Effects of SARA overexpression in PDGFR-β+ cells on histological changes in a mouse SSc model. Pericytes are defined by their anatomical location (38); however, their morphology and functions are diverse. To date, no specific surface markers that reflect their physiological diversity have been defined to our knowledge. The most common antigens used to identify pericytes are the platelet-derived growth factor receptor-β (PDGFR-β) together with the proteoglycan neurin glial antigen 2 (NG2), which is a coreceptor for PDGFR-β (39). We generated mice that overexpress SARA specifically in PDGFR-β+ cells (PDGFRb-Cre SARATg mice, SARATg hereafter) (40) (Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.160977DS1), then subjected them and their WT littermates (PDGFRb-Cre SARA–/– mice, SARAWT hereafter) to repeated subcutaneous injections of bleomycin, a widely used method to model SSc in mice (41–43). After 2 weeks of treatment, SARAWT mice developed a fibrotic phenotype, as detected by histological evaluation of the skin (Figure 1, A and B). In healthy SARAWT animals, as has been reported (44), male mice had significantly thicker dermal and thinner hypodermal skin layers compared with female mice (Figure 1C, SARAWT females treated with PBS in the left graph and males in the right graph). Therefore, we analyzed dermal thickness separately for male and female animals. Despite the differences, SARAWT animals treated with PBS as a vehicle showed a normal stratification of skin layers, whereas SARAWT mice treated with bleomycin showed a reduction of the adipose layer and a significant increase in dermal thickness. In contrast, the dermis of the SARATg mice was significantly less thick, and the fat layer was preserved in both males and females, even after bleomycin treatment (Figure 1C).

Effect of SARA overexpression in suppressing skin fibrosis in a mouse model of SSc. Representative images of hematoxylin-eosin (H&E) staining of the mouse skin from females (A) and males (B) subjected to PBS (left) or bleomycin (right) are shown. Dermal thickness is shown separately for female and male samples in the graphs (C). Each dot represents the value from a different mouse, and the average ± SEM for each condition is overlaid. mRNA expression for profibrotic gene Col1a1 and for activated myofibroblast marker Acta2 are shown (D). Masson’s trichrome staining and collagen protein deposition in skin are shown (E and F). Scale bar = 100 μm. SARAWT mice n = 14 (PBS treated n = 7 and bleomycin treated n = 7) versus SARATg mice n = 15 (PBS treated n = 7 and bleomycin treated n = 7). One-way ANOVA followed by Tukey’s multiple comparisons test: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Effects of SARA overexpression on profibrotic gene expression levels and collagen deposition. One of the main events in fibrogenesis is the unbalanced deposition of ECM components, which compromises tissue plasticity (45). Bleomycin-treated SARAWT mice showed a significant increase in mRNA levels of smooth muscle α actin (Acta2; α-SMA), a marker of activated myofibroblasts, and the α1 chain of type 1 collagen (Col1a1), one of the fibrotic ECM proteins (Figure 1D), compared with the SARAWT mice treated with PBS. In contrast, mRNA levels of these genes were not significantly different between SARATg mice treated with bleomycin or PBS. The increase in collagen deposition was further confirmed by Masson’s trichrome staining and by a collagen protein quantification assay on skin sections (Figure 1, E and F).

Effects of SARA overexpression on pericyte transdifferentiation toward myofibroblasts. We engineered a mouse model that expresses green fluorescent protein (GFP) in PDGFR-β+ cells (PDGFRb-Cre Z/EG) (40, 46) to trace the pericytes. In normal skin, α-SMA is expressed in vascular smooth muscle cells and pericytes specifically alongside the arterioles (38). As expected, the pericyte markers NG2 (Figure 2) and CD146 (Supplemental Figure 2) were only detected in a subpopulation of GFP+ cells that were localized along blood vessels, marked by α-SMA expression in vascular smooth muscle cells, in SARAWT mice treated with PBS (Figure 2A and Supplemental Figure 2A). In SARAWT mice treated with bleomycin, an additional population of NG2+ (or CD146+ in Supplemental Figure 2) cells coexpressed α-SMA, and these cells were different in shape and located in the dermal layer farther away from blood vessels (Figure 2A and Supplemental Figure 2A; arrowheads in Figure 2 indicate the NG2 and α-SMA double-positive cells), suggesting that these pericytes underwent phenotypic switch, expressing α-SMA as a myofibroblast marker. In contrast, in SARATg mice, NG2+ (or CD146+ in Supplemental Figure 2) cells were observed only adjacent to α-SMA+ blood vessels, and no additional α-SMA+ cells in the dermal layer were observed even after bleomycin treatment (Figure 2, A and B).

