PPAR-γ对B细胞调节功能的影响和机制研究

Immunosuppressive regulatory B cells (Breg) are potent inhibitors of antigen-specific inflamation and autoimmunity. Despite their important functions in immune regulation, the differentiation and activation of Breg are not well understood. Peroxisome proliferator-activated receptor γ (PPAR-γ) is a member of the nuclear hormone receptor superfamily that plays important roles not only in lipid metabolism but also in dampening inflammation and immune responses. PPAR-γ regulates B cell function and its haploinsufficiency enhances B cell proliferation and antibody production. Based on the important anti-inflammatory functions of PPAR-γ and its role in regulating B cell responses, we hypothesize that PPAR-γ also promotes the differentiation and activation of Breg. This hypothesis is based on our preliminary data that mice with B cell-specific PPAR-γ-deficiency exhibit increased oxazolone-induced ear swelling, suggesting that the regulatory functions of B cells are dependent on PPAR-γ. The goal of this application is to elucidate the mechanism by which PPAR-γ regulates Breg function. Specifically, we aim to: determine the alterations in Breg phenotype, survival, proliferation,secretion of IL-10, and suppresssive functions in the absence of PPAR-γ both in vitro and in vivo; determine the importance of B cell-intrinsic PPAR-γ in Breg-mediated immune suppression in a model of experimental autoimmune encephalomyelitis; determine the efficacy of using PPAR-γ agonists for the treatment of CHS via the induction of Breg. This study will yield novel insights into the regulation of Breg differentiation and function, serve as a new paradigm for the study of the regulation of immune tolerance in rsponse to metabolic cues, and lead to the identification of efficient therapeutic agents for the treatment of inflammatory disorders by leveraging the functions of Breg.

调节性B细胞（Breg）有明确的抗炎免疫抑制功能，但它的分化和激活仍不很清楚。PPAR-γ与其配体不仅在脂质代谢、还在抗炎免疫中发挥重要作用；PPAR-γ单倍缺陷能促进B细胞应答和抗体生成，提示它是否也能影响Breg? 本课题前期研究，已用条件基因敲除术，获得了仅在B细胞内敲除PPAR-γ的实验鼠，并发现其B细胞整体抑制恶唑酮诱导耳肿胀（CHS）的调节功能下降，部分Breg指标下调。以此为基础，项目深入研究PPAR-γ对Breg影响和作用机制：观察B细胞敲除PPAR-γ后，Breg相关亚群、表型、IL-10等抑制功能相关分子、增殖凋亡、Breg体外免疫抑制功能等的改变；并通过CHS、实验性免疫性脑炎模型，整体评价PPAR-γ缺失对B细胞调节功能、抑制分子、T细胞免疫等的作用，其激活剂与Breg。课题旨在研究PPAR-γ对B细胞调节功能的影响，发现免疫调节的新机制、脂质代谢与耐受异常的新关联

PPAR-γ激动后不仅能治疗糖尿病，还能抑制免疫炎症反应；调节性B细胞（Breg）是近年发现的具有免疫抑制功能的负向免疫调节细胞。然而PPAR-γ是否能通过影响Breg发挥免疫抑制功能还未有报道。PPAR-γ敲除导致小鼠胚胎死亡，我们首先建立了选择性地在小鼠B细胞内敲除PPAR-γ的实验鼠，探讨内源性PPAR-γ敲除对Breg的影响。结果发现B细胞内特异敲除PPAR-γ后其整体CHS耳肿胀升高、T细胞IFN-γ表达升高，提示其B细胞整体免疫调节功能降低；深入机制研究发现，B细胞PPAR-γ敲除后，并不影响非活化状态下脾脏CD5+CD1dhiBreg的数目和外周血CD4+Foxp3+Treg的数目；对Breg激活后与其免疫抑制相关的表面分子CD86、FasL表达无显著影响；但是B细胞特异敲除PPAR-γ后其脾脏CD5+CD1dhiBreg细胞亚群激活后IL-10表达下调，在CHS模型中活化后分泌IL-10的功能性CD5+CD1dhiBreg数目显著降低。该研究提示B细胞内敲除PPAR-γ后不通过影响未活化状态下的Treg和Breg的生成数目来影响其免疫调节功能，主要通过抑制活化后IL-10+CD5+CD1dhiBreg的分化生成来降低B细胞的免疫调节功能。PPAR-γ激动剂与PPAR-γ上游信号敲除对B10影响与前面结果一致。另外，B细胞内特异敲除PPAR-γ后，Breg细胞增殖降低，腹腔B细胞分泌IL-10降低，可能也是其B细胞整体免疫调节功能下调的机制。此外，在研究过程中，我们也发现了Breg的一些新特性，并建立了相关实验体系，发表论文，为进一步研究PPAR-γ和其它信号对B细胞调节功能的影响和机制提供了依据。总之，项目阐明了PPAR-γ对B细胞免疫调节作用的影响，探讨了其影响机制，并为相关Breg研究建立了基础。