Proliferating SPP1/MERTK-expressing macrophages in idiopathic pulmonary fibrosis

Abstract

A comprehensive understanding of the changes in gene expression in cell types involved in idiopathic pulmonary fibrosis (IPF) will shed light on the mechanisms underlying the loss of alveolar epithelial cells and development of honeycomb cysts and fibroblastic foci. We sought to understand changes in IPF lung cell transcriptomes and gain insight into innate immune aspects of pathogenesis.We investigated IPF pathogenesis using single-cell RNA-sequencing of fresh lung explants, comparing human IPF fibrotic lower lobes reflecting late disease, upper lobes reflecting early disease and normal lungs.IPF lower lobes showed increased fibroblasts, and basal, ciliated, goblet and club cells, but decreased alveolar epithelial cells, and marked alterations in inflammatory cells. We found three discrete macrophage subpopulations in normal and fibrotic lungs, one expressing monocyte markers, one highly expressing FABP4 and INHBA (FABP4^hi), and one highly expressing SPP1 and MERTK (SPP1^hi). SPP1^hi macrophages in fibrotic lower lobes showed highly upregulated SPP1 and MERTK expression. Low-level local proliferation of SPP1^hi macrophages in normal lungs was strikingly increased in IPF lungs.Co-localisation and causal modelling supported the role for these highly proliferative SPP1^hi macrophages in activation of IPF myofibroblasts in lung fibrosis. These data suggest that SPP1^hi macrophages contribute importantly to lung fibrosis in IPF, and that therapeutic strategies targeting MERTK and macrophage proliferation may show promise for treatment of this disease.

Figure 1.

Characterisation of normal lung macrophages. T-distributed stochastic neighbour embedding (T-SNE) of normal lung cells indicates 10 cell types including a large central grouping of macrophage/monocytes/dendritic cells composed of clusters 0, 1, 3 and 10 (panel a) All four of these clusters express AIF1 and all but the dendritic cell cluster (cluster 10) express CD163 (supplementary figure S1). The subject of origin for each of the samples in indicated by a different colour in panel b. The three different macrophage/monocyte clusters (clusters 0, 1 and 3) can be distinguished in feature plots (panel c, purple intensity indicates level of gene expression) and dot plots (panel d, dot size indicates percent of cells expressing and the intensity of purple the level of expression) by expression of FABP4 and INHBA (FABP4^hi macrophages, cluster 0); SPP1, MERTK, LGMN and SIGLEC10 (SPP1^hi macrophages, cluster 1); or IL1B and FCN1 (FCN1^hi monocyte/macrophages, cluster 3). Dendritic cells, expressing CD1C, are found in cluster 10 (see also supplementary figure S1). Macrophage monocyte cell types in bronchoalveolar lavage (BAL) clusters can be distinguished by gene expression (panels c and d), showing FABP4 and INHBA (FABP4^hi macrophages, cluster 0); SPP1, MERTK, LGMN and SIGLEC10 (SPP1^hi macrophages, cluster 1); or IL1B and FCN1 (FCN1^hi monocyte/macrophages, cluster 3). Double immunoflorescent staining for MERTK, FABP4 and FCN1 encoded proteins in serial sections show mostly discrete staining for these three macrophage populations, all three co-stain for CD163. Scale bar=500 μm (panel e). BAL cells from a healthy subject clustered by t-SNE show discrete clusters of macrophages (panel f).

Figure 2.