Effects of SARA on pericyte transdifferentiation. Representative images of immunofluorescence staining on skin sections for pericyte marker NG2 (purple) and myofibroblast marker α-SMA (red) are shown (A). Pericytes are also expressing GFP in our animal model. Single channels and merged images are shown in the panel. Scale bar = 20 μm. Higher magnification for merged images is shown (B). The arrowheads in the merged image indicate the NG2+α-SMA+ cells in WT bleomycin-treated samples. Scale bar = 10 μm. Representative images from 3 independent experiments are shown. Negative control images are shown in Supplemental Figure 2B. The corrected total cell fluorescence (CTCF) quantification for α-SMA signal and the percentage of double-positive area for NG2+ and α-SMA+ in dermal layer are shown in the graphs (C). One-way ANOVA followed by Tukey’s multiple comparisons test: *P < 0.05, **P < 0.01.

Quantification of fluorescence signal of the NG2 and α-SMA double-positive cells in the dermis demonstrated a significant increase in α-SMA signal, more specifically α-SMA and NG2 double-positive cells in SARAWT mice treated with bleomycin, but not in SARATg mice (see the graphs in Figure 2C). These results suggest that pericytes underwent a phenotypical switch toward myofibroblast-like cells in SARAWT mice but not in SARATg mice.

The location of the α-SMA+ cells was confirmed by visualizing blood vessels with vascular endothelial cells stained with the endothelial cell marker CD31 (Figure 3). In SARAWT mice treated with PBS, α-SMA+ cells were observed only in proximity to blood vessels, as expected, suggesting they are either healthy pericytes on arterioles or vascular muscle cells. By contrast, in SARAWT mice treated with bleomycin, α-SMA–expressing cells were also observed in the dermal area not physically associated with vascular structures marked by CD31, suggesting that those cells, presumably a subset of NG2+ pericytes according to the previous figure, underwent transdifferentiation to myofibroblasts and migrated out from the blood vessels. This change was not observed in SARATg mice treated with bleomycin (Figure 3).

Effects of SARA on α-SMA+ cell localization. Representative images of immunofluorescence staining on skin sections for blood vessel marker CD31 (purple) and α-SMA (red) are shown. Merged images are shown in the panel and higher magnification of the latter are provided. Scale bar = 20 μm and 10 μm in the higher magnification. Representative images from 3 independent experiments are shown.

During embryogenesis, the PDGFR-β promoter is also active in inflammatory cells (47). Indeed, in PDGFRb-Cre Z/EG mice, not all PDGFR-β+GFP+ cells expressed pericyte markers but did express the pan-leukocyte marker, CD45. Quite a few CD45+GFP+ cells were observed in the dermis of PDGFRb-Cre Z/EG mice treated with bleomycin as well as those treated with PBS (Figure 4, A and B). Flow cytometric analyses of the PDGFR-β+ cells isolated from healthy SARAWT or SARATg mouse skin showed that 30% of them were CD45+ cells (Figure 4C and Supplemental Figure 3). The CD45+ cell infiltration in healthy skin was expected and presumably due to inflammatory reactions caused by a subcutaneous injection, even with PBS. CD45+ cell numbers in the dermis were not significantly different between the SARAWT or SARATg treated with bleomycin or PBS; on the contrary, α-SMA+ cells were significantly increased (graphs in Figure 4D). Importantly, the CD45+ cells were negative for α-SMA (arrowheads in Figure 4, A and B, indicate the CD45+α-SMA– cells). In SARATg mice, we detected similar numbers of CD45+ cells as well, and they were not α-SMA+. Together, these results suggest that SARA overexpression in pericytes prevents their transdifferentiation toward ECM-producing myofibroblasts responsible for the fibrotic phenotype, whereas CD45+ cells do not undergo such transdifferentiation.

