Skin and lung fibrosis in systemic sclerosis (SSc) is driven by myofibroblasts, alpha-smooth muscle actin expressing cells. The number of myofibroblasts in SSc skin correlates with the modified Rodnan skin score, the most widely used clinical measure of skin disease severity. Murine fibrosis models indicate that myofibroblasts can arise from a variety of different cell types, but their origin in SSc skin has remained uncertain. Utilizing single cell RNA-sequencing, we define different dermal fibroblast populations and transcriptome changes, comparing SSc to healthy dermal fibroblasts. Here, we show that SSc dermal myofibroblasts arise in two steps from an SFRP2hi/DPP4-expressing progenitor fibroblast population. In the first step, SSc fibroblasts show globally upregulated expression of transcriptome markers, such as PRSS23 and THBS1. A subset of these cells shows markers indicating that they are proliferating. Only a fraction of SFRP2hi SSc fibroblasts differentiate into myofibroblasts, as shown by expression of additional markers, SFRP4 and FNDC1. Bioinformatics analysis of the SSc fibroblast transcriptomes implicated upstream transcription factors, including FOSL2, RUNX1, STAT1, FOXP1, IRF7 and CREB3L1, as well as SMAD3, driving SSc myofibroblast differentiation.

Skin and lung fibrosis in systemic sclerosis (SSc) is driven by myofibroblasts, alpha-smooth muscle actin expressing cells that arise from a variety of cell types in murine fibrosis models. Utilizing single cell RNA-sequencing to examine the transcriptome changes, we show that SSc dermal myofibroblasts arise from an SFRP2/DPP4-expressing progenitor fibroblast population that globally upregulates expression of transcriptome markers, such as PRSS23 and THBS1. Only a fraction of SSc fibroblasts differentiate into myofibroblasts, as shown by expression of additional markers, SFRP4 and FNDC1. The myofibroblast transcriptome implicates upstream transcription factors that drive myofibroblast differentiation.

Rationale Lung aging is characterized by a progressive functional impairment. One of the main features of aging is the accumulation of senescent cells, a phenotype that has been described in chronic lung diseases such as idiopathic pulmonary fibrosis (IPF). Senescent cells produce the senescence‐associated secretory phenotype (SASP), which is highly variable. Understanding SASP components would provide a new insight into the different pathologies of age‐related diseases. It is known that SASP has an effect in the immune system, and features of immunosenescence have been described in IPF patients. In this study, we point toward immunosenescence as a possible mechanism perpetrating the accumulation of senescent cells in the IPF lung and focus on the interaction between senescent fibroblast and natural killer lymphocytes (NK). Methods Human lung fibroblasts were isolated from explanted lungs and healthy donors. Conditioned media (CM) from lung fibroblasts were used to study the effects of the SASP on healthy fibroblasts and NKs. We used the BioID technique for the identification of the secreted proteins to study the fibroblast SASP. Fresh lung tissue was analyzed by flow cytometry to determine the lung immune response and senescence status. Single cell RNAseq was performed on 3 controls and 3 IPF. Blood from IPF and controls was collected for the study of plasma cytokines and PBMCs were analyzed by flow cytometry. Results CM from IPF fibroblasts induce a pro‐fibrotic and senescent phenotype on healthy control fibroblasts. Mass spectrometry of the secreted proteins in the SASP show increased secretion of extracellular matrix proteins and growth factors. Analysis of lung homogenates shows a decrease in the number of NK cells in the lower lobe of IPF patients compared to healthy controls. Looking into the NK subpopulations, there was a decrease in circulatory/cytotoxic NK cells. Single cell RNAseq analysis of the NK cluster in IPF patients revealed the upregulation of ER stress and oxidative stress response genes and downregulation of genes involved in the immune response or immunosenescence. Analysis of the fibroblast cluster reveals a profound decrease in the lung capacity to produce chemokines to recruit the circulating/cytotoxic NK population. There were no differences in the NK proportion in peripheral blood, but there was an increase in the circulating/cytotoxic NK population. The determination of the plasma cytokines confirms a reduced capacity to produce chemokines in IPF. The effect of the lung microenvironment over the NK cells is being assesed in vitro using CM from IPF fibroblasts to determine NK migration capacity, killing activity, and senescence status. Conclusions IPF fibroblasts induce senescence and a pro‐fibrotic phenotype in vitro through their SASP. IPF patients have reduced numbers of NK cells in the lung with an impaired phenotype that appears to be controlled by the lung microenvironment through the SASP of senescence cells. This deficiency in the cytotoxic NK activity and senescence status in t...

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