Elevated Fibronectin Levels in Profibrotic CD14+ Monocytes and CD14+ Macrophages in Systemic Sclerosis

Abstract

Background: Systemic sclerosis (SSc) is an autoimmune disease characterized by overproduction of extracellular matrix (ECM) and multiorgan fibrosis. Animal studies pointed to bone marrow-derived cells as a potential source of pathological ECM-producing cells in immunofibrotic disorders. So far, involvement of monocytes and macrophages in the fibrogenesis of SSc remains poorly understood.

Methods and results: Immunohistochemistry analysis showed accumulation of CD14⁺ monocytes in the collagen-rich areas, as well as increased amount of alpha smooth muscle actin (αSMA)-positive fibroblasts, CD68⁺ and mannose-R⁺ macrophages in the heart and lungs of SSc patients. The full genome transcriptomics analyses of CD14⁺ blood monocytes revealed dysregulation in cytoskeleton rearrangement, ECM remodeling, including elevated FN1 (gene encoding fibronectin) expression and TGF-β signalling pathway in SSc patients. In addition, single cell RNA sequencing analysis of tissue-resident CD14⁺ pulmonary macrophages demonstrated activated profibrotic signature with the elevated FN1 expression in SSc patients with interstitial lung disease. Peripheral blood CD14⁺ monocytes obtained from either healthy subjects or SSc patients exposed to profibrotic treatment with profibrotic cytokines TGF-β, IL-4, IL-10, and IL-13 increased production of type I collagen, fibronectin, and αSMA. In addition, CD14⁺ monocytes co-cultured with dermal fibroblasts obtained from SSc patients or healthy individuals acquired a spindle shape and further enhanced production of profibrotic markers. Pharmacological blockade of the TGF-β signalling pathway with SD208 (TGF-β receptor type I inhibitor), SIS3 (Smad3 inhibitor) or (5Z)-7-oxozeaenol (TGF-β-activated kinase 1 inhibitor) ameliorated fibronectin levels and type I collagen secretion.

Conclusions: Our findings identified activated profibrotic signature with elevated production of profibrotic fibronectin in CD14⁺ monocytes and CD14⁺ pulmonary macrophages in SSc and highlighted the capability of CD14⁺ monocytes to acquire a profibrotic phenotype. Taking together, tissue-infiltrating CD14⁺ monocytes/macrophages can be considered as ECM producers in SSc pathogenesis.

Keywords: CD14+ macrophages; CD14+ monocytes; TGF-β; fibronectin; fibrosis; systemic sclerosis.

Figure 1

Monocyte infiltration into the fibrotic lesion in SSc tissues. (A) Representative images of paraffin-embedded sections of the heart and lungs, and (B) corresponding quantification of IHC staining for CD14 (lungs HC N=7, SSc N=7, heart HC N=10, SSc N=11, unpaired t-test). (C) Representative images of paraffin-embedded sections of the heart, lungs and skin co-stained for pro-collagen I (red) and CD14 (brown). (A, C) Scale bar 100μm, scale bar in insert 20μm.

Figure 2

Characteristic of human SSc lung and heart tissues. Representative images of paraffin-embedded sections of the heart and lungs and corresponding quantification of IHC staining for αSMA (A), CD68 (B) and mannose-R (CD206) (C) (lung HC N=7, lung SSc N=7, heart HC N=10, heart SSc N=11, unpaired t-test; scale bar 100μm, scale bar in insert 20μm).

Figure 3

Transcriptomic analysis of CD14⁺ monocytes from SSc patients and HC. (A) Pathway enrichment analysis of SSc-related biological processes calculated based on differentially expressed gene sets (Metacore software). (B) Differentially expressed genes involved in fibrosis from RNAseq data. (C) qPCR confirmatory analyses of selected genes from RNAseq data (N=5-20, Mann Whitney-U-test). (D) qPCR analysis of FN1 mRNA levels in sorted CD14⁺⁺CD16^-, CD14^lowCD16⁺ and CD14⁺CD16⁺ monocyte from SSc patients and HC (n=3-6, Mann Whitney-U-test).