Inflammatory cells detected by PDGFR-β+GFP+ cells in the PDGFRb-Cre Z/EG mice during fibrogenesis. Representative images of immunofluorescence staining on skin sections for inflammatory cell marker CD45 and myofibroblast marker α-SMA are shown (A). Single channels and merged images are shown in the panel. Scale bar = 20 μm. Higher magnification for merged images is shown (B). The arrowheads in the merged image indicate the CD45+ and α-SMA– cells in the WT bleomycin-treated samples. Scale bar = 10 μm. The scatterplot of the flow cytometric analysis of the PDGFR-β+ cells isolated from healthy SARAWT or SARATg mouse skin and stained for CD45 is shown (C). Details for the flow analyses are provided in Supplemental Figure 3. The CTCF quantification for α-SMA and CD45 signal is shown in the graphs (D). One-way ANOVA followed by Tukey’s multiple comparisons test: **P < 0.01. Representative images from 3 independent experiments are shown.

Identification of pericyte subpopulations responsible for fibrogenesis and modulated by SARA overexpression. Our findings support the notion that preventing pericyte to myofibroblast transdifferentiation could be a novel therapeutic approach in SSc and that SARA inhibits the transdifferentiation. Since pericytes are heterogenous and diverse, we aimed to identify specific subpopulations of pericytes that transdifferentiate to myofibroblasts by investigating the molecular profiles of individual cells in fibrosing skin using single-cell RNA-Seq (scRNA-Seq) analysis. To include the majority, if not all, of pericytes, we used PDGFR-β as one of the most commonly expressed pericyte markers. PDGFR-β+GFP+ cells were isolated from skin of the PDGFRb-Cre Z/EG, SARATg, or SARAWT mice treated with PBS or bleomycin (Supplemental Figure 3) and subjected to single-cell transcriptome analysis. Quality controls of scRNA-Seq analyses are reported in Supplemental Figure 4.

Unsupervised uniform manifold approximation and projection (UMAP) clustering performed with Seurat package of the sequencing data revealed 25 distinct clusters (Figure 5A). The clusters were primarily divided in 2 groups, pericytes and inflammatory cells (Figure 5A). In addition, a small cluster of endothelial cells and keratinocytes were identified. Among the clusters, only a few showed a substantial change during fibrogenesis that was reverted with SARA overexpression. In more detail, cluster 10 was enriched with cells isolated from SARAWT mice treated with bleomycin while clusters 4 and 12 were depleted after bleomycin injection (circled areas in Figure 5B); with SARA overexpression these changes were abrogated. These dynamics were more evident when clusters were plotted separately for each condition (Figure 5C). The sample component of these clusters is also shown in the bar graph in Figure 5D.

scRNA-Seq analysis of PDGFR-β+ cells and subcluster distribution. PDGFR-β+ cells isolated from PDGFRb-Cre/GFP/SARATg or SARAWT mouse skin were sorted by flow cytometry and subjected to scRNA-Seq. Representative UMAP plot of the 25 different clusters revealed by Seurat analysis conducted in R Studio, with all the experimental conditions together, is shown (A). Cluster distribution in all 4 experimental conditions together, each represented by different colors in the plot (B). Representative UMAP plots for each condition are also shown (C). The clusters that changed the most by experimental conditions are circled. The sample component of these clusters in the 4 different experimental conditions is represented in the bar plot in D. Representative plots from 3 independent experiments are shown.

Consistent with our immunofluorescence evaluation, the clusters were largely divided into 2 large populations, pericytes and inflammatory cells (Figure 6A). A total of 6 clusters (clusters 4, 12, 16, 18, 21, 23; Figure 6A, circled by red line) were annotated as pericytes. A heatmap revealed that they share common markers, and those markers are not expressed by the Ptprc-expressing (coding for CD45) populations (Figure 6B). As shown in the feature plots and dot plots, some of the pericyte clusters (clusters 16, 23) expressed the canonical markers NG2 (encoded by Cspg4 gene), CD146 (encoded by Mcam gene), and nestin (encoded by Nes gene), whereas other clusters (clusters 4 and 12) in the vicinity of canonical pericyte clusters did not express these canonical markers (Figure 6C) and for this reason were annotated as “noncanonical” pericytes. The cells in noncanonical pericyte clusters 4 and 12 expressed Mfap5 (microfibril associated protein 5), which is involved in maintenance of vessel integrity (48), and Cthrc1 (collagen triple helix repeat containing 1), which is involved in vascular remodeling (49), indicating that these clusters are physiologically related to pericytes. As mentioned above, within the pericyte populations, clusters 4 and 12 diminished after bleomycin treatment in SARAWT mice, while they were preserved in SARATg mice (Figure 5C).

