Objective. To identify the epitopes recognized by autoantibodies targeting platelet-derived growth factor receptor a (PDGFRa) in systemic sclerosis (SSc) and develop novel assays for detection of serum antiPDGFRa autoantibodies.Methods. Epstein-Barr virus-immortalized B cells from 1 patient with SSc (designated PAM) were screened for expression of IgG binding to PDGFRa and induction of reactive oxygen species in fibroblasts. The variable regions of anti-PDGFRa IgG were cloned into an IgG expression vector to generate distinct recombinant human monoclonal autoantibodies (mAb), which were characterized by binding and functional assays. The epitopes of anti-PDGFRa recombinant human mAb were defined by molecular docking, surface plasmon resonance binding assays, screening of a conformational peptide library spanning the PDGFRa extracellular domains, and expression analyses of alanine-scanned PDGFRa mutants. Direct or competitive enzyme-linked immunosorbent assays were established to detect all serum anti-PDGFRa autoantibodies or, selectively, the agonistic ones.Results. Three types of anti-PDGFRa recombinant human mAb, with the same V H but distinct V L chains, were generated. Nonagonistic V H PAM-V k 13B8 recognized 1 linear epitope, whereas agonistic V H PAM-V l 16F4 and V H PAM-V k 16F4 recognized 2 distinct conformational epitopes. Serum anti-PDGFRa antibodies were detected in 66 of 70 patients with SSc, 63 of 130 healthy controls, 11 of 26 patients with primary Raynaud's phenomenon (RP), and 13 of 29 patients with systemic lupus erythematosus (SLE). Serum V H PAM-V k 16F4-like antibodies were found in 24 of 34 patients with SSc, but not in healthy controls, patients with primary RP, or patients with SLE. Peptides composing the V H PAM-V k 16F4 epitope inhibited PDGFRa signaling triggered by serum IgG from SSc patients.Conclusion. Agonistic anti-PDGFRa autoantibodies are enriched in SSc sera and recognize specific conformational epitopes that can be used to discriminate agonistic from nonagonistic antibodies and block PDGFRa signaling in patients with SSc.

Objective To describe a skin–SCID mouse chimeric model of systemic sclerosis (SSc; scleroderma) fibrosis based on engraftment of ex vivo–bioengineered skin using skin cells derived either from scleroderma patients or from healthy donors. Methods Three‐dimensional bioengineered skin containing human keratinocytes and fibroblasts isolated from skin biopsy specimens from healthy donors or SSc patients was generated ex vivo and then grafted onto the backs of SCID mice. The features of the skin grafts were analyzed by immunohistochemistry, and the functional profile of the graft fibroblasts was defined before and after treatment with IgG from healthy controls or SSc patients. Two procedures were used to investigate the involvement of platelet‐derived growth factor receptor (PDGFR): 1) nilotinib, a tyrosine kinase inhibitor, was administered to mice before injection of IgG from SSc patient sera (SSc IgG) into the grafts, and 2) human anti‐PDGFR monoclonal antibodies were injected into the grafts. Results Depending on the type of bioengineered skin grafted, the regenerated human skin exhibited either the typical scleroderma phenotype or the healthy human skin architecture. Treatment of animals carrying healthy donor skin grafts with SSc IgG resulted in the appearance of a bona fide scleroderma phenotype, as confirmed by increased collagen deposition and fibroblast activation markers. Results of the experiments involving administration of nilotinib or monoclonal antibodies confirmed the involvement of PDGFR. Conclusion Our results provide the first in vivo demonstration of the fibrotic properties of anti‐PDGFR agonistic antibodies. This bioengineered skin–humanized mouse model can be used to test in vivo the progression of the disease and to monitor response to antifibrotic drugs.

