Role of regulatory B-cells in pathogenesis and progression of autoimmune myocarditis

自身免疫性心肌炎可进展为慢性扩张型心肌病，并增加心力衰竭的风险，其发病机制尚不完全清楚。提示细胞免疫调节功能障碍可能导致促炎和抗炎过程的失衡，从而支持这种复杂的疾病。虽然T细胞在自身免疫性心肌炎中的关键作用已被广泛接受，但B细胞的作用仍不完全清楚。首先，研究局限于伴随疾病进展的自身抗体(自身抗体)。尤其是可能能够抑制炎症过程的调节性B细胞(BREG)亚群还没有被研究过。在我们的心肌肌钙蛋白I(CTnI)诱导的实验性自身免疫性心肌炎(EAM)小鼠模型中的初步工作已经指出，Breg在控制这种疾病方面具有强大的作用：虽然过继转移来自cTnI免疫小鼠的T细胞导致大多数未免疫小鼠发生EAM，但正如预期的那样，这些T细胞与自体B细胞过继共转移显著降低了EAM的发生率和程度。这意味着可能存在不同的B细胞亚群，具有支持或抵消心肌梗死的潜力。在本项目提案中，我们追求三个目标：1)表征EAM中Breg被激活和发挥功能的条件：为此，在将cTnI特异性T细胞转移到未免疫的小鼠之前，在不同的条件下刺激B细胞。进一步的Breg将在免疫之前或之后转移到EAM-小鼠身上，以评估它们是否能够保护EAM，甚至在炎症环境中下调症状。2)评估Breg对细胞环境的影响。在这里，将对cTnI免疫和Breg处理的小鼠的各种免疫细胞进行功能分析，以研究Breg对免疫反应的调节作用。此外，我们试图通过对可能代表Breg的B细胞亚型的分类来揭示Breg的表型，并测试它们的调节能力。3)评估Breg亚型的作用和自身抗体对EAM的影响。为此，将用cTnI免疫B细胞缺陷小鼠(JH-/-)，并与B细胞能力小鼠进行比较。此外，还添加了Breg单元(或第2部分中找到的Breg候选单元)。此外，将Breg与cTnI-T细胞共转移到Breg缺陷小鼠中。实验的补充是添加B细胞培养上清和EAM血清，并在去除和不去除包含抗体的情况下评估自身抗体在EAM进展中的影响。通过对BREG在EAM中作用的研究，我们旨在寻找恢复心肌炎免疫平衡的线索，这可能有助于未来开发新的免疫治疗药物。因此，我们计划将我们在cTnI-EAM(Kaya博士)方面的长期专业知识与Breg在慢性炎症方面的专业知识(Dr.Tretter)相结合。

The pathogenesis of autoimmune myocarditis that can progress into chronically dilated cardiomyopathy with an increased risk for heart failure is not completely understood. It is suggested that dysfunctional cellular immunoregulation might lead to an imbalance of pro- and anti-inflammatory processes and thereby support this complex disease. While the crucial impact of the T cells in autoimmune myocarditis is widely accepted, the role of B cells is still incompletely understood. Primarily, research is restricted to the auto-antibodies (auto-Abs) that accompany disease progression. Especially the subpopulation of regulatory B cells (Breg) that might be able to potentially counteract the inflammatory process has not been investigated, yet. Preliminary work in our mouse model of cardiac troponin I (cTnI) -induced experimental autoimmune myocarditis (EAM) already points to a strong impact of Breg in control of this disease: while adoptive transfer of T cells from cTnI immunized mice resulted in development of EAM in the majority of non-immunized mice, as expected, adoptive cotransfer of those T cells together with autologous B-cells strongly reduced incidence and extent of EAM. This implies that different subpopulations of B cells might exist, with the potential to support or to counteract myocardits. In this project proposal we pursue 3 aims: 1) Characterize the conditions under which Breg in EAM are activated and functional: To this end B cells will be stimulated under different conditions before transfer with cTnI specific T cells into non-immunized mice. Further Breg will be transferred to EAM- mice before or after their immunization to evaluate, whether they can protect from EAM or even down regulate symptoms in an inflammatory environment.2) Assess the influence of Breg on the cellular environment. Here functional assays with various immune cells from cTnI immunized and Breg treated mice will be performed to investigate the modulatory effects of Breg on immune responses. Further we attempt to reveal the Breg phenotype by sorting B-cell subtypes that might represent Breg and test their regulatory abilities.3) Evaluate the role of Breg subtypes and impact of auto-Abs on EAM. To this end B cell deficient mice (JH-/-) will be immunized with cTnI and compared to B cell competent mice. In addition Breg cells (or Breg candidates found in part 2) are added. Further, cotransfer of Breg, with cTnI-T cells into Breg deficient mice will be performed. The experiments are supplemented by addition of B cell culture supernatant and EAM-serum with and without depletion of containing antibodies to assess the impact of auto-Abs in progression of EAM. By investigating of the role of Breg in EAM we aim to find clues to restore the immunological balance in myocarditis This might help to develop novel immunotherapeutics in future. We therefore plan to merge our long-standing expertise in the cTnI-EAM (Dr.Kaya) with that of Breg in chronic inflammation (Dr.Tretter).