Figure 4

Characteristic of CD14⁺ macrophages in SSc-ILD lung tissues. Four human healthy control (HC) and 4 SSc-ILD lung tissue samples were used for a single-cell (sc) RNA-sequencing analysis according to Valenzi et al. (26). (A) UMAP plot visualization of CD14⁺ cells in macrophage clusters in HC and SSc-ILD samples. (B) Pathway expression in the specific GO biological process (Enrichr 2018) and the expression of selected fibrotic-related genes (C) upregulated in SSc-ILD CD14⁺fibronectin (FN1)⁺ cells compared to HC CD14⁺ cells in lungs. (D) FN1 expression in SSc-ILD CD14⁺ cells compared to HC CD14⁺ cells in lungs (N=4, Mann Whitney-U-test). (E) UMAP plot visualization of CD14⁺ cells in macrophage clusters in HC and SSc-ILD samples. UMAP, uniform manifold approximation and projection; SSc-ILD, systemic sclerosis-associated interstitial lung disease.

Figure 5

Profibrotic stimulation induces differentiation of monocytes into fibroblast-like cells. (A) mRNA expression of COL1A1, ACTA2 and FN1 after profibrotic cytokines stimulation (TGFβ, IL-4, IL-10, IL-13 [10 ng/ml each]) of HC and SSc monocytes (N=13, two-way ANOVA with Benjamini, Krieger and Yekutieli post-hoc test). (B) ELISA measurement of Pro-collagen 1α1 concentration in supernatants from CD14⁺ monocytes after profibrotic cytokines stimulation (TGFβ, IL-4, IL-10, IL-13, [10 ng/ml each]) (N=13, two-way ANOVA with Benjamini, Krieger and Yekutieli post-hoc test). (C) Western blot assessment of fibronectin level in cell lysates and supernatants from CD14⁺ monocytes stimulated with profibrotic cytokines (TGFβ, IL-4, IL-10, IL-13, [10 ng/ml each]) (N=3). (D) Time-dependent mRNA expression of COL1A1, ACTA2 and FN1 after profibrotic cytokines stimulation (TGFβ, IL-4, IL-10, IL-13 [10 ng/ml each]) of HC monocytes at following time points: 0, 8, 24, 47, 72 and 168h (N=4, two-way ANOVA with Tukey’s multiple comparison test, p values for row factor).

Figure 6

Profibrotic microenvironment induces differentiation of monocytes into fibroblast-like cells. (A) mRNA expression of ACTA2, COL1A1 and FN1 in CD14⁺ monocytes from HC and SSc patients after co-culture with HC and SSc skin fibroblasts (HC N=5, SSc N=6, two-way ANOVA with Uncorrected Fischer’s LSD post-hoc test, **p < 0.005). (B) Representative images of immunofluorescence staining of CD14⁺ monocytes with and without co-culture with fibroblasts (blue– DAPI nuclear staining, red– Phalloidin staining, green- α-SMA staining). (C) Representative images of immunofluorescence staining of HC and SSc CD14⁺ monocytes co-cultured with fibroblasts in 3D hydrogel model (blue– monocytes stained with Cell Trace Violet, green– fibroblasts stained with CFSE, red– α-SMA staining, grey- Phalloidin staining). ns, not significant.

Figure 7

Monocyte-to-fibroblast-like cells differentiation is dependent on TGFβ signalling pathways. (A) COL1A1 mRNA expression level and ELISA measurements of Pro-collagen 1α1 protein level in supernatants after profibrotic cytokine stimulation (TGFβ, IL-4, IL-10, IL-13 [10 ng/ml each]) and treated with TGFβ signalling pathway inhibitors [SD208 and (5Z)-7-Oxozeaenol (OXO) 1 μM, SIS3 2 μM] (N=4-7, one-way ANOVA with Benjamini, Krieger and Yekutieli post-hoc test). (B) FN1 mRNA expression level and Western Blot analysis, and corresponding densitometry of Fibronectin protein level in the supernatants after profibrotic cytokine stimulation (TGFβ, IL-4, IL-10, IL-13 [10 ng/ml each]) and treated with TGFβ signalling pathway inhibitors [SD208 and (5Z)-7-Oxozeaenol (OXO) 1 μM, SIS3 2 μM] (N=4-7, one-way ANOVA with Benjamini, Krieger and Yekutieli post-hoc test).