The role of protein arginine deiminase 4-dependent neutrophil extracellular traps formation in ulcerative colitis.

Neutrophil extracellular traps (NETs) play an important role in the development and progression of ulcerative colitis (UC). Peptidyl arginine deiminase 4 (PAD4) is essential for the formation of NETs via catalyzing histone citrullination. This study mainly to explore the role of PAD4-mediated NETs in intestinal inflammation of dextran sulfate sodium (DSS)-induced UC. Acute and chronic colitis mouse models were established by supplementing DSS in drinking water. Colon tissues from colitis mice were analyzed for the level of PAD4 expression, citrullinated histone H3(Cit-H3), intestinal histopathology, and inflammatory cytokines secretion. Serum samples were tested for systemic neutrophil activation biomarkers. Colitis mice administered with Cl-amidine, a PAD4 inhibitor, and PAD4 knockout mice were investigated to detect NETs formation, intestinal inflammation, and barrier function. We found the formation of NETs significantly increased in DSS-induced colitis mice and was correlated with disease markers. Blocking NETs formation by Cl-amidine or PAD4 genetic knockout could alleviate clinical colitis index, intestinal inflammation, and barrier dysfunction. This study provided a research basis for the role of PAD4-mediated NETs formation in the pathogenesis of UC and suggested that inhibition of PAD4 activity and the formation of NETs may be helpful for the prevention and treatment of UC.

Wang P ，Liu D ，Zhou Z ，Liu F ，Shen Y ，You Q ，Lu S ，Wu J ... -  《Frontiers in Immunology》

Berbamine ameliorates DSS-induced colitis by inhibiting peptidyl-arginine deiminase 4-dependent neutrophil extracellular traps formation.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease with immune dysregulation affecting colon inflammatory response. Recent studies have highlighted that neutrophil extracellular traps (NETs) play an important role in the pathogenesis of UC. Berbamine (BBM), one of the bioactive ingredients extracted from Chinese herbal medicine Berberis vulgaris L, has attracted intensive attentions due to its significant anti-inflammatory activity and a marketing drug for treating leukemia in China. However, the exact role and potential molecular mechanism of BBM against UC remains elusive. In the present study, our results showed that BBM could markedly improve the pathological phenotype and the colon inflammation in mice with dextran sulfate sodium (DSS)-induced colitis. Then, comprehensive approaches combining network pharmacology and molecular docking analyses were employed to predict the therapeutic potential of BBM in treating UC by peptidyl-arginine deiminase 4 (PAD4), a crucial molecule involved in NETs formation. The molecular docking results showed BBM had a high affinity for PAD4 with a binding energy of -9.3 kcal/mol Moreover, PAD4 expression and NETs productions, including citrullination of histone H3 (Cit-H3), neutrophil elastase (NE), myeloperoxidase (MPO) in both neutrophils and colonic tissue were reduced after BBM administration. However, in the mice with DSS-induced colitis pretreated with GSK484, a PAD4-specific inhibitor, BBM could not further reduce disease related indexes, expression of PAD4 and NETs productions. Above all, the identification of PAD4 as a potential target for BBM to inhibit NETs formation in colitis provides novel insights into the development of BBM-derived drugs for the clinical management of UC.

Tang W ，Ma J ，Chen K ，Wang K ，Chen Z ，Chen C ，Li X ，Wang Y ，Shu Y ，Zhang W ，Yuan X ，Shi G ，Chen T ，Wang P ，Chen Y ... -  《-》

Evaluation of protein arginine deiminase-4 inhibitor in TNBS- induced colitis in mice.

Many evidences indicated that neutrophil extracellular traps (NETs) are involved in the pathogenesis of inflammatory bowel disease (IBD). Citrullination of histones by Protein Arginine Deiminase-4 (PAD4) is central for NETs formation. This paper aimed to explore the definite role of NETs in mouse model of Crohn's disease (CD) with 2,4,6-trinitrobenzene sulfonic acid (TNBS). The expression of NETs-associated proteins and mRNAs in colon tissue were detected by immunohistochemistry and Real-time Quantitative PCR (QPCR) respectively. Neutrophils were isolated and stimulated in vitro to form NETs. In addition, we also administered Cl-amidine, PAD4 inhibitor, resulting in less NETs formation to investigate protective effect by measuring weight loss, gross bleeding, colon length, myeloperoxidase (MPO) activity, and cytokine expression in mice. The results showed enhanced expression of Ly6G, citrullinated histone H3 (CitH3), and PAD4 in TNBS-induced colitis mice and higher ability of neutrophil to produce NETs in vitro. Blocking NETs formation through Cl-amidine effectively alleviated the clinical colitis index and tissue inflammation in TNBS mice, regulated the expression of pro- or anti-inflammatory cytokines. In addition, Cl-amidine reduced the gene expression of PAD4 and the expression of NETs-associated proteins in the colon of TNBS mice and inhibited the formation of NETs in vitro. Our data showed that Cl-amidine could alleviate the clinical colitis index in TNBS mice to some extend and suggested blocking NETs formation through inhibition of PAD4 as therapeutic targets for the treatment of CD.

