Adeno-Associated Virus Type 5 Infection via PDGFRα Is Associated With Interstitial Lung Disease in Systemic Sclerosis and Generates Composite Peptides and Epitopes Recognized by the Agonistic Immunoglobulins Present in Patients With Systemic Sclerosis.

OBJECTIVE: The etiopathogenesis of systemic sclerosis (SSc) is unknown. Platelet-derived growth factor receptors (PDGFRs) are overexpressed in patients with SSc. Because PDGFRα is targeted by the adeno-associated virus type 5 (AAV5), we investigated whether AAV5 forms a complex with PDGFRα exposing epitopes that may induce the immune responses to the virus-PDGFRα complex. METHODS: The binding of monomeric human PDGFRα to the AAV5 capsid was analyzed by in silico molecular docking, surface plasmon resonance (SPR), and genome editing of the PDGFRα locus. AAV5 was detected in SSc lungs by in situ hybridization, immunohistochemistry, confocal microscopy, and molecular analysis of bronchoalveolar lavage (BAL) fluid. Immune responses to AAV5 and PDGFRα were evaluated by SPR using SSc monoclonal anti-PDGFRα antibodies and immunoaffinity-purified anti-PDGFRα antibodies from sera of patients with SSc. RESULTS: AAV5 was detected in the BAL fluid of 41 of 66 patients with SSc with interstitial lung disease (62.1%) and in 17 of 66 controls (25.75%) (P < 0.001). In SSc lungs, AAV5 localized in type II pneumocytes and in interstitial cells. A molecular complex formed of spatially contiguous epitopes of the AAV5 capsid and of PDGFRα was identified and characterized. In silico molecular docking analysis and binding to the agonistic anti-PDGFRα antibodies identified spatially contiguous epitopes derived from PDGFRα and AAV5 that interacted with SSc agonistic antibodies to PDGFRα. These peptides were also able to bind total IgG isolated from patients with SSc, not from healthy controls. CONCLUSION: These data link AVV5 with the immune reactivity to endogenous antigens in SSc and provide a novel element in the pathogenesis of SSc.

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin.

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models continue to be an essential tool for developing new antifibrotic strategies. Here we provide an effective method to investigate in vivo antifibrotic activity of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) in attenuating bleomycin-induced lung injury. C57BL/6 mice receive a single endotracheal injection of bleomycin (1.5 U/kg body weight) followed by a double infusion of hUC-MSC (2.5 x 105) into the tail vein, 24 h and 7 days after the bleomycin administration. Upon sacrifice at days 8, 14, or 21, inflammatory and fibrotic changes, collagen content, and hUC-MSC presence in explanted lung tissue are analyzed. The injection of bleomycin into the mouse trachea allows the direct targeting of the lungs, leading to extensive pulmonary inflammation and fibrosis. The systemic administration of a double dose of hUC-MSC results in the early blunting of the bleomycin-induced lung injury. Intravenously infused hUC-MSC are transiently engrafted into the mouse lungs, where they exert their anti-inflammatory and antifibrotic activity. In conclusion, this protocol has been successfully applied for the preclinical testing of hUC-MSC in an experimental mouse model of human pulmonary fibrosis. However, this technique can be easily extended both to study the effect of different endotracheally administered substances on the pathophysiology of the lungs and to validate new anti-inflammatory and antifibrotic systemic therapies.

Mesenchymal stromal cells from human umbilical cord prevent the development of lung fibrosis in immunocompetent mice.

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-TGFß 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.

Characterization of binding and quantification of human autoantibodies to PDGFRα using a biosensor-based approach.

Systemic sclerosis (SSc) is a chronic autoimmune disease of the connective tissue. The variety and clinical relevance of autoantibodies in SSc patients have been extensively studied, eventually identifying agonistic autoantibodies targeting the platelet-derived growth factor receptor alpha (PDGFRα), and representing potential biomarkers for SSc. We used a resonant mirror biosensor to characterize the binding between surface-blocked PDGFRα and PDGFRα-specific recombinant human monoclonal autoantibodies (mAbs) produced by SSc B cells, and detect/quantify serum autoimmune IgG with binding characteristics similar to the mAbs. Kinetic data showed a conformation-specific, high-affinity interaction between PDGFRα and mAbs, with equilibrium dissociation constants in the low-to-high nanomolar range. When applied to total serum IgG, the assay discriminated between SSc patients and healthy controls, and allowed the rapid quantification of autoimmune IgG in the sera of SSc patients, with anti-PDGFRα IgG falling in the range 3.20-4.67 neq/L of SSc autoantibodies. The test was validated by comparison to direct and competitive anti-PDGFRα antibody ELISA. This biosensor assay showed higher sensibility with respect to ELISA, and other major advantages such as the specificity, rapidity, and reusability of the capturing surface, thus representing a feasible approach for the detection and quantification of high affinity, likely agonistic, SSc-specific anti-PDGFRα autoantibodies.

Corrigendum: Agonistic Anti-PDGF Receptor Autoantibodies from Patients with Systemic Sclerosis Impact Human Pulmonary Artery Smooth Muscle Cells Function In Vitro.

[This corrects the article on p. 75 in vol. 8, PMID: 28228756.].

Agonistic Anti-PDGF Receptor Autoantibodies from Patients with Systemic Sclerosis Impact Human Pulmonary Artery Smooth Muscle Cells Function In Vitro.