scRNA-Seq analysis on pericyte subclusters. Feature plot and cluster annotation following scRNA-Seq analysis, conducted with Seurat package in R Studio, is shown. Inflammatory cells and noninflammatory cells, annotated as pericytes, were detected based on the expression level of Ptprc gene, coding for the pan-leukocyte marker CD45 (A). Heatmap reporting the different gene expression profiles of the inflammatory cells and pericytes is shown (B). Feature plots and dot plots for pericyte cluster identification, based on the expression of canonical markers Cspg4 coding for NG2, Mcam encoding for CD146, Nestin, and noncanonical markers Mfap5 and Cthrc1, are shown (C). gProfiler pathway enrichment analysis based on the differentially expressed genes of the pericyte clusters 4, 12, 16, 18, and 23, and dot plot of profibrotic gene expression, Col1a1, Col1a2, Fn1, Fbn1, Ctgf, Thbs2, and Lum, are shown (D and E). Representative plots from 3 independent experiments are shown.

gProfiler analysis of differentially expressed genes in all pericyte clusters revealed enrichment in pathways involved in vasculature development and morphogenesis, further supporting the notion that they are pericytes. In addition, genes associated with ECM assembly and organization were also enriched in some of the pericyte clusters (Figure 6D). Importantly, the expression of the profibrotic markers Col1a1, Col1a2, fibronectin 1 (Fn1), fibrillin-1 (Fbn1), connective tissue growth factor (Ctgf), thrombospondin 2 (Thbs2), and lumican (Lum) was seen mostly in the clusters of pericytes that do not express canonical pericyte markers (Figure 6E), suggesting that this subset of pericytes specifically assumes a myofibroblast-like phenotype.

Seurat reclustering of the 6 clusters identified as pericytes is shown in Figure 7A. The heatmap in Figure 7B clearly shows that the profibrotic genes were mainly expressed in the noncanonical pericyte populations, even if their expression was detected in the canonical pericytes at a lower level, confirming the evidence that a fraction of pericytes is more prone to transdifferentiate to myofibroblasts during fibrogenesis.

Trajectory inference analyses on pericyte subclusters. Reclustering of pericyte clusters (clusters 4, 12, 16, 18, 21, 23 in the original analyses) is shown (A). Heatmaps of the canonical markers and profibrotic markers in the pericyte clusters are shown (B). Trajectory inference analysis of the pericyte clusters between SARAWT and SARATg upon bleomycin treatment is shown (C). Representative plots from 3 independent experiments are shown.

Trajectory analysis of the pericyte clusters only confirmed that within SARAWT mice, the pericytes had a single-direction trajectory between the noncanonical pericyte cluster 4 and canonical cluster 6 (cluster 23 in the original plot) (Figure 7C). As previously mentioned, cluster 3 (cluster 12 in original analysis) was devoid in bleomycin-treated SARAWT mice, so there was this single directionality toward the canonical pericytes and an increase of them, probably due to a need to replace physiological pericytes in the tissue. In contrast, in SARATg samples, there was a branching point in cluster 3, which was preserved, that created an opposite, but truncated, direction toward cluster 4. These results suggest that SARA overexpression regulates the dynamics of the cells, specifically in the noncanonical pericyte clusters, preventing the fibrogenic path observed in the SARAWT mice.

Differentially expressed genes in the cluster located at the branching point of trajectory, cluster 3, play a critical role in determining the cellular fate. As shown in Table 1, among the top 5 significantly downregulated genes in SARATg samples, there are Nme2 and Gas5, and among the top 5 upregulated genes with SARA overexpression, there is Cyr61. NME2 is a histidine kinase involved in TGF-β–induced activation of hepatic stellate cells, a liver pericyte prototype, and CCl4-induced liver fibrosis (50), and Gas5 is a long noncoding RNA whose high levels are associated with liver fibrosis (51). In contrast, CYR61 attenuates the TGF-β signaling–mediated fibrosis (52), and it was described as an antifibrotic mediator in SSc (53).