Lung fibrosis is a severe condition resulting from several interstial lung diseases (ILD) with different etiologies. Current therapy of ILD, especially those associated with connective tissue diseases, is rather limited and new anti-fibrotic strategies are needed. In this study, we investigated the anti-fibrotic activity in vivo of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC). Adult immunocompetent C57BL/6 mice (n. = 8 for each experimental condition) were injected intravenously with hUC-MSC (n. = 2.5 × 105) twice, 24 hours and 7 days after endotracheal injection of bleomycin. Upon sacrifice at days 8, 14, 21, collagen content, inflammatory cytokine profile, and hUC-MSC presence in explanted lung tissue were analyzed. Systemic administration of a double dose of hUC-MSC significantly reduced bleomycin-induced lung injury (inflammation and fibrosis) in mice through a selective inhibition of the IL6-IL10-TGFβ axis involving lung M2 macrophages. Only few hUC-MSC were detected from explanted lungs, suggesting a “hit and run” mechanism of action of this cellular therapy. Our data indicate that hUC-MSC possess strong in vivo anti-fibrotic activity in a mouse model resembling an immunocompetent human subject affected by inflammatory ILD, providing proof of concept for ad-hoc clinical trials.

One of the earliest events in the pathogenesis of systemic sclerosis (SSc) is microvasculature damage with intimal hyperplasia and accumulation of cells expressing PDGF receptor. Stimulatory autoantibodies targeting PDGF receptor have been detected in SSc patients and demonstrated to induce fibrosis in vivo and convert in vitro normal fibroblasts into SSc-like cells. Since there is no evidence of the role of anti-PDGF receptor autoantibodies in the pathogenesis of SSc vascular lesions, we investigated the biologic effect of agonistic anti-PDGF receptor autoantibodies from SSc patients on human pulmonary artery smooth muscle cells and the signaling pathways involved. The synthetic (proliferation, migration, and type I collagen gene α1 chain expression) and contractile (smooth muscle-myosin heavy chain and smooth muscle-calponin expression) profiles of human pulmonary artery smooth muscle cells were assessed in vitro after incubation with SSc anti-PDGF receptors stimulatory autoantibodies. The role of reactive oxygen species, NOX isoforms, and mammalian target of rapamycin (mTOR) was investigated. Human pulmonary artery smooth muscle cells acquired a synthetic phenotype characterized by higher growth rate, migratory activity, gene expression of type I collagen α1 chain, and less expression of markers characteristic of the contractile phenotype such as smooth muscle-myosin heavy chain and smooth muscle-calponin when stimulated with PDGF and autoantibodies against PDGF receptor, but not with normal IgG. This phenotypic profile is mediated by increased generation of reactive oxygen species and expression of NOX4 and mTORC1. Our data indicate that agonistic anti-PDGF receptor autoantibodies may contribute to the pathogenesis of SSc intimal hyperplasia.

Systemic sclerosis (SSc) is a systemic, immune-mediated chronic disorder characterized by small vessel alterations and progressive fibrosis of the skin and internal organs. The combination of a predisposing genetic background and triggering factors that causes a persistent activation of immune system at microvascular and tissue level is thought to be the pathogenetic driver of SSc. Endothelial alterations with subsequent myofibroblast activation, excessive extracellular matrix (ECM) deposition, and unrestrained tissue fibrosis are the pathogenetic steps responsible for the clinical manifestations of this disease, which can be highly heterogeneous according to the different entity of each pathogenic step in individual subjects. Although substantial progress has been made in the management of SSc in recent years, disease-modifying therapies are still lacking. Several molecular pathways involved in SSc pathogenesis are currently under evaluation as possible therapeutic targets in clinical trials. These include drugs targeting fibrotic and metabolic pathways (e.g., TGF-β, autotaxin/LPA, melanocortin, and mTOR), as well as molecules and cells involved in the persistent activation of the immune system (e.g., IL4/IL13, IL23, JAK/STAT, B cells, and plasma cells). In this review, we provide an overview of the most promising therapeutic targets that could improve the future clinical management of SSc.