Single-cell profiling reveals immune aberrations in progressive idiopathic pulmonary fibrosis

Rationale: Changes in peripheral blood cell populations have been observed but not detailed at single-cell resolution in idiopathic pulmonary fibrosis (IPF). Objectives: To provide an atlas of the changes in the peripheral immune system in stable and progressive IPF. Methods: Peripheral blood mononuclear cells (PBMCs) from IPF patients and controls were profiled using 10x Chromium 5’ single-cell RNA sequencing (scRNA-seq). Flow cytometry was used for validation. Protein concentrations of Regulatory T-cells (Tregs) and Monocytes chemoattractants were measured in plasma and lung homogenates from patients and controls. Measurements and Main Results: Thirty-eight PBMC samples from 25 patients with IPF and 13 matched controls yielded 149,564 cells that segregated into 23 subpopulations, corresponding to all expected peripheral blood cell populations. Classical monocytes were increased in progressive and stable IPF compared to controls (32.1%, 25.2%, 17.9%, respectively, p<0.05). Total lymphocytes were decreased in IPF vs controls, and in progressive vs stable IPF (52.6% vs 62.6%, p=0.035). Tregs were increased in progressive IPF (1.8% vs 1.1%, p=0.007), and were associated with decreased survival (P=0.009 in Kaplan-Meier analysis). Flow cytometry analysis confirmed this finding in an independent cohort of IPF patients. Tregs were also increased in two cohorts of lung scRNA-seq. CCL22 and CCL18, ligands for CCR4 and CCR8 Treg chemotaxis receptors, were increased in IPF. Conclusions: The single-cell atlas of the peripheral immune system in IPF, reveals an outcome-predictive increase in classical monocytes and Tregs, as well as evidence for a lung-blood immune recruitment axis involving CCL7 (for classical monocytes) and CCL18/CCL22 (for Tregs). 38 peripheral blood mononuclear cells (PBMCs) samples from 25 patients with IPF (12 patients with progressive disease and 13 patients with stable disease) and 13 matched control subjects were profiled using 10x Genomics Chromium 5’ single-cell RNA sequencing (scRNA-seq).

研究背景：特发性肺纤维化（idiopathic pulmonary fibrosis, IPF）中外周血细胞群的变化已被观测到，但尚未在单细胞分辨率下进行详细阐释。
研究目的：构建稳定型与进展型特发性肺纤维化患者外周免疫系统变化的图谱。
研究方法：采用10x Chromium 5’端单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）对特发性肺纤维化患者及健康对照的外周血单个核细胞（peripheral blood mononuclear cells, PBMCs）进行测序分析，并通过流式细胞术进行验证。此外，检测患者及对照血浆与肺组织匀浆中调节性T细胞（regulatory T-cells, Tregs）及单核细胞趋化因子的蛋白浓度。
检测指标与主要结果：本研究共纳入25例特发性肺纤维化患者与13例匹配对照的外周血单个核细胞样本，最终获得149,564个细胞，可分为23个亚群，覆盖所有预期的外周血细胞群。与对照组相比，进展型与稳定型特发性肺纤维化患者的经典单核细胞比例均升高（分别为32.1%、25.2%、17.9%，P<0.05）。特发性肺纤维化患者总淋巴细胞比例较对照组降低，且进展型患者较稳定型患者进一步降低（52.6% vs 62.6%，P=0.035）。进展型特发性肺纤维化患者的调节性T细胞比例升高（1.8% vs 1.1%，P=0.007），且与患者生存率降低相关（Kaplan-Meier分析P=0.009）。独立队列的流式细胞术分析验证了上述发现。此外，在两个肺组织单细胞RNA测序队列中也观察到调节性T细胞比例升高。趋化因子CCL22与CCL18作为CCR4与CCR8型调节性T细胞的趋化受体配体，在特发性肺纤维化患者中表达升高。
研究结论：本研究构建的特发性肺纤维化外周免疫系统单细胞图谱显示，经典单核细胞与调节性T细胞的升高可预测患者预后，同时发现存在肺-血液免疫募集轴，该轴分别通过CCL7介导经典单核细胞募集、CCL18/CCL22介导调节性T细胞募集。
38例外周血单个核细胞样本来自25例特发性肺纤维化患者（其中12例为进展型疾病、13例为稳定型疾病）与13例匹配对照，采用10x Genomics Chromium 5’端单细胞RNA测序进行分析。