Top 5 downregulated and upregulated genes in cluster 3 (12 in original analysis) in SARATg compared with SARAWT mice upon bleomycin treatment

In vitro experiments were performed to verify the role of SARA in pericyte transdifferentiation. SARA downregulation by siRNA interference in cultured human pericytes (siZFYVE9) showed a substantial upregulation of profibrogenic genes such as COL1a1, ACTA2, and FN1. Moreover, we observed a modulation of the genes that were identified in our scRNA-Seq analysis, specifically in cluster 3 (cluster 12 in the original plot), where the critical branching point was observed. The antifibrotic gene CYR61 was downregulated, and the profibrotic genes NME2 and GAS5 were upregulated (Figure 8A) by SARA silencing. Contrariwise, SARA overexpression in cultured pericytes (ZFYVE9 overexpressing) attenuated upregulated expression of COL1a1, ACTA2, and FN1 as well as NME2 and GAS5 by TGF-β1. TGF-β1 treatment also slightly increased CYR61 expression. SARA overexpression also significantly downregulated expression CYR61 at baseline and after TGF-β1 treatment (Figure 8B). These findings imply that SARA prevents pericyte transition toward myofibroblast characteristics during fibrosis via modulating these molecules.

Effect of SARA downregulation and overexpression on human pericytes’ transdifferentiation. mRNA expression for profibrotic genes COL1a1, ACTA2, FN1, NME2, and GAS5 and for antifibrotic gene CYR61, after downregulation of endogenous SARA (ZFYVE9) levels by siRNA assay, is shown (A). Summary of 3 independent experiments is shown. Mann-Whitney test: *P < 0.05, **P < 0.01. mRNA expression for profibrotic genes COL1a1, ACTA2, FN1, NME2, and GAS5 and for antifibrotic gene CYR61, after overexpression of SARA (ZFYVE9) and upon 24-hour TGF-β treatment is shown (B). Summary of 3 independent experiments is shown. One-way ANOVA followed by Holm-Šídák multiple comparisons test: *P < 0.05, **P < 0.01. ZFYVE9 gene expression downregulation and overexpression are shown in the figure. scr, scrambled siRNA; PCDNA5, TGF-β1 plasmid.

Colocalization of noncanonical pericyte markers and α-SMA in fibrotic mouse skin and in SSc patient skin. To further investigate the role for the noncanonical pericyte markers identified in our scRNA-Seq analysis, we reanalyzed the mouse skin sections and performed double immunostaining for those markers and α-SMA. As shown in Figure 9, both CTHRC1 (Figure 9A) and MFAP5 (Figure 9B) were identified among the GFP+ cells in our animal model, and they both colocalized with α-SMA in SARAWT mouse skin treated with bleomycin, underlining that CTHRC1- and MFAP5-expressing cells are more prone to transdifferentiate toward myofibroblast lineage and are activated in fibrogenesis. The colocalization was not observed in healthy skin, and importantly, not in skin of the SARATg treated with bleomycin. To unravel the clinical relevance of those subsets of pericytes, we performed immunostaining for CTHRC1 and MFAP5 using SSc patient skin (Figure 10). Similar to the mouse skin, the colocalization of α-SMA with either CTHRC1 and MFAP5 was clearly observed in SSc patients but not in healthy skin tissue. In addition, scRNA-Seq of SSc patient skin demonstrated the expression of these genes in myofibroblast-like cells (J Varga and JE Gudjonsson at University of Michigan, unpublished observations).

Effect of SARA on noncanonical pericyte transdifferentiation. Representative images of immunofluorescence staining on skin sections for noncanonical pericyte markers CTHRC1 (A) and MFAP5 (B) and myofibroblast marker α-SMA are shown. The arrowheads in the merged image indicate the CTHRC1/α-SMA+ and the MFAP5/α-SMA+ cells in the WT bleomycin-treated samples. Single channels and merged images are shown in the panel. Scale bar = 10 μm. Representative images from 3 independent experiments are shown. Negative control images are shown in Supplemental Figure 2B.

Colocalization of noncanonical pericyte markers and α-SMA in SSc patient skin. Representative images of immunofluorescence staining on normal (left) and SSc patient (right) skin sections for noncanonical pericyte marker CTHRC1 (red) and MFAP5 (red) and myofibroblast marker α-SMA (purple) are shown. Single channels and merged images are shown in the panel. Scale bar = 10 μm. Representative images from 3 independent experiments are shown. Negative control images are shown in Supplemental Figure 2B.

