Single-Cell Profiling Reveals Immune Aberrations in Progressive Idiopathic Pulmonary Fibrosis

Abstract

Rationale: Changes in peripheral blood cell populations have been observed, but not detailed, at single-cell resolution in idiopathic pulmonary fibrosis (IPF). Objectives: We sought to provide an atlas of the changes in the peripheral immune system in stable and progressive IPF. Methods: Peripheral blood mononuclear cells (PBMCs) from patients with IPF and control subjects were profiled using 10× chromium 5' single-cell RNA sequencing. Flow cytometry was used for validation. Protein concentrations of regulatory T cells (Tregs) and monocyte chemoattractants were measured in plasma and lung homogenates from patients with IPF and control subjects. Measurements and Main Results: Thirty-eight PBMC samples from 25 patients with IPF and 13 matched control subjects yielded 149,564 cells that segregated into 23 subpopulations. Classical monocytes were increased in patients with progressive and stable IPF compared with control subjects (32.1%, 25.2%, and 17.9%, respectively; P < 0.05). Total lymphocytes were decreased in patients with IPF versus control subjects and in progressive versus stable IPF (52.6% vs. 62.6%, P = 0.035). Tregs were increased in progressive versus stable IPF (1.8% vs. 1.1% of all PBMCs, P = 0.007), although not different than controls, and may be associated with decreased survival (P = 0.009 in Kaplan-Meier analysis; and P = 0.069 after adjusting for age, sex, and baseline FVC). Flow cytometry analysis confirmed this finding in an independent cohort of patients with IPF. The fraction of Tregs out of all T cells was also increased in two cohorts of lung single-cell RNA sequencing. 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).

Keywords: IPF; immune system; monocytes; regulatory T cells; single-cell RNA sequencing.

Figure 1.

Study design and single-cell clustering results. (A) Banked, cryopreserved peripheral blood mononuclear cells (PBMCs) from 12 patients with progressive idiopathic pulmonary fibrosis (IPF), 13 matched patients with stable IPF, and 13 matched control subjects were included in this study. Blood had been drawn at the time of IPF diagnosis and was processed into cryopreserved PBMCs and plasma. Patients with IPF were followed for 36 months after the blood draw. Those subjects who were alive at the end of the 36-month period were considered “stable,” whereas those who died were termed “progressive,” as depicted in the Kaplan-Meier curve. Cryopreserved PBMCs were processed in five randomized batches and subjected to 5′ single-cell RNA sequencing (scRNA-seq), whereas plasma was used to determine the level of relevant cytokines and chemokines. Reanalysis of lung scRNA-seq data and cytokine levels in lung tissue homogenates supplemented the blood-based data. (B) Uniform manifold approximation and projection (UMAP) representation of 149,564 cells parceled into 23 cell types. All expected cell types were identified. (C) Same UMAP as in (B), with cells color coded according to disease and disease progression. DC = dendritic cells; NK = natural killer cells.

Figure 2.

Shifts in peripheral blood mononuclear cell (PBMC) subpopulations with disease severity. (A) Boxplots showing single-cell RNA sequencing (scRNA-seq)–based cell proportions for all monocytes and all lymphocytes (as percentage of all PBMCs), grouped by control (C), stable idiopathic pulmonary fibrosis (IPF) (S), and progressive IPF (P). The results are depicted in boxplots, in which the value for each subject is represented by a dot, and the upper and lower bounds represent the 75% and 25% percentiles, respectively. The center bars indicate the medians, and the whiskers denote values up to 1.5 interquartile ranges above the 75% or below the 25% percentiles. (B) Similar boxplots showing scRNA-seq–based cell proportions for each cell type. B = B cell; DC = dendritic cells; IM = intermediate; NC = nonclassical; NK = natural killer; T = T cell; Tregs = regulatory T cells. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3.

Differences in peripheral blood mononuclear cell (PBMC) gene expression profile between patients with idiopathic pulmonary fibrosis (IPF) and control subjects. (A) Heatmap showing the top differentially expressed genes (log₂ fold change [log₂FC] > 0.3; adjusted P values < 0.05 calculated by Wilcoxon rank-sum test with Bonferroni correction for multiple comparisons) for each major cell type, comparing patients with IPF to matched control subjects. T cell receptor and B cell receptor genes were excluded from this heatmap. The number of genes that are increased or decreased in patients with IPF (vs. control subjects), using a lower log₂FC cutoff of 0.25 and without exclusions, are listed in the legend for each cell type and presented in Table E4. The upper part of the heatmap depicts genes that are increased in IPF compared with control subjects (marked “increased in IPF”). (B) Violin plots focusing on selected genes that differ in monocytes and DCs between IPF and control. S100A8, S100A12, and CTSD are increased in IPF, whereas a composite score of HLA class II transcripts is decreased. (C) Violin plots depicting increased expression of the platelet-specific PPBP gene in monocyte clusters of IPF versus controls. (D) UMAP of PPBP expression in PBMCs, demonstrating increased expression in a subset of monocytes, potentially attributable to higher PMC formation in IPF. DC = dendritic cells; NK = natural killer; PMCs = platelet-monocyte complexes; UMAP = uniform manifold approximation and projection.

