Phenotypic and Functional Characterisation of Human B-cell Response in Pemphigus

Pemphigus is a rare autoimmune disease involving skin and mucous membranes with an incidence estimated as 0.7 to 7 new cases per million per year, responsible for non-negligible morbidity and mortality. Pemphigus is characterized by the production of pathogenic autoantibodies directed against two desmosomal proteins involved in keratinocyte adhesion (i.e. desmoglein 1 and desmoglein 3). These antibodies are responsible for the disruption of desmosomes leading to the acantholysis phenomenon, which results in the formation of skin and mucosal blisters. Two main classical subtypes of pemphigus have been individualized, pemphigus vulgaris (PV) and pemphigus foliaceus (PF), in which pathogenic autoantibodies are directed against desmosomal transmembrane glycoproteins belonging to the cadherin family, desmoglein 3, and desmoglein 1, respectively. As for many auto-immune diseases, high doses of corticosteroids (CS) remained the mainstay of first-line treatment for long time, alone or in conjunction with immunosuppressants. However, on these regimens, frequent relapses occurs when tapering CS dosage leading to long-term treatment and its associated risk of CS side effects.

Since ten years, the improvement in the knowledge of B-cell activation and survival, as well as their direct implication in auto-immune disease such as pemphigus through pathogenic auto-antibodies production, led to the identification of new therapeutic targets. New biological therapies based on B cell depletion were developed in severe autoimmune disorders, notably in pemphigus.

Several teams demonstrated the safety and efficacy of Rituximab, a chimeric immunoglobulin gamma-1(IgG1) monoclonal antibody(mAb) targeting the cluster of differentiation antigen 20(CD20) molecule expressed by normal B-cells from pre-B cells except for plasma cells, in severe pemphigus refractory to corticosteroids as well as in first line of treatment. Across these works, some of them concurred to improve the knowledge about B-cell populations responsible for pemphigus activity. In pemphigus patients, B-cell population was shown to comprise auto-reactive B lymphocytes producing antibodies targeting desmogleins, directly responsible for disease activity, and regulatory B lymphocytes (B-reg). After rituximab treatment of pemphigus patients, clinical activity was proved to be associated with circulating auto-antibodies high titers and an increase of auto-reactive B-cells, whereas clinical remission was associated with a change in B-cell populations, as B cell repertoire changed from oligoclonal to polyclonal when reconstituting after treatment, with an increase of immatures and transitional B-cells producing IL-10 (B-reg).

The mechanisms leading to autoreactive B-cells appearance, the precise role of B-reg in immune tolerance, and the factors triggering the imbalance between pro autoimmune and regulatory immune B-cells leading to pemphigus activity remain to be discovered. As for other auto-immune diseases, first flares and relapses were described following infectious events. Thus, infectious agents might trigger unknown mechanisms participating in the immune system imbalance by the activation of previously quiescent auto reactive B-cells or effector B-cells.

Polymorphonuclear neutrophil granulocytes (PMN) are the first responders of the immune system to threats by invading microorganisms; performing essential functions of innate immunity in host defence against a broad range of pathogens, notably by phagocytosis, intracellular degradation and extracellular discharge of antimicrobial factors. Since 2004, Polynuclear Neutral Neutrophil (PNN) were shown to produce neutrophil extracellular traps (NET), structures consisting of decondensed chromatin embedded with histones, granular and cytoplasmic proteins that trap and kill microbes. NET were initially consider to arise following cell death (NETosis) but NET formation was also described without PMN death with mitochondrial DNA extrusion. NET production during infectious events is responsible for the release of cytosolic, nuclear and mitochondrial antigens in the extracellular environment; this auto-antigen exposure constituting favourable conditions for autoimmune disease such as lupus erythematosus and AntiNeutrophil Cytoplasmic Antibody (ANCA) vasculitis, of which auto-antibodies targets belong to NET components (DNA, RNA, MPO, PR3). Besides antimicrobial function, an excessive production of NET or NET insufficient clearance were found to maintain tissue inflammation and excessive tissue damages that contribute to autoinflammatory and autoimmune diseases.

In lupus, recent works demonstrated evidences that NET components are found in immune complexes responsible for tissue inflammation and that polyclonal activation of B-cell as well as memory B-cell activation could be obtain in presence of immune complexes derived from NET.

Besides lupus, other works showed evidence of NET implication in inflammatory and auto-immune states in rheumatoid arthritis and small vessel vasculitis.

The investigators hypothesize that B-cell activation by NET might not be restricted to autoimmune diseases of which antibodies target NET components, as it is the case for lupus (DNA, RNA, LL37), Rheumatoid Arthritis(RA) (citrullinated proteins) or small vessel vasculitis (PR3, MPO).

Thus, the primary aim is to assess the effects on B-cell activation and the phenotypic changes in B-cell population from pemphigus patients after stimulation by neutrophil extracellular traps.

The secondary aim is to compare the results obtained in pemphigus patients to patients with other autoimmune diseases such as lupus, rheumatoid arthritis a Gougerot-Sjögren syndrome.