Th2 cell and macrophage activation in fibrosis and role for SARA. The other 19 clusters among the PDGFR-β+GFP+ cell population were represented by inflammatory cells that expressed Ptprc, which encodes the pan-leukocyte marker CD45 (Figure 6A, circled by blue line). Inflammation is one of the main manifestations of all autoimmune diseases. Skin from patients with SSc shows inflammatory infiltrates consisting of macrophages, T lymphocytes, and dendritic cells as the predominant cell types (54). Immune cells in skin can also be fibrogenic in SSc (55–59). However, the contributions of each cell type and the chemokines they produce in the profibrotic microenvironment are not well defined. scRNA-Seq analysis of our mouse model skin revealed characteristics of the CD45+ cells in fibrosing skin. Specifically, cluster 10 was enriched with cells derived from SARAWT mice treated with bleomycin but depleted in skin of the SARA-overexpressing mice treated with bleomycin (Figure 5C). This cluster showed enrichment in pathways involved in immune reactions, cytokine and chemokine production, and inflammation (Figure 11A), and one of the most highly upregulated genes was resistin-like molecule α (Retnla), a downstream target of Th2 cell–produced cytokines, including IL-31 (Figure 11B). Retnla is a regulator of Th2 driven inflammation, and it also regulates M1-M2 switch of macrophages and their activation (60, 61). IL-31 is a cytokine primarily produced by activated Th2 lymphocytes. In fact, mRNA expression levels of cytokines produced by Th2 lymphocytes, Il-31 and Il-13, and the transcription factor involved in Th2 activation, Gata3, were significantly increased in SARAWT mouse skin, but not in SARA-overexpressing mouse skin, after bleomycin treatment (Figure 11C). IL-31 protein levels measured in skin tissue homogenates by ELISA were also increased in SARAWT samples, but not in SARATg samples, after bleomycin treatment (Figure 11D). We then examined Th2 cell activation in skin biopsy sections from diffuse cutaneous SSc patients and from healthy volunteers (demographics of the samples are reported in Supplemental Figure 5A). As expected, normal skin tissue did not show any signal for IL-31 or phosphorylated STAT3 (p-STAT3), a well-known downstream mediator for IL-31 (62–64). In contrast, SSc skin sections were positive for both IL-31 and p-STAT3. Variability in staining intensity was detected, reflecting the phenotypic heterogeneity of patients with SSc (Figure 12).

Th2 cell activation during fibrogenesis. gProfiler pathway enrichment analysis of cluster 10 is shown in A. Cluster 10, circled in the plot and enriched in cells in fibrotic animals, had the highest expression of Retnla, a downstream effector of IL-31 pathway, a well-known polarized Th2 cytokine (B). mRNA expression for Il-31, Il-13, and Gata3 (C) and protein expression of IL-31 (D) evaluated by quantitative PCR and ELISA, respectively, are shown. Each dot represents the expression level from a different mouse. SARAWT mice n = 15 (PBS treated n = 8 and bleomycin treated n = 7) versus SARATg mice n = 15 (PBS treated n = 8 and bleomycin treated n = 6). One-way ANOVA followed by Holm-Šídák multiple comparisons test: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Th2 cell activation in SSc patient skin. Representative images of IL-31 (A) and p-STAT3 (B) in 4 different SSc skin biopsies, compared with normal skin, are shown. The CTCF quantification for IL-31 and p-STAT3 is shown in the graph. Nuclei were detected with DAPI and the merged snapshots are shown in the panel. Scale bar = 20 μm. SSc skin patients n = 4, healthy volunteer skin n = 4. Mann-Whitney test: ***P < 0.001.

In accordance with our hypothesis, SARA was abundantly expressed in normal skin but decreased specifically in the dermis layer of SSc skin (Supplemental Figure 5B). We also evaluated published data sets (GSE9285) analyzing gene expression profiles in SSc skin by Gene Array (33) and found that SARA mRNA (ZFYVE9) levels were significantly lower in patients with SSc (Supplemental Figure 5C).

Moreover, scRNA-Seq analysis on SSc patients showed that SARA expression was relatively diffused, and it was decreased in SSc patients when compared with healthy volunteers (J Varga and JE Gudjonsson at University of Michigan, unpublished observations).