Figure 4.

Gene expression changes in stable versus progressive idiopathic pulmonary fibrosis (IPF), and the increased regulatory T cells (Tregs) in blood and lung in progressive disease. (A) Heatmap showing the top differentially expressed genes (log₂ fold change [log²FC > 0.3; adjusted P values < 0.05 calculated by Wilcoxon rank-sum test with Bonferroni correction for multiple comparisons) for each major cell type, comparing patients with stable and progressive IPF. T cell receptor and B cell receptor genes were excluded from this heatmap. The number of genes that are increased or decreased in patients with progressive IPF (vs. stable IPF), using a lower log₂FC cutoff of 0.25 and without exclusions, is listed in the color key for each cell type and also presented in Table E5. The upper part of the heatmap enumerates genes that are increased in progressive IPF compared with stable IPF (marked “increased in progressive”), whereas the lower part lists genes increased in stable IPF. Each row indicates a gene, and each column indicates a patient. Note the color codes for IPF severity at the top and cell type on the right. (B) Expression of IL1B in monocytes of all subjects. On average, IL1B expression is higher in patients with stable IPF compared with patients with progressive IPF. (C) Box plot showing an increased level of Tregs (presented as percentage of all T cells) in patients with progressive IPF. Each dot represents an individual subject. (D) Flow cytometry validation in an independent cohort of patients with IPF, demonstrating an increase in Tregs in progressive IPF (P = 0.039). (E) Kaplan-Meier survival curves in patients with IPF, showing a clear split of the curves that is based on the single-cell RNA-sequencing (scRNA-seq) levels of Tregs (cutoff for high Tregs [as percentage of T cells], >5%; P = 0.009). (F) Same as in (D) but split according to the scRNA-seq levels of all monocytes (cutoff for high monocytes, >39% of all PBMCs; P = 0.046). (G) Tregs are increased in progressive IPF/ILD in two lung scRNA-seq datasets by Adams and colleagues (27) and Habermann and colleagues (28), after the removal of outliers. DC = dendritic cells; ILD = interstitial lung disease; NK = natural killer cells. *P < 0.05 and **P < 0.01.

Figure 5.

Potential chemoattractants of monocytes and regulatory T cells (Tregs) in idiopathic pulmonary fibrosis (IPF) and a proposed lung–blood recruitment model in progressive disease. (A) Expression of the monocyte chemokine receptor CCR2, and of the Treg chemokine receptors CCR4 and CCR8 in IPF peripheral blood mononuclear cells (PBMCs). Note that CCR4 and CCR8 are also expressed in memory CD4 T cells, especially Th2 cells (see Figure E8) and that nonclassical monocytes lack the expression of CCR2. (B) Chemokine ligands (CCL7, CCL22, and CCL18) of the above respective chemokine receptors (CCR2, CCR4, and CCR8) that were increased in the plasma of patients with IPF (n = 12 with stable IPF, and 11 with progressive IPF) compared with control subjects (n = 9). Black bars indicate the mean values. These chemokines are potential candidates that may be involved in chemoattraction of classical monocytes and Tregs from the blood into the IPF lung. Figure E9 shows levels of other cytokines and chemokines measured in plasma and lung tissue homogenates. (C) Proposed integrative lung–blood recruitment model in progressive IPF. The left side of the figure depicts Tregs and classical monocytes in the peripheral blood, whereas the right side depicts lung cells. Lung macrophages (Mφ) and myofibroblasts (MF) secrete CCL7 (and, potentially, CCL13) into the blood, which stimulates CCR2-mediated recruitment of classical monocytes. These monocytes migrate to the IPF lung (black arrow) and are thought to be precursors of lung macrophages. Lung macrophages and dendritic cells (DC) secrete CCL18 and CCL22, respectively. These chemokines may drive CCR8- and CCR4-mediated recruitment of Tregs and Th2 cells, which may migrate to the fibrotic niche and induce a profibrotic secretory profile in macrophages. Figure 5C was created with BioRender.com. *P < 0.05 and **P < 0.01.

Figure 6.

Cellular origin of the outcome-predictive 52-gene signature. Dot plot showing the cellular origin of the 52-gene signature in our single-cell RNA-sequencing cohort. Each row represents a binned cell type, and each column represents a gene. Dot size indicates the percentage of cells expressing a gene; its color intensity represents the average expression. Fifty of the 52 genes were detected in our dataset. The seven increased genes that correlate with shorter TFS (green) were mainly of monocyte origin, whereas the 43 decreased genes (red) originated mainly from T, B, and NK cells. NK = natural killer; TFS = transplant-free survival.