Zhang T ，Mei Y ，Dong W ，Wang J ，Huang F ，Wu J ... -  《-》

Histones of Neutrophil Extracellular Traps Directly Disrupt the Permeability and Integrity of the Intestinal Epithelial Barrier.

Increased neutrophil extracellular trap (NET) formation and abundant NET-associated proteins are frequently found in the inflamed colon of patients with inflammatory bowel disease. Peptidyl arginine deiminase 4 (PAD4) activation is essential for the generation of NET and NET-mediated pathogenesis. However, the role of PAD4-dependent NET formation in murine inflammatory bowel disease models and the molecular mechanisms responsible for the altered gut barrier function are unknown. Wild-type and Pad4 knockout (Pad4-/-) mice were administrated 3% dextran sulfate sodium (DSS) in their drinking water. Caco-2 monolayers were used to test the effect of NETs on intestinal barrier function and cytotoxicity. Histones were intrarectally administrated to wild-type mice to determine their effects on intestinal barrier function and cytotoxicity in vivo. PAD4 deficiency reduced the severity of DSS-induced colitis with decreased intestinal NET formation and enhanced gut barrier function and integrity in mice. NETs disrupted the barrier function in intestinal epithelial Caco-2 monolayers through their protein, rather than DNA, components. Pretreatment of NETs with histone inhibitors abrogated the effects on epithelial permeability. Consistent with these observations, adding purified histone proteins to Caco-2 monolayers significantly damaged epithelial barrier function, which was associated with the abnormal distribution and integrity of tight junctions as well as with increased cell death. Furthermore, intrarectal administration of histones damaged the intestinal barrier integrity and induced cytotoxicity in the mouse colon epithelium. PAD4-mediated NET formation has a detrimental role in acute colitis. NET-associated histones directly inhibit intestinal barrier function, resulting in cytotoxicity in vitro and in vivo.

Lai HJ ，Doan HT ，Lin EY ，Chiu YL ，Cheng YK ，Lin YH ，Chiang HS ... -  《-》

Dihydromyricetin ameliorates experimental ulcerative colitis by inhibiting neutrophil extracellular traps formation via the HIF-1α/VEGFA signaling pathway.

Dihydromyricetin (DHM), which has various biological functions, possesses therapeutic potential for ulcerative colitis (UC). Neutrophil extracellular traps (NETs) and their components play a crucial role in several pathological processes in UC. However, whether DHM alleviates UC by regulating NETs remains unclear. Mice with dextran sulfate sodium (DSS)-induced acute colitis were treated with DHM at different concentrations, and the severity of colitis was evaluated by assessing body weight, colon length, histological scores, cytokine production, and epithelial barrier integrity. To quantify and visualize NETs, the expression of cell free-DNA (cf-DNA) in serum and Cit-H3 in colonic tissue was analyzed via western blotting and immunofluorescence analysis. HL-60 cells and mouse bone marrow-derived neutrophils (BMDNs) were used to evaluate the effects of DHM on NETs in vitro. NETs were treated with DHM at varying concentrations or DNase I and used to repair the intestinal epithelial barrier in a Caco-2/HIEC-6 cell monolayer model. Furthermore, the genes targeted by DHM through neutrophils for alleviating UC were identified by screening online databases, and the results of network pharmacological analysis were verified via western blotting and quantitative real-time polymerase chain reaction. DHM alleviated DSS-induced colitis in mice by reversing weight loss, increased DAI score, colon length shortening, enhanced spleen index, colonic pathological damage, cytokine production, and epithelial barrier loss in a dose-dependent manner. In addition, it inhibited the formation of NETs both in vivo and in vitro. Based on the results of network pharmacological analysis, DHM may target HIF-1α and VEGFA through neutrophils to alleviate UC. Treatment with PMA increased the expression of HIF-1α and VEGFA in D-HL-60 cells and BMDNs, whereas treatment with DHM or DNase I reversed this effect. Treatment with DMOG, an inhibitor of HIF prolyl hydroxylase (HIF-PH), counteracted the suppressive effects of DHM on NETs formation in D-HL-60 cells and BMDNs. Accordingly, it partially counteracted the protective effects of DHM on the intestinal epithelial barrier in Caco-2 and HIEC-6 cells. These results indicated that DHM alleviated DSS-induced UC by regulating NETs formation via the HIF-1α/VEGFA signaling pathway, suggesting that DHM is a promising therapeutic candidate for UC.

Ma X ，Li M ，Wang X ，Xu H ，Jiang L ，Wu F ，Wei L ，Qi G ，Zhang D ... -  《-》