reHMGB1诱导NKp46(+)ILC3扩增分泌IL-22调控EAM小鼠心肌纤维化的研究

The chronic inflammation will lead to cardiac fibrosis and then progress into dilated cardiomyopathy (DCM). Therefore, it is of great significance to elucidate the mechanism of cardiac inflammatory resolution for prevention the progress of myocarditis. Our data showed that the IL-22 producing NKp46+ILC3 cells were expanded and reduced HMGB1 (reHMGB1) was up-regulated in the spleen and myocardium of EAM mice. HMGB1 blockade could significantly decrease the lymphocytes infiltration and ameliorate fibrosis. IL-22 could inhibit collagen expression in fibroblasts in vitro. In view of this, we speculate that "IL-22 producing NKp46+ILC3 cells expansion induced by reHMGB1 regulates cardiac fibrosis". To confirm this hypothesis, in vitro, firstly, the mechanism of reHMGB1 promoting activation and expansion of NKp46+ILC3 will be demonstrated. Secondly, how does IL-22 inhibit IL-17 and TGF-β induced collagen expression? In vivo, wild type, IL-22 deficient and IL-22BP deficient mice will be used to induce EAM model. Recombinant HMGB1 (rHMGB1), rIL-22BP, anti-HMGB1 antibody and anti-CD90.2 antibody will be used, respectively, as the intervention factors to further confirm the above hypothesis. The inflammatory resolving and cardiac remodeling mechanisms of cardiac injury will be clearly demonstrated by the present project.

心肌炎症损伤的迁延不愈会导致心肌纤维化进而发展为DCM。因此，阐明心肌炎症损伤转归的机制对于预防该病的进展具有重要意义。预实验显示EAM小鼠脾脏和心肌组织NKp46+ILC3增加,产生IL-22；reHMGB1分泌上调；体内中和HMGB1能减少淋巴细胞浸润，缓解心肌纤维化；IL-22体外可抑制成纤维细胞胶原表达。有鉴于此，提出“reHMGB1促进NKp46+ILC3源性IL-22分泌,调节心肌纤维化”的假说。为证实这一假说，体外分别阐明reHMGB1对NKp46+ILC3活化、扩增、IL-22分泌影响；IL-22体外抑制IL-17、TGF-β诱导的心肌胶原表达的机制；体内使用WT、IL-22-/-、IL-22BP-/-小鼠诱导EAM，并使用reHMGB1、rIL-22BP、抗HMGB1和抗CD90.2抗体进行干预，进一步证实上述假说。通过以上研究有望阐明心肌炎症损伤转归与心功能重塑的机制。

心肌损伤后炎症的迁延不愈而引发的DCM，是导致青少年心衰、心源性休克与心脏猝死的重要原因。因此，阐明心肌损伤微环境下炎症转归的机制，对于积极预防心肌炎疾病的进展，具有重要意义。本项目在前期工作的基础上提出假说，旨在阐明reHMGB1通过促进NKp46+ILC3源性IL-22分泌，直接或间接影响心肌纤维化的进程与心肌病的发生，通过本项目的实施以期为心肌炎症的转归寻找到实验证据，也为心肌炎向DCM 进展的干预寻找到新的靶点。对照项目目标，开展了以下几方面的工作：① 阐释清楚了不同形式的HMGB1、IL-33、IL-17等诱导ILC3扩增的机制；尤其阐释清楚了在心肌损伤背景下NKp46+ILC3扩增的机制（迁移、分化）；②证实了IL-22对心肌细胞的保护以及对心肌纤维化的抑制作用，并从代谢的角度解释了IL-22抑制血管紧张素和TGF-β介导纤维化的机制；同时发现Reg3通过抑制TGF-β介导的Smad等信号途径缓解了心脏的纤维化。③ 解析清楚了在心脏稳态环境下免疫细胞的构成、演变规律，并探讨了新生期驻留巨噬细胞对心脏再生的影响以及炎症损伤环境下心脏巨噬细胞更新的新机制（心脏损伤后活化的成纤维细胞可以使得炎症巨噬细胞转分化为心脏驻留巨噬细胞）；探究了在心脏损伤环境下反馈调节导致的B10细胞对于心脏炎症和心肌纤维化的调控机制；④阐释了稳态下心脏驻留T、B细胞的生理调节作用。本项目的实施对于全面了解心脏区域免疫特征，寻找有效地心脏损伤干预靶点具有重要的指导意义。