Antibodies against specific extractable nuclear antigens (ENAs) as diagnostic and prognostic tools and inducers of a profibrotic phenotype in cultured human skin fibroblasts: are they functional?

Claudio Corallo,Daniela Franci,Maurizio Cutolo,Sara Cheleschi,Ranuccio Nuti,Stefano Soldano,Antonella Fioravanti,Nila Volpi,Nicola Giordano

doi:10.1186/s13075-019-1931-x

Claudio Corallo, Daniela Franci + Show 7 more

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https://doi.org/10.1186/s13075-019-1931-x

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Abstract

BackgroundThe importance of systemic sclerosis (SSc) autoantibodies for diagnosis has become recognized by their incorporation into the 2013 ACR/EULAR classification criteria. Clear prognostic and phenotypic associations with cutaneous subtype and internal organ involvement have been also described. However, little is known about the potential of autoantibodies to exert a direct pathogenic role in SSc. The aim of the study is to assess the pathogenic capacity of anti-DNA-topoisomerase I (anti-Topo-I) and anti-centromeric protein B (anti-Cenp-B) autoantibodies to induce pro-fibrotic markers in dermal fibroblasts.MethodsDermal fibroblasts were isolated from unaffected and affected skin samples of (n = 10) limited cutaneous SSc (LcSSc) patients, from affected skin samples of diffuse cutaneous (DcSSc) patients (n = 10) and from healthy subjects (n = 20). Fibroblasts were stimulated with anti-Topo-I, anti-Cenp-B IgGs, and control IgGs in ratios 1:100 and 1:200 for 24 h. Cells were also incubated with 10% SSc anti-Topo-I+ and anti-Cenp-B+ whole serum and with 10% control serum for 24 h. Viability was assessed by MTT test, while apoptosis was assessed by flow cytometry. Activation of pro-fibrotic genes ACTA2, COL1A1, and TAGLN was evaluated by quantitative real-time PCR (qPCR), while the respective protein levels alpha-smooth-muscle actin (α-SMA), type-I-collagen (Col-I), and transgelin (SM22) were assessed by immunocytochemistry (ICC).ResultsMTT showed that anti-Cenp-B/anti-Topo-I IgGs and anti-Cenp-B+/anti-Topo-I+ sera reduced viability (in a dilution-dependent manner for IgGs) for all the fibroblast populations. Apoptosis is induced in unaffected LcSSc and control fibroblasts, while affected LcSSc/DcSSc fibroblasts showed apoptosis resistance. Basal mRNA (ACTA2, COL1A1, and TAGLN) and protein (α-SMA, Col-1, and SM22) levels were higher in affected LcSSc/DcSSc fibroblasts compared to LcSSc unaffected and to control ones. Stimulation with anti-Cenp-B/anti-Topo-I IgGs and with anti-Cenp-B+/anti-Topo-I+ sera showed a better induction in unaffected LcSSc and control fibroblasts. However, a statistically significant increase of all pro-fibrotic markers is reported also in affected LcSSc/DcSSc fibroblasts upon stimulation with both IgGs and sera.ConclusionsThis study suggests a pathogenic role of SSc-specific autoantibodies to directly induce pro-fibrotic activation in human dermal fibroblasts. Therefore, besides the diagnostic and prognostic use of those autoantibodies, these data might further justify the importance of immunosuppressive drugs in the early stages of the autoimmune disease, including SSc.

Highlights

The importance of systemic sclerosis (SSc) autoantibodies for diagnosis has become recognized by their incorporation into the 2013 American College of Rheumatology (ACR)/European league against rheumatism (EULAR) classification criteria
The clinical phenotype of SSc varies between two main distinct subsets according to the extent of the skin involvement [3]: limited cutaneous systemic sclerosis (LcSSc) in which skin thickening is mainly restricted to the face, fingers, and forearms [4] and diffuse cutaneous systemic sclerosis (DcSSc) in which skin lesions are observed on the trunk and over the elbow and/or knee [5]
We have support from literature: it has been demonstrated that ubiquitous nuclear protein Cenp-B is the main target of anti-endothelial cell antibodies (AECA) in patients with LcSSc and that AECA from DcSSc patients bind to endothelial cell topoisomerase I, suggesting that classical autoantibodies such as anti-Cenp-B and antiTopo-I antibodies could act as AECA inducing cellmediated toxicity and apoptosis in the early stages of the disease [49]

Summary

Introduction

The importance of systemic sclerosis (SSc) autoantibodies for diagnosis has become recognized by their incorporation into the 2013 ACR/EULAR classification criteria. SSc-specific ANAs are detected in SSc patients and rarely found in other connective tissue diseases or in healthy subjects [9]. They include anti-centromere (ACAs) and anti-DNA topoisomerase I (Topo-I) antibodies mainly, and anti-RNA polymerase III (RNAP), anti-U3 ribonucleoprotein (RNP), anti-Th/To, anti-U11/U12 RNP, anti-eukaryotic initiation factor 2B (eIF2B), anti-U1 RNP, anti-PM-Scl, anti-Ku, and antiRuvBL1/2 antibodies for the minor component. The two main subsets of SSc (LcSSc and DcSSc) do not reflect only a clinical classification [3], but they are usually associated with a precise autoimmune pattern: ACAs and in particular anti-centromere B (anti-Cenp-B) antibodies are predominantly associated with LcSSc, while antiTopo-I with DcSSc [12]. Taken into account the abovementioned diagnostic and prognostic utilities of SSc-specific antibodies, the goal of the present study is to investigate whether those antibodies could have a direct pathogenic effect on in vitro cultured human fibroblasts

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