A potential crosstalk between the pericytes and immune cells in fibrogenesis. Activated lymphocytes may also regulate other immune cell behavior and pericyte transdifferentiation through the chemokines they produce in SSc (55). Indeed, our single-cell RNA-Seq data suggested a potential crosstalk between immune cells and pericytes during fibrogenesis. In SARAWT mice treated with bleomycin, Th2 lymphocytes were activated and induced macrophage polarization, as shown by Retnla expression in cluster 10. In addition, ligand-receptor analysis showed a strong interaction between cluster 10 and 12 subsets of pericytes in SARAWT mice (Figure 13A). Among the ligands produced by cluster 10, IL-6 and TGF-β1 were found to significantly drive gene expression in the receiver cluster 12. In the receiver cluster 12, genes related to morphogenesis were associated with the ligand produced by cluster 10 (Figure 13B), in addition to the receptors of IL-31, IL-6, IL-11, and TGF-β (Table 2). IL-6 is a cytokine mainly produced by macrophages (65), and together with IL-31 and IL-11, it has been shown to be involved in skin fibrosis and cutaneous wound healing (66), as well as in SSc progression (67). These data show that the chemokines produced by cluster 10 might act directly on cluster 12, inducing the molecular changes and the transdifferentiation of the subset of pericytes toward a myofibroblast phenotype, as shown by the upregulation of Col1a1 and Col1a2 gene expression in cluster 12 (Table 2). In contrast, in SARATg mice, many more interactions between clusters 10 and 12 were detected (Figure 13C). Many target genes detected in cluster 12 are encoding ECM proteins, and their expression levels were significantly decreased in SARATg mice even after bleomycin treatment (Table 3), while significant interactions with the genes associated with morphogenesis detected in SARAWT mice were not detected in SARATg mice.

Crosstalk between pericytes and immune cells. Ligand-receptor NicheNet analysis in SARAWT bleomycin versus SARAWT PBS is shown. Ligands from cluster 10 are represented on the bottom of the plot, and target genes in cluster 12 (original analysis) are represented on the top of the plot (A). gProfiler pathway analysis of the target gene expression in cluster 12 is shown (B). Circos plot of the interaction between cluster 10 (sender) and cluster 12 (receiver) based on ligand-receptor NicheNet analysis in SARATg bleomycin versus SARATg PBS is shown. Ligands from cluster 10 are represented on the bottom of the plot, and target genes in cluster 12 are represented on the top of the plot (C). Representative plots from 3 independent experiments are shown.

Expression levels for chemokine receptors and extracellular matrix–related genes in the receiver cluster in SARAWT after NicheNet analysis

Expression levels for ECM-related genes in the receiver cluster in SARATg after NicheNet analysis

The crosstalk between lymphocytes and pericytes was further evaluated in vitro. Immortalized mouse pericytes treated with IL-31 showed a significant increase in the expression of the profibrotic markers Col1a1, Acta2, and vimentin (Vim). A positive trend for Fn1 was observed after 8 hours of the treatment, and it remained substantially higher compared with the vehicle-treated cells after 24 hours of stimulation. After 48 hours, Acta2 expression level was also increased. These results were confirmed by immunofluorescence for α-SMA (Figure 14). To better investigate the involvement of SARA in regulating IL-31 profibrotic activity on pericytes, mouse pericytes were transfected with siZfyve9 to downregulate endogenous SARA expression level, and then they were treated with IL-31 for 24 hours. As shown in Figure 15A, with SARA downregulation we observed a strong induction in Col1a1, Fn1, Vim, and Acta2 expression, which was not observed with IL-31 treatment only. In contrast, when we overexpressed SARA in mouse pericytes (ZFYVE9 overexpression) and treated them with IL-31 for 24 hours, we observed that the induction of profibrotic genes was attenuated (Figure 15, B and C). These results verified that SARA regulates and prevents pericyte-myofibroblast transdifferentiation mediated by IL-31 stimulation.

