macrophages. A similar result was obtained when human monocyte-derived macrophages (HMDMs) were treated with BthTX-I. These findings demonstrated the inflammatory effect of BthTX-I on muscle tissue, pointing out a role for the ATP released by damaged cells for the NLRP3 activation on macrophages, contributing to the understanding of the microenvironment of the tissue damage of the Bothrops envenomation.

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Evaluating the Expression of Candidate Homeobox Genes and Their Role in Local-Site Inflammation in Mucosal Tissue Obtained from Children with Non-Syndromic Cleft Lip and Palate

Journal of Personalized Medicine ◽

10.3390/jpm11111135 ◽

2021 ◽

Vol 11 (11) ◽

pp. 1135

Author(s):

Nityanand Jain ◽

Mara Pilmane

Keyword(s):

Gene Expression ◽

Protein Expression ◽

Cleft Lip ◽

Cleft Lip And Palate ◽

Homeobox Genes ◽

Craniofacial Development ◽

Mucosal Tissue ◽

Local Site ◽

Gene And Protein Expression

Craniofacial development including palatogenesis is a complex process which requires an orchestrated and spatiotemporal expression of various genes and factors for proper embryogenesis and organogenesis. One such group of genes essential for craniofacial development is the homeobox genes, transcriptional factors that are commonly associated with congenital abnormalities. Amongst these genes, DLX4, HOXB3, and MSX2 have been recently shown to be involved in the etiology of non-syndromic cleft lip and palate. Hence, we investigated the gene and protein expression of these genes in normal and cleft affected mucosal tissue obtained from 22 children, along with analyzing their role in promoting local-site inflammation using NF-κB. Additionally, we investigated the role of PTX3, which plays a critical role in tissue remodeling and wound repair. We found a residual gene and protein expression of DLX4 in cleft mucosa, although no differences in gene expression levels of HOXB3 and MSX2 were noted. However, a significant increase in protein expression for these genes was noted in the cleft mucosa (p < 0.05), indicating increased cellular proliferation. This was coupled with a significant increase in NF-κB protein expression in cleft mucosa (p < 0.05), highlighting the role of these genes in promotion of pro-inflammatory environment. Finally, no differences in gene expression of PTX3 were noted.

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Therapeutic Potential of Melatonin in the Regulation of MiR-148a-3p and Angiogenic Factors in Breast Cancer

MicroRNA ◽

10.2174/2211536608666190219095426 ◽

2019 ◽

Vol 8 (3) ◽

pp. 237-247 ◽

Cited By ~ 8

Author(s):

Jéssica Zani Lacerda ◽

Lívia Carvalho Ferreira ◽

Beatriz Camargo Lopes ◽

Andrés Felipe Aristizábal-Pachón ◽

Marcio Chaim Bajgelman ◽

...

Keyword(s):

Breast Cancer ◽

Protein Expression ◽

Real Time ◽

Tumor Cells ◽

Therapeutic Potential ◽

Xenograft Model ◽

Angiogenic Factors ◽

Migration And Invasion ◽

Gene And Protein Expression

Background: The high mortality rate of breast cancer is related to the occurrence of metastasis, a process that is promoted by tumor angiogenesis. MicroRNAs are small molecules of noncoding mRNA that play a key role in gene regulation and are directly involved in the progression and angiogenesis of various tumor types, including breast cancer. Several miRNAs have been described as promoters or suppressors angiogenesis and may be associated with tumor growth and metastasis. Melatonin is an oncostatic agent with a capacity of modifying the expression of innumerable genes and miRNAs related to cancer. Objective: The aim of this study was to evaluate the role of melatonin and the tumor suppressor miR- 148a-3p on angiogenesis of breast cancer. Method: MDA-MB-231 cells were treated with melatonin and modified with the overexpression of miR-148a-3p. The relative quantification in real-time of miR-148a-3p, IGF-IR and VEGF was performed by real-time PCR. The protein expression of these targets was performed by immunocytochemistry and immunohistochemistry. Survival, migration and invasion rates of tumor cells were evaluated. Finally, the xenograft model of breast cancer was performed to confirm the role of melatonin in the tumor. Results: The melatonin was able to increase the gene level of miR-148a-3p and decreased the gene and protein expression of IGF-1R and VEGF, both in vitro and in vivo. In addition, it also had an inhibitory effect on the survival, migration and invasion of breast tumor cells. Conclusion: Our results confirm the role of melatonin in the regulation of miR-148a-3p and decrease of angiogenic factors.

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Abstract 127: Txnip-Mediated NLRP3 Inflammasome Activation as a Novel Mechanism of Myocardial Ischemia-Reperfusion Injury

Circulation Research ◽

10.1161/res.111.suppl_1.a127 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Yi Liu ◽

Lijian Zhang ◽

Yan Qu ◽

Chao Gao ◽

Jingyi Liu ◽

...

Keyword(s):

Myocardial Ischemia ◽

Infarct Size ◽

Nlrp3 Inflammasome ◽

Ischemia Reperfusion ◽

Inflammatory Cells ◽

Inflammasome Activation ◽

Caspase 1 ◽

Nlrp3 Inflammasome Activation ◽

Myocardial Ischemia Reperfusion

As an inhibitor of the antioxidant thioredoxin, thioredoxin-interacting protein (Txnip) is linked to insulin resistance. NLRP3 inflammasome, a major regulator of innate immunity, has been reported to be activated by Txnip, thus contributing to the pathogenesis of type 2 diabetes mellitus. However, the role of Txnip and its NLRP3 inflammasome activation in the myocardial ischemia/reperfusion (MI/R) injury has not been previously investigated. C57BL/6J mice were subjected to 30 min of ischemia and 3 or 24 hrs of reperfusion. The ischemic heart exhibited increased Txnip and NLRP3 expressions, increased interaction between Txnip and NLRP3 (by immunoprecipitation, 1.8-fold increase over sham), and increased IL-1β, IL-18 and caspase-1 expressions (%increase: 80%, 77% and 110%, respectively) (n=8, all P <0.05). Compared with vehicle group, those mice either receiving intramyocardial small-interfering RNA (siRNA) injection to specifically knockdown the myocardial NLRP3 or intraperitoneal injection of the inflammasome inhibitor (BAY 11-7082) exhibited significantly improved cardiac function (by 28% and 25%), decreased the infarct size (by 40% and 38%), and decreased the cardiomyocytes apoptosis (all P <0.05). NLRP3 knockdown or inflammasome inhibitor also decreased the inflammatory cells infiltration (macrophages and neutrophils) and cytokines (TNF-α, INF-γ and IL-6) production (all P <0.05). To elucidate the role of Txnip in the NLRP3 activation in MI/R, intramyocardial injection of Txnip siRNA was performed to specifically knockdown the myocardial Txnip expression. Compared with vehicle, the Txnip knockdown significantly decreased Txnip/NLRP3 interaction and NLRP3activation as evidenced by lower expressions of IL-1β and caspase-1, decreased inflammatory cells infiltration and cytokines expressions, and consequently decreased the myocardial infarct size and increased the heart function (all P <0.05). Collectively, we demonstrated for the first time that Txnip mediatedNLRP3 inflammasome activation is a novel mechanism of MI/R injury. Interventions targeted to blocking the activation of NLRP3 by inhibiting Txnip may have therapeutic potential for preventing MI/R injury.

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