Background: Systemic sclerosis (SSc) is a severe autoimmune disease characterized by immune dysregulation, vascular abnormalities and tissue fibrosis. Early identification and diagnosis, particularly in the preclinical phase termed VEDOSS (Very Early Diagnosis of Systemic Sclerosis), are crucial to improve outcomes through timely and individualized therapies capable of preventing organ damage. Immune dysregulation is thought to occur early in disease progression, driving inflammation and subsequent fibrosis [1, 2]. The Th17/IL-17 axis plays a pivotal role in SSc pathogenesis, shown by increased Th17 cell frequencies and elevated serum levels of IL-17A and IL-17D associated with disease activity and collagen overproduction [3]. Elevated Th17-related cytokines correlate with higher skin scores in SSc patients, and a strong link has been identified between IL-17A and the development of pulmonary arterial hypertension (PAH), a severe complication in SSc [4]. Despite these findings in established SSc, little is known about the involvement of the Th17/IL-17 axis in the preclinical VEDOSS phase. Identifying IL-17-associated immune changes during this stage could provide critical insights for early intervention and biomarker discovery.

Objectives: To better understand immune dysregulation and to identify potential biomarkers in early stages of SSc, we determined frequencies of Th17 cells and expression of proteins associated with the activation of the IL-17 pathway in VEDOSS patients in comparison to healthy controls (HC) or patients with established SSc.

Methods: Peripheral blood mononuclear cells (PBMC) were collected from 23 clinically characterized VEDOSS patients, 54 SSc patients (ACR/EULAR 2013 criteria fulfilled), and age- and sex-matched HCs, as well as serum samples from 32 VEDOSS and 56 SSc patients. Frequencies of Th17 cells (defined as: CD3+CD4+FoxP3-CD45RO+CCR4+CCR6+) were analyzed by multi-color flow cytometry and a random forest prediction algorithm was developed to classify HC and VEDOSS cohorts based on immunophenotype frequencies to evaluate the importance of Th17 cells. Disease activity was determined for all patients using the EUSTAR Activity Index (EUSTAR AI). Proteomic profiling of 92 inflammatory proteins was performed on serum samples from VEDOSS and SSc patients using the proximity extension assay (PEA) method.

Results: Th17 cell frequencies among CD3+CD4+ T cells were significantly elevated in SSc patients (median: 19.71%, p = 0.0004) and unexpectedly also in VEDOSS patients (median: 20.81%, p = 0.0034) both compared to HC (median: 11.28%), while there was no significant difference between the VEDOSS and SSc cohort. The Random Forest prediction algorithm based on immunophenotypic data for classification of HC and VEDOSS patients highlighted a proven relevance of Th17 cells in the decision process of the algorithm with an overall area under the curve (AUC) of 0.83. Further, proteomic analysis showed that IL-17A and IL-17C levels in serum were significantly higher in SSc patients compared to VEDOSS patients (p = 0.0001; p = 0.0334, respectively). Several additional inflammatory and matrix remodeling proteins, including MMP1, CCL2, CXCL10, CCL7, CCL20, and CXCL8 showed the same significant trend and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed their contribution within the IL-17 pathway downstream the cascade. In addition, MMP1, CXCL10 and CCL2 levels showed a significant positive correlation with the EUSTAR AI in VEDOSS patients (p = 0.027, r = 0.492; p = 0.028, r = 0.491; p = 0.034, r = 0.476, respectively), highlighting their association with disease activity in the early phase of disease. Interestingly, this correlation pattern differed substantially from this in patients with established SSc.

Conclusion: This study proposes that activation of the Th17/IL-17 axis is one hallmark of immune dysregulation in the early VEDOSS phase. The comparable elevation of Th17 cell frequencies in VEDOSS and SSc patients compared to HC underlines the continuum of immune activation from preclinical to established disease. In line with this, the differential secretion of IL-17A, IL-17C, and downstream inflammatory mediators such as MMP1 as major matrix remodeling promoter in VEDOSS patients reveals an evolving inflammatory landscape, where early IL-17 pathway activation may drive disease progression. In particular, the positive correlation of MMP1, CCL2 and CXCL10 with the EUSTAR AI in VEDOSS patients highlights their potential as biomarkers for early disease activity and progression. These results open up new possibilities for the use of components of the IL-17 cascade as diagnostic and therapeutic targets in the earliest stages of SSc. Future studies should further functionally explore these biomarkers to refine early diagnostic strategies and explore targeted interventions, potentially altering the course of this debilitating disease.

REFERENCES: [1] Bellando-Randone S, Matucci-Cerinic M. Very early systemic sclerosis. Best Practice & Research Clinical Rheumatology (2019). Doi: 10.1016/j.berh.2019.101428.

[2] Ross R et al. Biological hallmarks of systemic sclerosis are present in the skin and serum of patients with Very Early Diagnosis of SSc (VEDOSS). Rheumatology (2024). Doi: 10.1093/rheumatology/keae698.

[3] Yang X et al. Increased frequency of Th17 cells in systemic sclerosis is related to disease activity and collagen overproduction. Arthritis Res Ther (2014). Doi: 10.1186/ar4430.

[4] Seki N et al. Th17/IL-17A axis is critical for pulmonary arterial hypertension (PAH) in systemic sclerosis (SSc): SSc patients with high levels of serum IL-17A exhibit reduced lung functions and increased prevalence of PAH. Cytokine (2024). Doi: 10.1016/j.cyto.2024.156534.

Acknowledgements: NIL.

Disclosure of Interests: Justus Ohmes: None declared, Hanna Grasshoff Honoraria for lectures from Abbvie, Grant support from the German Research Foundation (DFG) within the EXC2167, clinical demonstrator project 1, John Grube Foundation, Deutsche Rheumaliga und Deutsche Psoriasis Bund e.V., Sara Comduehr: None declared, Reza Akbarzadeh: None declared, Antje Müller: None declared, Timo Gemoll: None declared, Gabriela Riemekasten Honoraria for lectures from Abbvia, Boehringer Ingelheim, Chugai, Novartis, Pfizer, Astra Zeneca, BMS, Galapagos, MSD, UCB Pharma, Medupdate and Forum für Medizinische Bildung, Grants from Mitsubishi, Jens Y. Humrich Honoraria for lectures from AstraZeneca, GSK, MSD Sharp & Dohme, Otsuka Pharma, Chugai Pharma and Sanofi-Aventis, Consulting fees from MSD Sharp & Dohme and AstraZeneca, Grant support from the German Research Society (DFG, EXC 2167), the European Commission (Horizon Europe 2022-2027), MSD Sharp & Dohme and Sanofi-Aventis.

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.