Effects of IL-31 treatment on mouse pericytes. mRNA expression for profibrotic genes Col1a1, Acta2, Vim, and Fn after 8 hours, 24 hours, and 48 hours of 50 ng/mL IL-31 treatment, are shown (A). Mann-Whitney test: *P < 0.05, **P < 0.01. Representative images of immunofluorescence staining on mouse pericytes for α-SMA after 8 hours, 24 hours, and 48 hours of 50 ng/mL IL-31 stimulation are shown (B). Nuclei were detected with DAPI and the merged snapshots are shown in the panel. Scale bar = 20 μm. Representative image and summary from 3 independent experiments are shown.

Effect of SARA downregulation and overexpression on IL-31 activity in mouse pericytes’ transdifferentiation. mRNA expression for profibrotic genes Col1a1, Acta2, Vim, and Fn1, after downregulation of endogenous SARA (Zfyve9) levels by siRNA assay and after 24-hour IL-31 stimulation, is shown (A). Summary of 3 independent experiments is shown. One-way ANOVA followed by Holm-Šídák multiple comparisons test: *P < 0.05, **P < 0.01, and ***P < 0.001. mRNA expression for profibrotic genes Col1a1, Acta2, Vim, and Fn1, after overexpression of SARA (ZFYVE9) and upon 24-hour IL-31 treatment, is shown (B). Zfyve9 gene expression downregulation and ZFYVE9 overexpression are shown (C). Summary of 3 independent experiments is shown. One-way ANOVA followed by Holm-Šídák multiple comparisons test: *P < 0.05, **P < 0.01, and ***P < 0.001.

Interaction between cluster 12 noncanonical pericytes and lymphocytes was also suggested. Cells in cluster 12 strongly expressed the Lgal1 gene, a well-known inducer of lymphocyte T apoptosis (68) (Supplemental Figure 6). In SARAWT mice treated with bleomycin, the Lgal1-expressing cluster was depleted, leading to the abnormal activation of T cells. Importantly, in bleomycin-treated SARATg mouse skin, cluster 12 and Lgal1 expression were preserved, suggesting that this cluster prevents the Th2 cell expansion, the M2 macrophage polarization, and consequent fibrogenic changes.

Effects of pericyte-specific, inducible SARA overexpression on skin fibrosis. The data reported so far were generated using a PDGFRb-Cre SARATg mouse, where Cre recombinase was constitutively expressed under the control of PDGFRb promoter. As our data show, in agreement with literature (47), the PDGFRb promoter is active during embryogenesis in pericytes as well as inflammatory cells, revealing the interesting involvement of SARA in immune cell activation and in pericyte transdifferentiation. However, the Cre recombinase activity in both pericytes and inflammatory cells in the PDGFRb-Cre SARATg mice raised a possibility that antifibrotic effects we observed with the PDGFRb-Cre SARATg mice could be due to SARA overexpression in inflammatory cells in addition to pericytes. To test the specificity of the effect of SARA expression in pericytes, we repeated the experiments using an inducible Cre system (PDGFRb-CreERT2) (Supplemental Figure 7). In this mouse, Cre recombinase is expressed only after tamoxifen treatment after birth and therefore is active specifically in pericytes but not in inflammatory cells in which PDGFRb promoter is active only during embryogenesis (47).

Bleomycin-induced skin fibrosis was significantly less severe in the inducible SARA-overexpressing mice, suggesting that the protective effect of SARA was specific to pericytes, especially in females, and this was independent from the role of SARA overexpression in the inflammatory cells. In male mice, the protective effect of SARA on skin morphology was not as prominent as that observed in the constitutive Cre model, but the levels of Col1a1 and Acta2 mRNA in skin were still significantly reduced (Figure 16). These results suggest that SARA overexpression in the inflammatory cells may provide additional protection in fibrosis.

Effects of pericyte-specific inducible SARA overexpression on skin fibrosis. Representative images of H&E staining and dermal thickness measurement from female and male skin of tamoxifen-inducible Cre animal model, PDGFRb-CreERT2/GFP/SARATg or WT, subjected to PBS or bleomycin are shown. (A). Each dot represents the expression level from a different mouse. mRNA expression for profibrotic gene Col1a1 and for activated myofibroblast marker Acta2 in skin tissue evaluated by quantitative PCR is shown (B). Each dot represents the expression level from a different mouse. SARAWT animals n = 14 (PBS treated n = 7 and bleomycin treated n = 7) versus SARATg mice n = 7 (bleomycin treated n = 7). One-way ANOVA followed by Tukey’s multiple comparisons test: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.