Background/Purpose: Circulating monocytes with pro-inflammatory and pro-fibrotic phenotype have been implicated in SSc-ILD pathogenesis, but their potential trajectory to mature cell lineage macrophages driving organ-specific damage is not well defined.Evidence suggests that osteopontin expressing macrophages likely contribute to fibrotic mechanisms in SSc-ILD. We sought to characterize specific myeloid subpopulations associated with SSc-ILD progression and establish potential precursor relationships with lung macrophages.

Methods: Single-cell RNA-seq (scRNA-seq) was performed on peripheral blood obtained from 60 SSc patients meeting 2013 ACR/EULAR criteria. Unsupervised clustering identified two subpopulations of monocytes with type I interferon signatures. Differential expression and subtype correlation analyses against %change in forced vital capacity (FVC, liter) per year from baseline were conducted in subjects with SSc-ILD (n=40). Transcription factor (TF) analysis was conducted for progressive ( >10% decrease in FVC) and non-progressive SSc-ILD patients. Reprocessed publicly available scRNA-seq data from SSc-ILD lungs were used to perform trajectory analysis.

Results: Clustering and annotation of myeloid cells identified 11 myeloid cell types and two subclusters (CD74+ and CD74-) of an “ISG15-hi” monocyte population expressing high levels of interferon (IFN)-inducible markers (Fig.1A). The CD74+ subpopulation showed further enrichment for genes involved in “Response to type I IFN signaling” (FDR = 6.82 x 10-4) whilst the CD74- subpopulation upregulated genes involved in “Leukocyte cell-to-cell adhesion” (FDR = 3.61 x 10-3) (Fig.1B,D). Correlation analysis of cell type proportions against FVC %-change for SSc-ILD patients (Fig.1C) revealed a Pearson correlation of 0.60 (p=1.9×10-4) in the CD74+ “ISG15-hi” monocyte population. In the CD74+ subpopulation, progressive SSc-ILD patients upregulated genes involved in “Myeloid differentiation” (FDR = 2.90 x 10-2) (Fig.2A) and further showed increased activity for TFs previously reported as specific regulators in osteopontin expressing lung macrophages (Mac-SPP1-lung) from SSc-ILD patients including ATF5 and MAFF (Fig.2B). A reanalysis of SSc-ILD lung scRNA-seq data identified a population of “ISG15-hi” classical monocytes CMos-ISG15-lung and the previously reported Mac-SPP1-lung (Fig.3A). Trajectory analysis predicted a differentiation trajectory from CMos-ISG15-lung to Mac-SPP1-lung (Fig. 3B).

Conclusion: We identified a novel CD74⁺ subcluster within ISG15-high circulating monocytes, marked by strong type I IFN-inducible gene expression and significantly negatively associated with progressive SSc-ILD. This population may serve as a precursor to pro-fibrotic, osteopontin-expressing lung macrophages, suggesting a pathogenic role in SSc-ILD.

Fig. 1. A. UMAP of myeloid cells showing subclustering of monocyte cluster with type I IFN signature. B. Volcano plot of subcluster markers. C. Correlation of cluster frequencies with change in %fvc from baseline for SSc-ILD patients. D. Pathways for classical monocyte ISG15 subclusters.

Fig. 2. A. Volcano plot for differential expression of progressive vs non-progressive SSc-ILD in CD74+ subpopulation. B. SCENIC TF analysis of subclusters split by disease progression.

Fig. 3. A. DotPlot for markers of lung monocytes and osteopontin expressing macrophages. B. UMAP of clusters and trajectory analysis. Ref 1: Ann Rheum Dis. 2019 Aug 12;78(10):1379_1387

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/circulating-monocytes-with-high-interferon-signature-as-precursors-to-osteopontin-expressing-lung-macrophages-in-patients-with-systemic-sclerosis-and-progressive-interstitial-lung-disease/