| [Objective] To investigate the effect and mechanism of NLRP3/ Interleukin--1β(IL-1β)signaling pathway in acute graft versus host disease(a GVHD)after allogenic hematopoietic stem cell transplantation.[Methods] First of all,the concentrations of IL-1β and IL-18 of patients receiving allogeneic hematopoietic stem cell transplantation were detected by Elisa.The overall survival and event-free survival were evaluated in two groups according to the concentration levels.Secondly,preliminary exploration of the role of NLRP3/IL-1β signaling pathway in the pathogenesis of acute graft-versus-host disease was performed,and the expression levels of NLRP3 and the downstream molecules in the early stage of conditioning and a GVHD were investigated in a mouse model of a GVHD.Furthermore,a mouse model of a GVHD based on NLRP3 knockout mice was established and the survival,GVHD score and pathological changes of the target organ were evaluated.The cell subpopulations and effective molecules in the NLRP3/IL-β signaling pathway were compared with wild-type mice by flow cytometry,Luminex microarray assay and RT-PCR,to further verify the role of the NLRP3 inflammasome in the pathogenesis of a GVHD.RNA-seq was applied for differential gene analysis in NLRP3 KO mice and WT mice,and GO enrichment and KEGG enrichment were applied to explore the possible pathways and mechanism for NLRP3 in the pathogenesis of a GVHD.In addition,MCC950 was applied to explore the treatment effect for a GVHD.[Results] The plasma concentrations of IL-1β and IL-18 in patients with a GVHD after alloHSCT were higher than those without a GVHD,which were related with the prognosis of patients.The overall survival and event-free survival of patients were worse in the high-risk cohort than the low-risk cohort.In addition,the expression of NLRP3 m RNA in PBMCs of patients with a GVHD significantly increased,which positively correlated with expression of IL-1β m RNA.In the mouse model of acute graft-versus-host disease,the expression of molecules in the NLRP3/IL-1β pathway were significantly upregulated in the mice receiving conditioning and developing a GVHD.Knockout of the NLRP3 gene can significantly improve the survival and alleviate the a GVHD performance as well as the targeted organ damage.The expression of proinflammatory factors related a GVHD were downregulated by NLRP3 knockout.Furthermore,the polarization of T cells to Th1 and Th17 was inhibited due to the downregulation of transcription factors of Th1 and Th17 in NLRP3 knockout mice,and the macrophage infiltration was reduced in NLRP3 knockout mice.The present study analyzed the differential genes of NLRP3 KO mice and WT mice by RNA-seq,and found that NLRP3 inflammasome significantly upregulate the chemokinechemokine receptor interaction pathway as well as the chemokines of CCL family and CXCL family through GO enrichment and KEGG enrichment analysis.Finally,the survival was improved and the GVHD performance was alleviate in the mice treated with MCC950.The molecules of NLRP3/IL-1β pathway,transcription factors of Th1/Th17 and the chemokines of CCL family and CXCL family were downregulated in the mice treated with MCC950,indicating a significant treatment effect for a GVHD and validating the mechanism of NLRP3 in a GVHD[Conclusion] The NLRP3/IL-1β signaling pathway activates innate immune cells and promotes the release of IL-1β and IL-18.IL-1β and IL-18 could stimulate epithelial cells and other immune cells to release chemokines of CCL and CXCL family,trafficking Th1,Th17,CD8+ T cells and more inflammatory monocyte-macrophages to target organs.Furthermore,IL-1β and IL-18 promote the polarization of T cell to Th1 and Th17 by upregulating the transcription factors of Th1 and Th17,and then release more proinflammatory cytokines,aggravating the cascade inflammatory response of a GVHD.MCC950,a selective inhibitor of the NLRP3 inflammasome,can inhibit the NLRP3/IL-1βpathway to alleviate a GVHD in mice,providing new targets and new ideas for the treatment of a GVHD in the future. |
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