Systemic sclerosis (SSc) is a heterogeneous autoimmune disease with inflammatory – fibrotic changes in skin and internal organs, especially lungs (1). Lung fibrosis and hypertension are the main causes of morbidity and mortality in SSc (2). Despite the diversity of fibrotic disease and initiating molecular pathways, the biochemical and cellular mechanisms that lead to the fibrosis process are stereotypical. Cell injury and death provoke immune system activation, production of cytokines and growth factors such as TGF-β and CTGF (connective tissue growth factor), and finally transdifferentiation of different cell types to myofibroblasts. As fundamental elements in fibrosis, myofibroblasts produce extracellular matrix (ECM), especially collagen type 1 and matrix metalloproteinases (MMPs). An imbalance between ECM production and degradation causes excessive deposition of ECM and fibrosis in organs (1).

Systemic sclerosis is a female predominate autoimmune disease (3, 4). Women have more robust immune responses to infections and vaccinations at the cost of increased prevalence of autoimmune diseases, but the reasons for these events are not clear. Gender-related factors such as sex-dependent genetic variations and sex hormones contribute to the immune system’s development and responses. Both parenchymal and immune system cells bear receptors for sex hormones and respond to hormonal inputs (5). Progesterone (P) is the primary hormone of the luteal phase and pregnancy, and it influences autoimmune diseases through action on the immune system and parenchymal cells. We investigated progesterone’s effect on cell apoptosis in our previous work because cell injury and death are the primary mechanisms involved in the fibrosis cascade (6).

In the following study, we evaluate progesterone’s impact on other molecular mechanisms of fibrosis in a systemic sclerosis mouse model.

Our results for the first time showed that progesterone at pregnancy-related physiological concentrations could exacerbate fibrosis in a mouse model of systemic sclerosis.

The results of our study are summarized in Table 2 , which includes some new findings concerning the bleomycin mouse model (column 1), the effect of progesterone on bleomycin-induced lung fibrosis (column 2,3), and the input of progesterone in normal lung tissues (column 4,5).

Our findings confirm other achieved information regarding the bleomycin model. In this model, bleomycin injection induced stereotypical fibrotic molecular cycle ( Figure 6 ). Bleomycin induces cellular apoptosis in lung tissue (6), inflammation in the lung and BAL (bronchoalveolar lavage) (10), increases myofibroblasts, TGF-β, and finally, collagen deposition (gene and protein expression), which promotes fibrosis in this tissue. Bleomycin increased MMP9 in lung tissue (9); however, in this method, CTGF and ET-1 did not alter in BALB/c mice ( Table 2 ). Subcutaneous administration of bleomycin (BLM) for four weeks caused pulmonary apoptosis (6) and fibrosis which was started from the pleura and subpleural area and extended to the lung parenchyma compatible with the most apoptotic areas (6). On the other hand, in this model, central areas of the lung were not affected. The same pattern of initiation and progression of fibrosis was seen in interstitial lung disease of systemic sclerosis and resembled human lung fibrosis in SSc (2, 6, 7).

In control groups, progesterone augmented gene and protein expression of collagen I in normal lung tissues, while the amount of TGF-β and myofibroblasts did not alter until the 21^st day of progesterone administration. However, with increasing progesterone administration from day 21 to day 28, due to unknown etiology, the number of myofibroblasts and TGF-β increased in normal lung tissues ( Table 2 ). This finding indicates that the repair or fibrosis process starts with increasing the days of progesterone administration. Progesterone without influencing cell injury and apoptosis (6) induced repair factors in normal lung tissues.

Progesterone in BLM injured lung tissue did not alter the cell apoptosis (4) but increased the number of myofibroblasts and TGF-β in this tissue. Progesterone in BLM injured mice decreased MMP9 in lung tissue, inflammatory cells in BAL (10), and TNF-α in serum with a simultaneous increment of serum cortisol. These progesterone effects caused extracellular matrix (ECM) expansion in mice who received bleomycin plus progesterone for 28 days ( Figure 6 ).

Myofibroblasts are activated fibroblasts with α-SMA fiber in their cytoplasm. These cells appear transiently during the wound healing process and are removed by apoptosis after the process is completed, while in pathological fibrosis, these cells persist in the fibrotic tissues (11). In accordance with previous studies, in our investigation, the number of myofibroblasts increased in parallel with fibrosis progression, and the highest expression of α-SMA was indicated in the group with the most severe lung fibrosis, namely the BLM+P28 group. Progesterone increased myofibroblasts in normal and fibrotic lung tissues. Progesterone activates and proliferates liver fibroblasts (12) but inhibits heart fibroblasts’ proliferation (13). Progesterone inhibits the proliferation of fibroblasts in the skin but causes activation and production of collagen type one and MMP1 by these cells (14). The conflicting finding concerning progesterone’s effect on fibroblasts could be due to the heterogeneity of fibroblasts, different microenvironment in tissues, or different study methods.

TGF-β is the cardinal cytokine in fibrosis development; the cooperative interaction of TGF-β with CTGF and ET-1 aggravates the fibrosis process (15). We detected the highest concentration of TGF-β in groups with the highest amount of fibrosis (BLM+P28 group). However, in this study, CTGF and ET-1 did not increase in fibrotic groups; even CTGF was decreased in the presence of progesterone in BLM+P28 group. In a study by Lasky et al. the amount of CTGF and ET-1 did not increase in bleomycin-treated BALB/c mice, which can explain the relative resistance of BALB/c mouse strain to the induction of fibrosis (16, 17). Several investigations showed that the increment of CTGF and ET-1 expression required the activation of smad1 (mothers against decapentaplegic homolog 1) and ERK1 (extracellular signal-regulated kinase 1) through TGF-β receptors I, II, and ALK1 (18–20). These signaling pathways must be further evaluated in BALB/c mice for understanding the involved mechanisms in the production of CTGF and ET-1. In a recent study by Kunzmann et al. Progesterone dose-dependently inhibited TGF-β activated Smad signaling and suppressed TGF-β1/Smad-induced upregulation of CTGF, transgelin, and plasminogen activator inhibitor-1 (PAI-1) in lung epithelial cells (21). Progesterone administration in 28 days increased TGF-β and decreased CTGF in normal and fibrotic lung tissues. Progesterone’s inhibition of Smad signaling can explain why increased TGF-β cannot induce CTGF production in progesterone received mice, but further investigation needs in this regard.

