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

自身免疫性心肌炎可发展为慢性扩张型心肌病并增加心衰风险，其发病机制尚不完全清楚。这表明功能失调的细胞免疫调节可能导致促炎和抗炎过程的不平衡，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重要作用已被广泛接受，但B细胞的作用仍未完全了解。首先，研究仅限于伴随疾病进展的自身抗体（auto-Abs）。特别是调节性B细胞（Breg）亚群，可能能够潜在地对抗炎症过程，但尚未被研究。我们在心肌肌钙蛋白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的强大影响：虽然来自cTnI免疫小鼠的T细胞过继性转移导致大多数未免疫小鼠发生EAM，正如预期的那样，这些T细胞与自体b细胞的过继性共转移大大降低了EAM的发生率和程度。这意味着可能存在不同的B细胞亚群，具有支持或对抗心肌的潜力。在本项目提案中，我们追求3个目标：1)表征Breg In EAM被激活和功能的条件：为此，B细胞将在不同条件下被刺激，然后与cTnI特异性T细胞一起转移到未免疫的小鼠中。在EAM-小鼠免疫前或免疫后，进一步将Breg转移到EAM-小鼠身上，以评估它们是否能保护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。本研究将对cTnI免疫和Breg处理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。进一步，我们试图通过对可能代表Breg的b细胞亚型进行分类并测试它们的调节能力来揭示Breg表型。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细胞正常小鼠进行比较。此外，还添加了Breg细胞（或在第2部分中发现的候选Breg细胞）。此外，将Breg与cTnI-T细胞共转移到Breg缺陷小鼠中。实验通过添加B细胞培养上清和EAM血清（含和不含含抗体）来补充，以评估自身抗体在EAM进展中的影响。通过研究Breg在心肌炎EAM中的作用，我们希望找到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方法。因此，我们计划将我们在cTnI-EAM方面的长期专业知识（Dr.Kaya）与Breg在慢性炎症方面的专业知识（Dr.Tretter）合并。

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).