Combined t-distributed stochastic neighbour embedding (t-SNE) analysis of single-cell transcriptomes from three normal, three idiopathic pulmonary fibrosis (IPF) upper and three IPF lower lobes. a) Tissue sections of i) three control lung samples show preserved lung architecture with no significant pleural, subpleural or interstitial fibrosis and no increase in inflammatory cells. Tissue sections from patients with IPF showed iii) usual interstitial pneumonia lower lobes with a predominant cicatricial process and complete replacement of the lung architecture by scar tissue with honeycomb change, increased inflammatory cells and fibroblastic foci. ii) In comparison, the upper lobes showed relatively uninvolved lung tissue with preserved architecture and only mild interstitial organisation and mildly increased interstitial mononuclear cells suggestive of acute/subacute inflammation in samples SC88 and SC94 with more extensive fibrosis in SC154. Scale bar=1 mm. b) The t-SNE plot shows 20 clusters, with cell types identified by marker genes (supplementary figures S13 and S14). c,e) The tissue origin of the cells stratified by the type of tissue (control, IPF upper or IPF lower lobes) is indicated by different colouring of the cells. d) Reclustering of epithelial cells, showing the discrete clusters of club, ciliated (cluster 1) goblet and AT1 and AT2 cells, as identified by marker genes (supplementary figure S15).

Figure 3.

Percentages of cell types captured in control, upper idiopathic pulmonary fibrosis (IPF) and lower IPF lung samples. The average proportion of cell subgroups as a percentage of total cells analysed, in control, upper IPF lobes and lower IPF lobes are shown for each cluster as shown in figure 2a and subclusters as shown in figure 2d. *: p<0.05.

Figure 4.

Macrophage populations in idiopathic pulmonary fibrosis (IPF) lungs. a) SPP1 and FABP4 expression define two discrete populations in IPF and normal lungs. b) (and supplementary figure S18) SPP1^hi macrophages express more SPP1 and make up a higher percentage of cells in lower lobes, while FABP4^hi macrophages are a higher percentage of the cells in healthy and IPF upper lobes. c) Violin plots of combined IPF/control lung data show expression of FABP4, SPP1, LGMN, MERTK, FCN1 and IL1B limited mainly to macrophage populations (clusters 0, FABP4^hi macrophages; cluster 1, SPP1^hi macrophages and cluster 6, FCN1^hi monocyte/macrophages; figure 2). d) (and supplementary figure S19) FABP4 expression is increased in FABP4^hi macrophages, although it is also expressed at lower levels in SPP1^hi macrophages; SPP1, MERTK and LGMN show highly increased expression in SPP1^hi macrophages; FCN1 and IL1B are most highly expressed in FCN1^hi monocyte/macrophages. Immunofluorescent staining for SPP1 shows macrophages embedded in the SPP1/osteopontin matrix; while staining for MERTK and FABP4 reveals these largely two discrete macrophage populations with increased FABP4-staining macrophages in upper lobes and increased MERTK macrophages in lower lobes. Scale bar=100 μm.

Figure 5.

SPP1 macrophages and myofibroblasts in idiopathic pulmonary fibrosis (IPF) lungs. a) Fibroblastic foci in IPF lungs were stained with α-smooth muscle actin (SMA; red). Macrophages were stained with SPP1 or MERTK as indicated (green). b) Violin plots, indicating expression of several genes associated with fibrosis in control, IPF upper and IPF lower lungs.

Figure 6.

Proliferating cells in idiopathic pulmonary fibrosis (IPF) and normal lungs. a) Proliferating cells from control and IPF lungs form a distinct cluster (cluster 15, green G2/M phase cells marked by arrow; see also figure 2b, cluster 15). b, c) Cluster 15 cells highly express MKI67 (Ki-67), BIRC5, UBE2C and KIAA0101. d) Reclustering of cluster 15 cells shows macrophages expressing either FABP4 or SPP1. Using KIAA as a marker for proliferating cells, both FABP4 and SPP1^hi cells are proliferating in this cluster. e) MERTK-expressing macrophages from IPF lower lobe lung explant co-stained with MERTK and proliferation marker KIAA0101. Macrophages labelled in vitro by incubation with EdU (red), co-stained by immunofluorescent with MERTK (green). f) Examining gene expression by only the proliferating macrophage subset shows low level proliferation of FCN1/(IL1B-expressing) macrophages in control lungs; primarily proliferation of FCN1/(IL1B-expressing) macrophages and FABP4^hi macrophages in IPF upper lobes and primarily proliferation of SPP1^hi macrophages in IPF lower lobes (size of dot indicates the proportion of cells and intensity of purple the relative level of gene expression). Scale bar=100 μm.