We previously demonstrated the increased gelatinases (MMP2 and MMP9) activity in bleomycin-induced systemic sclerosis, and MMP9 was the main gelatinase in the lung tissues (9). In this study, progesterone does not alter MMP9 in 21 days of administration; however, progesterone administration for 28 days decreased the amount of MMP9 in fibrotic lung tissues. Interactions between progesterone, TGF-β, and MMPs are complex and may be different in various tissues. Progesterone is declared as a repressor of MMPs (22–24); this hormone by reduction of MMP9 and dephosphorylation of focal adhesion kinase (FAK) in lung adenocarcinoma cells inhibits migration and invasion of lung cancer cells (25). In endometrial tissue, progesterone increased gene and protein expression of TGF-β1, and this cytokine in negative feedback downregulates progesterone receptors. TGF-β has different effects on MMPs; it reduces matrilysin (MMP7) but augments gelatinases (MMP2, MMP9) in endometrial tissues. Progesterone reduced TGF- β mediated increment in gelatinases; thus, declining progesterone levels may result in loss of progesterone’s inhibitory effect on TGF- β mediated up-regulation of MMPs. After the mid-luteal phase in the uterine, progesterone level and action decreased, which would cause upregulation of proteases, the permeability of vessels, entrance of the inflammatory cells into the uterine, and production of inflammatory cytokines such as TNF-α. These events cause more activation of MMPs and, finally, loss of endometrial tissue (26–29).

The mentioned scenario concerning progesterone’s action in endometrial tissue is repeated by progesterone in fibrotic lung tissues. Bleomycin injection augments TGF-β in lung tissues then this cytokine escalates gelatinases (9). Progesterone injection in fibrotic mice further raises TGF-β level but declines the amount of MMP9. On the other hand, progesterone as an anti-inflammatory agent (30) in fibrotic mice with an increment of serum cortisol and decrement of TNF-α diminishes inflammation, reducing the activation of MMPs. MMPs have a significant role in ECM homeostasis; hence, reducing their function prevented ECM and collagen degradation (31). As a result, progesterone in fibrotic lung tissues with boosting myofibroblasts, TGF-β, and remitting MMPs induces ECM deposition and fibrosis ( Figure 6 ).

Gharaee- Kermani et al. indicated the reduction of lung fibrosis in ovariectomized rats and replacement of Estradiol returned the lung collagen deposition and fibrotic changes (32). Our obtained results, incoherent with this study about the estrogen effect on lung fibrosis, demonstrate that female hormones (estrogen and progesterone) can exacerbate lung fibrosis in susceptible women. This event may occur in susceptible women during pregnancy, in which serum and tissue levels of female hormones reach their highest concentration. Clinical studies concerning the influence of pregnancy in women with systemic sclerosis emphasize that pregnancy in the early course of diffuse SSc increased the risk of developing cardiopulmonary and renal problems. Pregnancy appears to exacerbate organ involvement or adversely affects the 10-year survival of scleroderma patients (33).

It is known that progesterone is a fibrotic agent in the liver (34). Progesterone activates rat hepatic stellate cells (the primary cell type involved in liver fibrosis), and in a dose-dependent manner, enhances ROS generation and TGF-β1 expression by these cells while acting oppositely to the favorable effects of Estradiol (12). In heart tissue, 17β-estradiol, its metabolites, and progesterone inhibit rat cardiac fibroblasts’ growth in a gender-independent fashion (13). Progesterone, with producing repair factors such as TGF-β and amphiregulin in the lungs, reduces alveolar penetration of proteins and decreases influenza infection mortality (35). Also, this hormone with an anti-inflammatory effect reduces mortality in COVID – 19 infections (36). Progesterone repairs myelin fibers in the central nervous system and inhibits the progression of multiple sclerosis (37). In a simple review of these experiments, progesterone increases tissues’ abilities for healing through anti-inflammatory action or repairing process. However, the discrepancy between various effects of progesterone can be due to the tissue pathology. Progesterone reduces mortality in severe infections by accelerating the repair process, prepares uterine cavity for embryonic implantation, increases fibrosis in the fibrotic liver and lungs, and is a beneficial factor in heart and brain ischemia.

This study cannot explain why progesterone has a dual effect in fibrotic and normal lung tissue in the expression of collagen mRNA. It is unknown whether progesterone in negative feedback control mechanisms, such as downregulation of progesterone receptors and affecting collagen mRNA expression, regulates collagen content in tissues. Also, it is recommended to analyze the fibrotic factors and progesterone receptors in time courses to understand progesterone action mechanisms better.

In conclusion, our study showed progesterone aggravates fibrosis in bleomycin-induced systemic sclerosis.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by Animal Care Committee from Iran University of Medical Sciences.

FV, AH, SP and NM carried out the experiments, FV, KM, and NM interpreted the data and wrote the manuscript, and FV, KM, HP, and NM conceived and designed the study. All authors contributed to the article and approved the submitted version.

This study was supported financially by Iran University of Medical Sciences under the grant No. (93–03–30–25094).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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