Systemic Sclerosis (SSc) is a rare autoimmune connective tissue disease characterized by microvascular injury, immune activation, and progressive fibrosis of the skin and internal organs. Diffuse cutaneous SSc (dcSSc), particularly in its early phase, is associated with aggressive fibrotic evolution and high morbidity. Despite advances in immunomodulatory therapy, no treatment has proven capable of consistently halting or reversing fibrosis, highlighting the need for new mechanistic targets.

TL1A (TNF-like ligand 1A) is a cytokine of the TNF superfamily expressed by endothelial and immune cells, which interacts with death receptor 3 (DR3) to regulate immune activation, endothelial dysfunction, and tissue remodeling. Elevated TL1A levels have been observed in SSc patients and correlate with disease activity. TL1A has also been shown to promote lung fibrosis in preclinical models.

The FIBROSTOP study is a proof-of-concept, in vitro, interventional study on biological samples aimed at elucidating the immunological and fibrotic mechanisms regulated by TL1A, and at assessing whether TL1A inhibition can reverse pathogenic processes in SSc.

Ten (n=10) patients with early diffuse cutaneous SSc and ten (n=10) age- and sex-matched healthy controls will be enrolled at a single center (Fondazione Policlinico Universitario Campus Bio-Medico, Rome). Each participant will undergo a single visit (T0) involving peripheral blood sampling and skin biopsy. No follow-up visits are planned.

The study pursues three co-primary objectives: (1) evaluating the role of TL1A and its inhibition in modulating T lymphocyte activity; (2) assessing the effects of TL1A and its inhibition on endothelial cell activation and function; (3) investigating the impact of TL1A and its inhibition on fibrotic remodeling in ex vivo SSc skin cultures using single-cell RNA sequencing.

The findings may inform future targeted antifibrotic interventions in SSc and other fibrotic diseases.

BACKGROUND AND RATIONALE

Systemic sclerosis (SSc) is an immune-mediated connective tissue disease characterized by fibrosis and vasculopathy. SSc is a heterogeneous orphan disease with a broad spectrum of organ involvement and life-threatening manifestations. The hallmark feature is the presence of thickened and hardened skin (scleroderma). The disease is divided into limited cutaneous and diffuse cutaneous SSc subsets based on the extent of skin involvement. Diffuse cutaneous SSc (dcSSc) is associated with a higher risk of interstitial lung disease, renal crisis, and cardiac involvement.

The pathophysiology of SSc involves a progressive self-amplifying process starting with microvascular/endothelial damage, followed by autoimmune response, inflammation, and fibrosis. T lymphocytes, particularly CD4+ T cells with a predominant Th2 cytokine profile, play an essential role in SSc pathogenesis. Regulatory T cells (Tregs) show decreased functional ability to suppress effector T cells.

TL1A (TNF-like cytokine 1A) is a member of the TNF superfamily constitutively expressed in endothelial cells and upregulated in response to TNF-α stimulation. TL1A binds to its receptor death receptor 3 (DR3), activating downstream signaling involved in innate and adaptive immune homeostasis. Elevated TL1A levels have been detected in serum of SSc patients compared to healthy subjects and correlate with disease activity. TL1A has been demonstrated to promote lung fibrosis in bleomycin-induced mouse models. The ATHENA-SSc-ILD study is currently evaluating Tulisokibart, a TL1A inhibitor, for SSc-associated interstitial lung disease.

STUDY DESIGN

FIBROSTOP is a monocentric, non-profit, interventional proof-of-concept in vitro study on biological samples. The study is funded by an unconditional grant from MSD Italia S.r.l. and conducted at the Fondazione Policlinico Universitario Campus Bio-Medico, Rome (Principal Investigator: Prof. Roberto Giacomelli).

OBJECTIVES AND ENDPOINTS

Co-Primary Objectives:

• OBJ1: To evaluate the role of TL1A and its inhibition in modulating T lymphocyte activity in SSc. Peripheral blood T-cell subsets (CD4+, CD8+, and Tregs) will be isolated from SSc patients and controls, and cultured with TL1A ± DR3 silencing. Effects on cytokine production, cellular activation, autophagy, apoptosis, and necroptosis will be analyzed through multiplex assays, flow cytometry, and Western blot.
• OBJ2: To assess the effects of TL1A and its inhibition on endothelial cell activation and function. Human Umbilical Vein Endothelial Cells (HUVEC) cultured with SSc serum will be treated with TL1A ± DR3 silencing to evaluate adhesion molecule expression, angiogenic capacity, and cell death/autophagy pathways. Bulk RNA sequencing will profile TL1A-regulated transcriptional signatures.
• OBJ3: To investigate the impact of TL1A and its inhibition on cellular interactions and fibrotic remodeling in SSc skin. Ex vivo skin biopsies from SSc patients and controls will be cultured with TL1A ± neutralizing antibody, and single-cell RNA sequencing (scRNA-seq) will identify transcriptional reprogramming in fibroblasts, lymphocytes, and endothelial cells.

