The Applied Basic Research Of Prevention And Treatment Of Acute GVHD On Targeting NLRP3 Inflammasome

Objective:To study the effect and mechanism of action of Thiolutin（THL）,which is a targeted inhibitor of NLRP3 inflammasome on the prevention and treatment of acute graft-versus-host disease（aGVHD）in mice.Methods:1.Male C57BL/6J mice aged 8-10 weeks were used as donors and male BALB/c mice aged 8-10 weeks were used as recipients.On the day of allo-HSCT,BALB/c mice received 6.5 Gy total body irradiation（TBI）and were then randomly divided into control group and aGVHD group,receiving 1×107 donor’s bone marrow cells or 1×107 donor’s bone marrow cells combination with 5×106 spleen cells respectively,by tail vein 6 hours after irradiation.Survival rates,clinical scores and loss of body weight were assessed.Pathology of target organs include liver,colon and small intestine was detected by H＆E staining at day 11 post-transplatation.Chimerism rate in peripheral blood,spleen,liver and small intestine were detected by flow cytometry marked with anti-mouse H-2Kb and anti-mouse H-2Kd at day 11 post-transplant.2.The aGVHD mice group was given different doses of THL（0.25 mg/kg,0.5 mg/kg and 1 mg/kg）by intraperitoneal injection the day before transplantation,and administered once every other day to 20 days after transplantation,while the control group was given the same volume o11 post-transplant,the liver,small intestine and colon were taken for tissue fixationf THL Drug vehicle DMSO.The survival of mice was observed daily,clinical scores and body weights were recorded three times a week.At day,embedding,sectioning,H＆E staining and pathological examination.Mouse spleen lymphocytes,liver lymphocytes and small intestine lymphocytes were separated on day 6 and 11 post-transplant,then the chimerism rate,lineage differentiation,T-lymphocyte subpopulation,proliferation and activation were investigated by flow cytometry.The serum concentration of cytokines were measured by ELISA and Cytometric Bead Array（CBA）.Detection of cytokine mRNA expression levels in target organ tissues by qPCR.3.Knockout of BRCC3 in donor or recipient to explore the role of BRCC3 deficiency on aGVHD.Specific knockout the BRCC3 in aGVHD mouse model.Examine the target organ damage through histopathology.Measure the serum cytokines levels by ELISA and Cytometric Bead Array（CBA）.Spleen,liver and small intestine lymphocytes were isolated on the 9 day post-transplantation,and the infiltration and activation of T cells in the target organs were assayed using flow cytometry.Results:1.Under the irradiation conditions of 6.5 Gy 60Co total body irradiation and 1×107 bone marrow cells plus 5×106 splenocytes,the recipients mice could develop severe aGVHD,a stable and reliable aGVHD mouse model was successfully established.In the aGVHD model group,obvious aGVHD manifestations such as weight loss,skin lesions,piloerection,arched back and diarrhea were observed,and all died on day 19 after transplantation.However,the control group had only mild weight loss,piloerection and arched back on day 10 after transplantation,and long-term survival was achieved after recovery.Pathological sections of the liver and small intestine of the aGVHD group showed obvious pathological manifestations of aGVHD,and the control group had no obvious pathological features of aGVHD.The chimerism rate of peripheral blood,spleen,liver and small intestine in the aGVHD model group reached about 95%on day 11 after transplantation.2.THL-treated group had prolonged survival,less weight loss,alleviated tissue damage of target organs,and lower clinical scores compared to control group.The levels of serum IL-10,IL-17A,IL-2 and TNF were significantly lower in THL-treated group on day 6 post-transplantation,while the levels of IL-10,IFN-γ,IL-1β and IL-2 cytokines were significantly lower in the administration group on day 11 posttransplantation.Flow cytometric analysis revealed that THL did not affect donor cell lineage differentiation and the proportion of CD4+T cells and CD8+T cells in the spleen,but decreased T cell activation and increased Treg cell differentiation in the spleen and liver,then reducing the infiltration of donor CD4+T cells and CD8+T cells into the liver and small intestine.The qPCR resulted that THL reduced the mRNA expression of cytokines such as IL-1β,IFN-γ and TNF-α and IL-18 in target organs.3.The deletion of BRCC3 in the donor made no significant difference in aGVHD,but the absence of BRCC3 in the recipient exacerbated aGVHD.The absence of BRCC3 in the recipient aggravates liver damage and promotes the infiltration,proliferation and activation of CD8+T cells in target organs.Conclusion:Our results showed that THL promoted the survival of aGVHD recipients with decreased weight loss and clinical scores,and attenuates the pathological changes of GVHD target organs including liver,small intestine,and colon.Moreover,THL reduces the donor T-cell activation,infiltration to target organs and expansion of Treg cells.The activation of NLRP3 inflammasome and proinflammatory cytokines production（TNF-α,IFN-γ,IL-2,IL-17A and IL-1β）were also inhibited by THL treatment.Our findings therefore indicate that THL is associated with marked suppression of aGVHD,supporting its clinical development as an anti-inflammatory treatment for therapeutic intervention in allogeneic HSCT.Knockout of BRCC3,a target molecule of THL,aggravates aGVHD,indicating that THL attenuates aGVHD may not through the BRISC complex.