Purpose: Lung transplant (LT) recipients experience episodes of immune-mediated acute lung allograft dysfunction (ALAD). We have applied single-cell RNA sequencing (scRNAseq) to bronchoalveolar lavage (BAL) cells of stable and ALAD patients to determine key cellular elements in dysfunctional lung allografts. Our particular focus here is on studying alveolar macrophages (AMs) as scRNAseq enables us to elucidate their heterogeneity and possible association with ALAD where our knowledge from cytometry-based assays is very limited. Methods: Fresh bronchoalveolar lavage (BAL) cells from 6 LT patients, 3 with stable lung function and 3 undergoing an episode of ALAD were used for scRNAseq. R Bioconductor and Seurat were used to perform QC, dimensionality reduction, annotation, pathway analysis, and trajectory. Donor and recipient deconvolution was performed using single nucleotide variations. Results: Our data revealed that AMs are highly heterogeneous (12 transcriptionally distinct subsets in stable). We identified two AM subsets uniquely represented in ALAD. Based on pathway analysis and the top differentially expressed genes in BAL we annotated them as pro-inflammatory interferon-stimulated genes (ISG) and metallothioneins-mediated inflammatory (MT). Pseudotime analysis suggested that ISG AMs represent an earlier stage of differentiation which may suggest them as monocyte drive macrophages. Our functional analysis on an independent set of BAL samples shows that ALAD samples have significantly higher expression of CXCL10, a marker of ISG AM, as we as higher secretion of pro-inflammatory cytokines. Single nucleotide variation calling algorithm has allowed us to identify macrophages of donor origin and demonstrated that donor AMs are lost with time post-transplant. Conclusion: Using scRNAseq, we observed AMs heterogeneity and identified specific subsets that may be associated with allograft dysfunction. Further exploration with scRNAseq will shed light on LT immunobiology and the role of AMs in allograft injury and dysfunction.

中文翻译：

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肺移植后与同种异体功能不全相关的促炎性肺泡巨噬细胞

目的：肺移植（LT）受者会经历免疫介导的急性肺同种异体移植功能障碍（ALAD）的发作。我们已经对稳定和ALAD患者的支气管肺泡灌洗（BAL）细胞应用了单细胞RNA测序（scRNAseq），以确定功能异常的肺移植物中的关键细胞成分。由于scRNAseq使我们能够阐明它们的异质性以及与ALAD的可能联系，因此我们在这里的重点是研究肺泡巨噬细胞（AM），而基于细胞计数的检测知识非常有限。方法：将6例LT患者，3例肺功能稳定和3例ALAD发作的新鲜支气管肺泡灌洗（BAL）细胞用于scRNAseq。R Bioconductor和Seurat用于执行质量控制，降维，标注，路径分析和轨迹。供体和受体的解卷积使用单核苷酸变异进行。结果：我们的数据显示AM高度异质（稳定存在12个转录上不同的子集）。我们确定了ALAD中唯一表示的两个AM子集。基于途径分析和BAL中最差表达的基因，我们将其注释为促炎性干扰素刺激基因（ISG）和金属硫蛋白介导的炎症（MT）。伪时间分析表明，ISG AMs代表了分化的早期阶段，这可能表明它们是单核细胞驱动的巨噬细胞。我们对一组独立的BAL样品的功能分析表明，ALAD样品的CXCL10（ISG AM的标志物）表达明显较高，因为促炎性细胞因子的分泌较高。单核苷酸变异调用算法已使我们能够鉴定出供体来源的巨噬细胞，并证明了供体AM随移植时间的流逝而丢失。结论：使用scRNAseq，我们观察了AMs的异质性，并鉴定了可能与同种异体移植功能障碍相关的特定亚型。用scRNAseq进行的进一步探索将阐明LT免疫生物学以及AMs在同种异体移植物损伤和功能障碍中的作用。