Co-Primary Endpoints:

• EP1: Changes in cytokine production (IFN-gamma, IL-1beta, IL-6, IL-10, IL-17A, IL-35, TNF-alpha, TGF-beta), cellular activation, autophagy, apoptosis, and necroptosis in T-cell subsets following TL1A stimulation and inhibition.
• EP2: Endothelial sprouting and angiogenic capacity; changes in expression of P-selectin, E-selectin, ICAM-1, VCAM-1, and Matrigel tube formation, as well as bulk RNA-seq profiles in HUVECs following TL1A stimulation and inhibition.
• EP3: Expression of fibrotic and inflammatory markers (Col-1, alphaSMA) in ex vivo SSc skin cultures following TL1A stimulation and inhibition.

STUDY POPULATION AND PROCEDURES

Ten (n=10) patients with SSc (ACR/EULAR 2013 criteria, diffuse cutaneous subset per LeRoy et al. 1988, disease onset ≤5 years from first non-Raynaud symptom) and ten (n=10) age- and sex-matched healthy controls will be enrolled consecutively over approximately 12 months.

Each participant undergoes a single study visit (T0) involving:

• Peripheral blood sampling: PBMCs will be isolated by Ficoll-Paque density gradient centrifugation; CD4+, CD8+, and Treg subsets purified using MACS separation kits and cultured under four experimental conditions (untreated, TL1A stimulation, DR3 silencing by siRNA, TL1A + DR3 silencing).
• Skin biopsy: A 3×3 mm punch biopsy from the forearm of the most affected side (SSc patients) or from residual surgical tissue/voluntary donation (controls). Ex vivo cultures will be treated with TL1A ± neutralizing antibody and analyzed by scRNA-seq.
• Endothelial cell assays: HUVECs cultured with SSc serum under five experimental conditions will be assessed for activation markers, angiogenic function, and bulk RNA sequencing.

STATISTICAL CONSIDERATIONS

The FIBROSTOP study is exploratory in nature; no formal sample size calculation is required. Statistical analyses will include Student's t-test or Mann-Whitney U test for two-group comparisons, one-way/two-way ANOVA or Kruskal-Wallis test for multiple-group comparisons, and Spearman's rank correlation for continuous variables. Transcriptomic data will be analyzed using DESeq2 (bulk RNA-seq) and Seurat (scRNA-seq) pipelines. Pathway enrichment analyses will use Reactome, Gene Ontology (GO), and KEGG databases.

DURATION

Total study duration: 24 months (12 months enrollment + 12 months data generation and analysis). Each participant takes part in a single visit only.

Inclusion Criteria:

For both SSc and healthy control populations:

• Signed written informed consent
• Age ≥18 years at the time of consent

For SSc population only:

• Diagnosis of SSc according to ACR/EULAR 2013 classification criteria
• Diffuse cutaneous SSc subtype (LeRoy et al., 1988)
• Disease onset (defined as the first non-Raynaud symptom) within 5 years prior to recruitment
• No new initiation of immunosuppressive therapy (including methotrexate, mycophenolate mofetil, cyclophosphamide, rituximab, tocilizumab, or prednisone >20 mg/day) within the 2 months preceding recruitment
• Ongoing immunosuppressive therapy must be at a stable dose for at least 1 month prior to enrollment

Exclusion Criteria:

• Coexisting active or treated skin diseases (e.g., atopic dermatitis), except for SSc
• Diagnosis of other systemic autoimmune rheumatic diseases, including overlap syndromes (e.g., SSc + dermatomyositis)
• History or current signs/symptoms of severe or uncontrolled non-rheumatic diseases
• Active malignancy or history of malignancy within the previous 5 years (except adequately treated non-melanoma skin cancer or in situ cervical carcinoma)
• Chronic or recurrent infections, including viral hepatitis B/C, HIV, or untreated tuberculosis
• Severe active infection or major surgery within 8 weeks before enrollment
• Non-serious skin infections within 8 weeks before enrollment
• Limited cutaneous SSc or SSc sine scleroderma
• SSc patients unable to undergo therapeutic stabilization due to severe organ complications
• Pregnancy or lactation
• Inability to provide informed consent

Ten patients with early diffuse cutaneous Systemic Sclerosis (dcSSc) fulfilling the 2013 ACR/EULAR classification criteria and the LeRoy subset definition, with disease onset ≤5 years from the first non-Raynaud symptom, consecutively recruited from the Scleroderma Unit of Fondazione Policlinico Universitario Campus Bio-Medico, Rome. Ten age- and sex-matched healthy controls without SSc diagnosis, donating peripheral blood and residual skin tissue from elective surgical procedures or voluntary donation.