To the best of our knowledge, this is the first study in which the direct effect of antibodies targeting SSc-specific ENAs that are anti-Cenp-B and anti-Topo-I has been evaluated on the pro-fibrotic activation of cultured human dermal fibroblasts and their subsequent differentiation into a myofibroblast phenotype in vitro. SSc-specific antibodies have been used mainly as indicators of clinical subsets of the disease [
27]. Moreover, these antibodies are as important tools for the prediction of possible organ involvement [
28]. However, very little is known about their direct pathogenic effect on different cell phenotypes in the disease [
29]. What is known in the population of SSc antibody patients is that patients with anti-Cenp-B antibodies (usually LcSSc patients) more frequently develop pulmonary arterial hypertension (PAH) and prolonged gastrointestinal transit time [
30], while SSc patients with anti-Topo-I antibodies (usually DcSSc patients) are linked with a higher probability of interstitial lung disease (ILD), renal vascular damage, renal crisis, and heart fibrosis [
31]. All these internal organ complications involve the fibroblast as the key effector cell phenotype driving the fibrotic process in SSc [
32]: therefore, there must be a direct and/or indirect link between the presence of anti-Cenp-B/anti-Topo-I antibodies and the pro-fibrotic activation of fibroblasts. In literature, there are some hypotheses on how those antibodies could indirectly mediate the fibrotic development in SSc [
33,
34]. Among these, the hypothesis that SSc-specific antibodies could trigger the fibrotic development by inducing microvascular alterations and subsequent tissue remodeling is one of the most reliable [
35]. Another important hypothesis is that SSc-specific antibodies form immune complexes (ICs) upon their interaction with soluble target antigens [
36]: it has been demonstrated that ICs containing anti-Cenp-B/anti-Topo-I antibodies induce a pro-fibrotic and pro-inflammatory phenotype in dermal fibroblasts [
37]. Particularly, scientists demonstrated that Topo-I binding to fibroblast surfaces is both necessary and sufficient for anti-Topo-I binding [
38]. Second, Topo-I/anti-Topo-I complex binding can then trigger the adhesion and activation of monocytes, thus providing a plausible model for the amplification of the fibrogenic cascade in anti-Topo-I-positive SSc patients [
39]. To some extent, this model looks very artificial since Topo-I should be an intracellular antigen (it is usually located in the nucleus) and not an extracellular one [
40]. However, subsequent in vivo studies demonstrated that Topo-I released from injured endothelial cells could bind to bystander fibroblasts thus displaying chemoattractant activity toward immature dendritic cells and human monocytes [
41]. These results represent a clear demonstration that Topo-I could display an extracellular role that can affect fibroblast physiology. Similar results were found for Cenp-B antigen [
42]: in particular, scientists demonstrated that Cenp-B released from apoptotic endothelial cells in vivo binds more specifically to the surface of human pulmonary artery smooth muscle cells (SMCs) than fibroblasts [
43]; in particular, Cenp-B binds preferentially to SMCs of the contractile type rather than to SMCs of the synthetic type [
44]. The different Cenp-B selectivity of binding to SMCs rather than to fibroblasts could explain our results showing that anti-Topo-I antibodies have a stronger effect on fibroblasts than anti-Cenp-B ones. The different target cells of Cenp-B (SMCs) and Topo-I (fibroblasts) autoantigens released by apoptotic endothelial cells could also partially explain the different complications and organ involvement between LcSSc and DcSSc [
45]. Both Cenp-B and Topo-I models assume an already established endothelial damage as a source of autoantigens binding to targeting cells and generating a specific pathogenic autoantibody (anti-Cenp-B and anti-Topo-I) response. However, these models are quite in disagreement with the timing of the disease evolution since those specific circulating autoantibodies could be detected in SSc patients before an established endothelial damage [
46]. This is also the reason why in the past the use of Topo-I inhibitors for the treatment of SSc patients resulted unsuccessful [
47]. With the results of the present study, we assume that anti-Cenp-B and anti-Topo-I antibodies could exert a direct pathogenic role, and therefore, they could be considered “functional antibodies.” In fact, an autoantibody is considered “functional” if its direct interaction with an identified target antigen leads to a molecular pathway activation or inhibition that can be replicated in an experimental setting [
48]. Therefore, the results of the present study suggest a new interpretation of the role of anti-Cenp-B and anti-Topo-I antibodies in the disease in terms of disease drivers and not only representing an epiphenomenon and/or useful diagnostic and prognostic tools. In fact, we believe that these main autoantibodies, when present in the circulation even years before the clinical involvement, need to be tackled to slow down or prevent disease development. To prove this hypothesis, 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 anti-Topo-I antibodies could act as AECA inducing cell-mediated toxicity and apoptosis in the early stages of the disease [
49]. In this scenario, the use of immunosuppressive drugs and/or the development of more specific drugs targeting anti-Cenp-B and anti-Topo-I antibodies should be recommended at an early stage of the disease to prevent future organ damage or decrease the fibrotic evolution. In this regard, we underline that in the literature, to our knowledge, there are no studies on the progress and the possible decrease of the autoantibody titer during immunosuppressive therapy. Another important finding evidenced in this work is the different responsiveness of the fibroblasts to those autoantibodies according to their differentiation stage: healthy and unaffected LcSSc fibroblasts were more prone to be activated upon stimulation with anti-Cenp-B and anti-Topo-I antibodies than affected LcSSc and DcSSc fibroblasts that resulted already differentiated into activated myofibroblasts, thus secreting the maximum level of pro-fibrotic proteins. On the other hand, healthy and unaffected LcSSc fibroblasts were also more susceptible to apoptosis than affected LcSSc and DcSSc ones, suggesting that the fate of fibroblasts depends not only on autoantibodies but on a combination of specific autoantibodies (e.g., anti-histone antibodies did not induce apoptosis) and other soluble factors: in fact what we call healthy or unaffected LcSSc fibroblasts are cells that, upon stimulation with autoantibodies, they secrete a maximum amount of pro-fibrotic proteins and then undergo apoptosis. On the other hand, what we call affected LcSSc and DcSSc fibroblasts are cells that already express high amount of pro-fibrotic proteins and, upon stimulation with autoantibodies, they keep producing collagen and other contractile proteins but with apoptosis resistance, so the self-sustained pro-fibrotic loop is established [
50]. This theory is in line with recently published literature in which it has been demonstrated that mitochondria in activated myofibroblasts, but not quiescent fibroblasts, are primed by death signals (proximity to the apoptotic threshold) which creates a requirement for tonic expression of the antiapoptotic proteins to ensure myofibroblast survival [
51]. In this irreversible loop, the inefficient removal of nuclear components of cells targeted by the autoantibodies (defective cellular “waste disposal” theory) may also lead to the release and prolonged exposure of nuclear components and thus to the generation of new autoantibodies with increased or stable titers in sera of SSc patients.