Cyclosporine A alleviates colitis by inhibiting the formation of neutrophil extracellular traps via regulating the pentose phosphate pathway

Abstract

Abstract Background: The aberrant formation of neutrophil extracellular traps (NETs) has been implicated in ulcerative colitis (UC), a chronic recurrent intestinal inflammation. Cyclosporine A (CsA) is now applied as rescue therapy for acute severe UC. In addition, it has been curtained that CsA inhibits the formation of NETs in vitro and the mechanism of which was still vague. The study aimed to explore the mechanism CsA inhibits the NETs formation of colitis in vivo and in vitro. Methods: NETs enrichment in clinical samples was analyzed using databases from Gene Expression Omnibus and verified in our center. Dextran sulfate sodium (DSS)-induced acute colitis mice model was used to investigate the effect of CsA on NETs of colonic tissue expression. To clarify the mechanism, intracellular energy metabolites were examined by Liquid Chromatograph Mass Spectrometer, and ROS levels were examined by fluorescence intensity in neutrophils treated with CsA after LPS stimulation. The transcriptional level and activity of G6PD of neutrophils were also assessed using qRT-PCR and WST-8. RNA-sequence was used to detect differentially expressed genes of neutrophils stimulated by LPS with or without CsA. The expression levels of related proteins were detected by western blot. Results: NETs enrichment was especially elevated in moderate-to-severe UC patients compared to HC. NETs expression in the colon from DSS colitis was decreased after CsA treatment. Compared with neutrophils stimulated by LPS, NETs formation and cellular ROS levels were decreased in LPS+CsA group. Cellular guanosine monophosphate and NADPH/NADP+ related to the pentose phosphate pathway (PPP) were reduced in LPS+CsA group. In addition, CsA could decrease G6PD（rate-limited enzyme of PPP） activity in neutrophils stimulated with LPS, and the results were further verified by inhibiting G6PD activity. At last, P53 protein was highly expressed in LPS+CsA group compared with the LPS group. Intracellular G6PD activity, ROS level and NETs formation, which were downregulated by CsA, could be reversed by a P53 inhibitor. Conclusion: Our results indicated CsA could alleviate the severity of colitis by decreasing the formation of NETs in vivo. In vitro, CsA reduced NETs release via downregulating PPP and cellular ROS levels by decreasing G6PD activity directly by activating the P53 protein.