Over the last years, the great worldwide interest in inherited EB has led to the publication of studies concerning EB clinical features and its genetic correlation, which are important for the disease classification and the prediction of organ involvement. However, no studies about serological features of patients with different subtypes of inherited EB are available. Recently, recombinant proteins have been used as antigenic targets in ELISA methods for detection of anti-skin autoantibodies. These methods have also confirmed that the sensitivity and the specificity are analogous to those of the method of reference.
In our study, we analysed for the first time in the literature the presence of circulating anti-skin autoantibodies in patients suffering from RDEB and EBS. In other autoimmune blistering skin diseases (as pemphigus and pemphigoid), it has been shown that anti-skin specific autoantibodies can take part in the formation process of the bullous lesion . Regarding EBA, the cross-reacting autoantibodies are responsible for the incorrect assembly of type VII collagen into anchoring fibrils, thus altering both the natural maintenance and turnover of fibrils or interfering with the ability of the anchoring fibrils to attach to the basement membrane at dermal level .
The pathogenic role of these autoantibodies has been demonstrated in experimental models .
As for DEB, relatively little is known about the mechanistic consequences of the mutations in relation to blistering, which has led us to look for autoantibodies directed against cutaneous structures also in these patients. Some very interesting data have emerged from our study about the prevalence of anti-skin positivity in patients with RDEB.
The relationship between a monogenic disease and autoimmunity is difficult to clarify; we tried to explain it through different hypothesis.
In RDEB patients, a possible explanation for the development of autoantibodies recognizing type VII collagen epitopes could be based on an altered immunologic recognition of "self" consequence of altered proteins synthesis due to genetic mutation.
We know that autoimmune diseases are characterized by aberrant immune responses against healthy cells and tissues, in which a given individual's genetic susceptibility may play a central role. However, the exact mechanisms underlying the development of these conditions remain for the most part unknown. In recent years, growing evidence has demonstrated that, in addition to genetics, other complementary mechanisms are involved in the pathogenesis of autoimmunity; in particular, epigenetics. Epigenetics is defined as stable and heritable patterns of gene expression that do not entail any alterations to the original DNA sequence. Epigenetic mechanisms primarily consist of DNA methylation, histone modifications and small non-coding RNA transcripts .
Another hypothesis for this autoimmune condition may relate to the "molecular mimicry", based on the assumption that a foreign antigen holds similar sequence or structure with self-antigens. Molecular mimicry has typically been characterized at antibody or T cell level. Moreover, the exposure to antigen protein derived from bacterial and viral infection can induce this phenomenon with the activation of T and B cells . RDEB patients, as a consequence of chronic wound formation, are often exposed to bacterial infection; therefore, molecular mimicry is likely to occur. However, it is possible that the initial insult to the anchoring fibrils, mediated by an antibody recognizing a single epitope, could lead to the exposure of additional epitopes and subsequent development of polyvalent antisera.
However, it has recently been shown that the reactivity versus the NC-1 and NC-2 domains of type VII collagen protein is more predictive of an inflammatory rather than an autoimmune disease. This hypothesis is supported by the presence of anti-type VII collagen autoantibodies in inflammatory diseases involving the intestine, where the presence of this protein has been demonstrated but not featured by skin blistering such as Crohn’s disease and inflammatory bowel disease. However, the correlation between the presence of those antibodies and their pathogenic significance in that illness remains elusive [33, 34]. It has recently been reported that the major determinant of autoantibody pathogenicity relies on the different IgG subclass, and within the anti-type VII collagen autoantibodies the IgG4 sub-class was the most represented. IgG4 may induce inflammation and tissue damage by activating leukocytes with a non-complement fixing pattern or just binding collagen VII in a Fc-independent fashion leading to dermal-epidermal separation . Although in our study we have not identified the subclass of IgG-autoantibodies, we observed very high levels of IgG4 among patients with RDEB; this observation might explain the dissimilar clinical evolution not only relative to skin manifestation, but also in relation to extracutaneous complication, such as organ damage.
In this study, we have also found autoantibodies against NC-16A domain of BP180 and C- and N-terminal domains of BP230. Another current model of chronic autoimmune skin diseases predicts that “dominant” self epitopes induce the inflammatory cascade and tissue damage in the target organ. Recruitment and activation of autoreactive T lymphocytes specific for “secondary” epitopes of the nominal autoantigen or non distinct self-proteins subsequently contribute to the disease course. This phenomenon is called epitope spreading (ES). There is growing evidence that inter- and intramolecular ES events are not simply an epiphenomenon but are critical in the evolution and progression of diseases . Our data confirm this hypothesis and demonstrate that anti-type VII collagen autoantibodies strictly correlate with BP180 and BP230 autoantibodies.
Our study could be limited by the lack of Direct Immunofluorescence (DIF) data on skin biopsies for detection of tissue-bound autoantibodies. We believe that this detection, which is very important to define the role of these autoantibodies, should be carried out by more specific epitope-related methods rather than by fluorescence pattern observed with DIF.
Our opinion is that the most significant finding can be seen in the correlation between antibodies titres and the skin disease activity score. However, we also think that this finding should be confirmed on a wider number of DEB patients, considering the natural history of the disease. These correlations can also be assessed by means of other clinical scores [37–39]. On the other hand, the determination of anti-skin autoantibodies by means of the ELISA method has many advantages: it can be easily introduced in the clinical routine, it is little traumatic for the patient, therefore repeatable, and quantitative.