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.

Reduced type I collagen gene expression by skin fibroblasts of patients with systemic sclerosis after one treatment course with rituximab.

OBJECTIVES: There is evidence that B lymphocytes play a role in the pathogenesis of systemic sclerosis (scleroderma). Stimulatory autoantibodies targeting and activating normal human fibroblasts in vitro have been demonstrated in sera from scleroderma patients. Rituximab is a monoclonal antibody which selectively targets and depletes CD20+ B lymphocytes. We investigated the biological effects of rituximab in six patients affected by scleroderma with severe skin involvement. METHODS: Six patients with severe skin fibrosis, unresponsive to immunosuppressive treatment, were treated with 375 mg/m2 per week of intravenous rituximab for a total of four doses. Serum stimulatory autoantibodies to the PDGF receptor were detected. Fibroblast activation was evaluated in fibroblasts grown from skin biopsies performed at baseline and at months 3 and 6 post-treatment. The modified Rodnan's skin score, health assessment questionnaire (HAQ) and visual analogic scale (VAS) for global wellness and B lymphocyte count were performed monthly. RESULTS: A significant reduction of anti-PDGF receptor autoantibodies was observed in the serum of all patients 3 months after treatment. Fibroblasts showed a significant downregulation of type I collagen gene expression and of the intracellular signalling triggered by anti-PDGFR autoantibodies. A decrease of the skin score and an improvement of disability indexes matched with the in vitro results. A single course of rituximab reduced scleroderma fibroblast activation in vitro and the serum levels of anti-PDGFR stimulatory autoantibodies. CONCLUSIONS: These data provide further evidence of B-cell involvement in the pathogenesis of scleroderma. Targeting B cells may be a promising treatment for scleroderma patients, and controlled clinical trials are warranted.

Epitope Specificity Determines Pathogenicity and Detectability of Anti-Platelet-Derived Growth Factor Receptor α Autoantibodies in Systemic Sclerosis.

OBJECTIVE: To identify the epitopes recognized by autoantibodies targeting platelet-derived growth factor receptor α (PDGFRα) in systemic sclerosis (SSc) and develop novel assays for detection of serum anti-PDGFRα autoantibodies. METHODS: Epstein-Barr virus-immortalized B cells from 1 patient with SSc (designated PAM) were screened for expression of IgG binding to PDGFRα and induction of reactive oxygen species in fibroblasts. The variable regions of anti-PDGFRα 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-PDGFRα recombinant human mAb were defined by molecular docking, surface plasmon resonance binding assays, screening of a conformational peptide library spanning the PDGFRα extracellular domains, and expression analyses of alanine-scanned PDGFRα mutants. Direct or competitive enzyme-linked immunosorbent assays were established to detect all serum anti-PDGFRα autoantibodies or, selectively, the agonistic ones. RESULTS: Three types of anti-PDGFRα recombinant human mAb, with the same VH but distinct VL chains, were generated. Nonagonistic VH PAM-Vκ 13B8 recognized 1 linear epitope, whereas agonistic VH PAM-Vλ 16F4 and VH PAM-Vκ 16F4 recognized 2 distinct conformational epitopes. Serum anti-PDGFRα 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 VH PAM-Vκ 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 VH PAM-Vκ 16F4 epitope inhibited PDGFRα signaling triggered by serum IgG from SSc patients. CONCLUSION: Agonistic anti-PDGFRα autoantibodies are enriched in SSc sera and recognize specific conformational epitopes that can be used to discriminate agonistic from nonagonistic antibodies and block PDGFRα signaling in patients with SSc.

Role of viral infections in the etiopathogenesis of systemic sclerosis.

Systemic sclerosis or scleroderma (SSc) is a clinically heterogeneous disease of the connective tissue characterised by vascular, immune/inflammatory and fibrotic manifestations. Despite extensive investigations, the key pathogenic links between these disease hallmarks remain obscure, as well as the etiology underlying the beginning of this complex disorder. As for other diseases characterised by prominent autoimmune phenomena, the search for infectious agents responsible for immune tolerance breaks or molecular mimicry events has been a long-pursued issue. In this review, we summarise the current knowledge regarding the association of different viral infections with SSc, focusing mainly on those reports describing a mechanistic interplay between the viral agents and the pathogenesis of SSc. Moreover, we speculate on how viral infections may trigger additional pathogenic mechanisms recently proposed to contributing to SSc phenotype.

Stimulatory autoantibodies to the PDGF receptor: a link to fibrosis in scleroderma and a pathway for novel therapeutic targets.

Systemic sclerosis (scleroderma) is a complex disease characterized by excessive deposition of collagen and abnormalities of blood vessels. In addition, activation of the immune system is a central feature of scleroderma as shown by mononuclear cell infiltration of the skin, autoantibody production and release of inflammatory cytokines. The pathogenesis of the disease is poorly understood and the molecular events underlying the main clinical features are not known. The detection of agonistic autoantibodies targeting PDGF receptor in serum of patients with scleroderma may indicate a novel link between phenotypic features of the disease and a specific signalling pathway. Agonistic PDGF receptor antibodies induce in vitro the scleroderma phenotype in normal human fibroblasts and, thus, link autoimmunity to fibrosis. These findings pave the way to novel therapeutic strategies.