Transcriptomic profiling of peripheral blood mononuclear cells in sarcoidosis and matched controls in A Case Controlled Etiologic Study of Sarcoidosis (ACCESS)

Introduction: In sarcoidosis, peripheral lymphopenia and anergy have been associated with increased inflammation and maladaptive immune activity, likely promoting development of chronic and progressive disease. However, the molecular mechanisms that lead to reduced lymphocyte proportions, particularly CD4+ T-cells, have not been fully elucidated. We posit that paradoxical peripheral lymphopenia is characterized by a dysregulated transcriptomic network associated with cell function and fate that results from altered transcription factor targeting activity. Methods: Messenger RNA-sequencing (mRNA-seq) was performed on peripheral blood mononuclear cells (PBMCs) from ACCESS study subjects with sarcoidosis and matched controls and findings validated on a sarcoidosis case-control cohort and a sarcoidosis case series. Preserved PBMC transcriptomic networks between case-control cohorts were assessed to establish cellular associations with gene modules and define regulatory targeting involved in sarcoidosis immune dysregulation utilizing weighted gene co-expression network analysis and differential transcription factor involvement analysis. Network centrality measures identified master transcriptional regulators of subnetworks related to cell proliferation and death. Predictive models of differential PBMC proportions constructed from ACCESS target gene expression corroborated the relationship between aberrant transcription factor regulatory activity and imputed and clinical PBMC populations in the validation cohorts. Results: We identified two unique and preserved gene modules significantly associated with sarcoidosis immune dysregulation. Strikingly, increased expression of a monocyte-driven, and not a lymphocyte-driven, gene module related to innate immunity and cell death was the best predictor of peripheral CD4+ T-cell proportions. Within the gene network of this monocyte-driven module, TLE3 and CBX8 were determined to be master regulators of the cell death subnetwork. A core gene signature of differentially over-expressed target genes of TLE3 and CBX8 involved in cellular communication and immune response regulation accurately predicted imputed and clinical monocyte expansion and CD4+ T-cell depletion. Conclusions: Altered transcriptional regulation associated with aberrant gene expression of a monocyte-driven transcriptional network likely influences lymphocyte function and survival. Although further investigation is warranted, this indicates that crosstalk between hyperactive monocytes and lymphocytes may instigate peripheral lymphopenia and underlie sarcoidosis immune dysregulation and pathogenesis. Future therapies selectively targeting master regulators, or their targets, may mitigate dysregulated immune processes in sarcoidosis and disease progression. Examination of PBMC gene expression comparing treatment naive cases of sarcoidosis to matched controls from the ACCESS study.

引言：结节病（sarcoidosis）患者中，外周淋巴细胞减少症（peripheral lymphopenia）与免疫无应答（anergy）常与炎症加剧及适应性免疫失衡相关，可能推动慢性进行性疾病的发生发展。然而，导致淋巴细胞比例降低（尤其是CD4阳性T细胞（CD4+ T-cells）比例下降）的分子机制尚未完全阐明。我们提出假说：矛盾性外周淋巴细胞减少的特征为转录调控网络紊乱，该网络与细胞功能及命运相关，其成因是转录因子靶向活性发生改变。
方法：本研究对来自ACCESS研究（ACCESS study）的结节病患者及匹配对照受试者的外周血单个核细胞（PBMCs）实施信使RNA测序（mRNA-seq），并在结节病病例对照队列及结节病病例系列中验证研究结果。通过加权基因共表达网络分析（weighted gene co-expression network analysis）与差异转录因子参与分析（differential transcription factor involvement analysis），评估病例对照队列间保守的PBMC转录组网络，以明确基因模块与细胞表型的关联，并解析结节病免疫失调相关的调控靶向机制。借助网络中心性分析，鉴定出与细胞增殖、死亡相关亚网络的核心转录调控因子（master transcriptional regulators）。基于ACCESS队列靶基因表达构建的外周血单个核细胞比例差异预测模型，验证了异常转录因子调控活性与验证队列中推算及临床检测的PBMC群体之间的关联。
结果：本研究鉴定出两个独特且保守的基因模块，二者与结节病免疫失调显著相关。值得注意的是，与先天免疫及细胞死亡相关的单核细胞驱动型（而非淋巴细胞驱动型）基因模块的表达上调，是外周CD4阳性T细胞比例的最佳预测因子。在该单核细胞驱动型模块的基因网络中，TLE3与CBX8被鉴定为细胞死亡亚网络的核心调控因子。TLE3与CBX8的差异高表达靶基因构成核心基因特征，这些靶基因参与细胞通讯与免疫应答调控，可精准预测推算及临床检测到的单核细胞扩增与CD4阳性T细胞耗竭情况。
结论：单核细胞驱动型转录网络的基因表达异常所伴随的转录调控改变，可能影响淋巴细胞的功能与存活。尽管尚需进一步研究，但该结果提示：过度活化的单核细胞与淋巴细胞间的串扰可能诱发外周淋巴细胞减少，并成为结节病免疫失调与发病机制的基础。未来选择性靶向核心调控因子或其靶标的治疗手段，或可缓解结节病中失调的免疫进程及疾病进展。本研究还对ACCESS研究中未接受治疗的结节病病例与匹配对照的PBMC基因表达进行了对比分析。