Background: Systemic sclerosis (SSc) is characterized by the unique pathogenetic triad of microvasculopathy, (auto)immune activation and fibrosis. Capillaroscopic evidence shows erythrocyte extravasation even at the earliest stages of SSc, before the development of skin fibrosis. Given that 70% of total body iron is stored in erythrocytes, this extravasation can lead to the hemorrhagic deposition of iron in SSc tissues. Iron is involved in central cellular processes including oxygen transport, while its uptake and storage are tightly regulated to avoid deficiency or excess. Labile (Fe ²⁺ ) iron is the strongest oxidant in the human body, inducing proinflammatory responses and cellular mesenchymal transition [1,2]. Moreover, fibrotic tissues in preclinical models of liver, lung, kidney, and heart fibrosis show iron excess [2], while mice with iron overload spontaneously develop lung fibrosis [3]. Notably, iron chelation prevents or even reverts lung fibrosis in bleomycin experimental models [3]. However, the possible role of tissue iron accumulation in the pathogenesis of SSc remains unknown.

Objectives: To test the hypothesis that iron accumulates in tissues of SSc patients and contributes to profibrotic cellular transformation.

Methods: We developed a magnetic resonance imaging (MRI)-based protocol to quantify the possible presence of iron in the soft tissues of fingers of SSc patients. T2* cardiac magnetic resonance (CMR) algorithms were utilized to detect iron in the heart of SSc patients and examine its association with local edema and fibrosis. Perl’s Prussian blue staining was used to detect iron in punch skin biopsies. Bulk RNA-seq data derived from SSc patients’ skin (GSE130955) were analysed to examine the enrichment of iron-related gene signatures and their association with profibrotic gene expression. A re-analysis of spatial RNA-seq data from SSc skin (GSE249279) was also performed to examine the enrichment of iron-related genes within fibrotic foci. Markers of systemic iron metabolism were measured in the serum of SSc patients and age-/ sex-matched healthy controls. Finally, healthy and SSc-derived skin fibroblasts were treated in vitro with iron or the iron chelator deferiprone and transforming growth factor beta (TGF-b), to examine their effect on proinflammatory and profibrotic cell responses.

Results: MRI of fingers showed significantly reduced T2* values in multiple areas of all examined SSc patients (n=40) consistent with soft tissue iron deposition in SSc vs none of the healthy controls (n=10, except from one finger with previous surgery due to sports injury). Repeated measurements in controls after 1 week and 1 year showed good repeatability of the method. Similarly, myocardial iron deposition, detected by decreased T2* values in at least one myocardial segment, was evident in 21/34 SSc patients with clinically suspected primary heart disease versus none of patients with acute myocarditis (n=20) and none of healthy controls (n=8). While iron deposition did not colocalize preferentially with myocardial edema or fibrosis at the segmental level, fibrotic changes were more frequent than edema within iron-positive segments. Iron deposition in the dermis of SSc patients was confirmed by immunohistochemistry. Gene Set Enrichment Analysis of bulk RNA-seq from early diffuse SSc skin biopsies revealed that the KRAS signaling pathway was the most enriched; this pathway is strongly associated with the presence of labile iron [4]. Moreover, expression of Ferritin subunits ( FTH1, FTL ), the protein acting as universal iron storage, was increased in SSc skin (1.5-fold, P<0.001) and positively correlated with the expression of profibrotic genes such as IL6 , SERPINE1 , ACTA2 (αSMA) and COL1A1 . Notably, spatial RNA-seq from SSc skin revealed co-localization of FTH1 / FTL with COL1A1 , suggesting the enrichment of iron in fibrotic foci of SSc skin. On the other hand, parameters of systemic iron metabolism, such as serum Heme and Heme Oxygenase 1, were comparable between 20 SSc patients and 20 age-/sex-matched controls. Moreover, in vitro treatment of skin fibroblasts with iron increased the expression of proinflammatory/ profibrotic genes including IL6 and SERPINE1 . Co-treatment of skin fibroblasts with iron and TGF-b led to synergistic increase in IL6 , probably through the TGF-b-induced upregulation of the iron importer TFRC . Conversely, treatment of skin fibroblasts with deferiprone reduced TGF-b-induced profibrotic responses, even in the absence of exogenous iron administration.

Conclusions: These findings suggest that hemorrhagic iron deposition is a link between microvasculopathy and progressive fibrosis in SSc. Additional studies are warranted to examine the potential pathogenic role of iron at different stages of SSc, as well as the value of iron chelators as anti-fibrotic agents.

REFERENCES: [1] Sfikakis PP, Vlachogiannis NI, Ntouros PA, et al. Microvasculopathy-Related Hemorrhagic Tissue Deposition of Iron May Contribute to Fibrosis in Systemic Sclerosis: Hypothesis-Generating Insights from the Literature and Preliminary Findings. Life. 2022;12:430. doi: 10.3390/life12030430

[2] Maus M, López-Polo V, Mateo L, et al. Iron accumulation drives fibrosis, senescence and the senescence-associated secretory phenotype. Nat Metab . 2023;5:2111–30. doi: 10.1038/s42255-023-00928-2

[3] Ali MK, Kim RY, Brown AC, et al. Critical role for iron accumulation in the pathogenesis of fibrotic lung disease. J Pathol . 2020;251:49–62. doi: 10.1002/path.5401

[4] Jiang H, Muir RK, Gonciarz RL, et al. Ferrous iron-activatable drug conjugate achieves potent MAPK blockade in KRAS-driven tumors. J Exp Med . 2022;219:e20210739. doi: 10.1084/jem.20210739

Acknowledgments: NIL.

Disclosure of Interests: None declared.