Mucous Membrane Pemphigoid is a group of hetereogenous autoimmune subepidermal blistering multisystem disorders that affects the skin and orificeal mucous membranes including the ocular (OcMMP), oral, aero-digestive tract, anogenital and genitourinary mucosae. OcMMP occurs in about 70% of cases and is typically characterized by a chronic relapsing conjunctival inflammation associated with a progressive scarring process that is bilaterally blinding in up to 20%, if left untreated [1,2]. While the prevalence of MMP is 1:40,000 [3], the incidence of ocular involvement ranges from 0.8 to 2.5 per million population [4,5]. The disease is more aggressive when it presents in younger populations [6]; early recognition and diagnosis is essential to reduce sight-threatening complications. Positive direct and/or indirect immunofluorescence studies detailing immunoprecipitation along the basement membrane zone (BMZ) are diagnostic of MMP with the exception of ocular monosite MMP, that can have negative immunofluorescence studies in 50% and more likely to have central corneal involvement and visual impairment [[7], [8], [9]].

The ocular features are characterized by chronic autoimmune-driven conjunctival inflammation leading to progressive scarring, debilitating symptoms and blinding sequelae. Systemic immunotherapy is the mainstay and modifies inflammation but does not limit progression of fibrosis either in inflamed eyes, or in 50% of clinically quiescent eyes. Progression of fibrosis results in trichiasis, lid malposition, severe dry eye, keratinization, ocular surface failure and recurrent infections leading to irreversible blindness. Understanding the pathogenic mechanism underpinning progressive conjunctival fibrosis is essential to facilitate development of novel anti-scarring therapies for which there is a clear unmet clinical need. The pathogenesis includes loss of tolerance to conjunctival mucosal epithelial BMZ proteins resulting in the development of pathogenic autoreactive T cells that trigger antibody (IgG,IgA) production from plasma cells to the BMZ. Antibody-BMZ interaction activates the complement cascade, acute inflammation, accumulation of inflammatory effector cells (neutrophils, dendritic cells, mast cells, eosinophils, macrophages) and associated cytokines/growth factors (IL2, IL5, IL13, IFNγ,TNFα) resulting in expansion of both Th1 and Th2 cells in the chronic inflammatory-fibrotic phase of OcMMP. Autoreactive T cells may mediate an antibody-independent pathway with similar effects [10,11].

Macrophages, T cells, mast cells and eosinophils release profibrotic mediators (PDGF, IL-13, TGFβ and HSP47). In addition, dendritic cell aldehyde dehydrogenase (ALDH1A3) catalyzes retinaldehyde to retinoic acid which in turn activates conjunctival fibroblasts leading to extracellular matrix deposition (e.g. Type 1 and 3 collagen, fibronectin, thrombospondin). Fibroblasts release proinflammatory and profibrotic factors (IL-1, IL-6, VEGF) perpetuating the chronic inflammatory response [[12], [13], [14]]. Furthermore, pemphigoid fibroblasts remain profibrotic after clinical inflammation resolves, through an autocrine ALDH-mediated retinoic acid effect driving conjunctival fibrosis [11,15]. i.e. during both active inflammation and after resolution, the activated OcMMP fibroblasts continue to scar. The induction of the TGFβ1 gene, and/or activation of latent TGFβ1 seem to play an important role [10]. TGFβ driven critical changes in fibroblast metabolism in addition with pro-inflammatory cytokines and growth factors lead to an activation to a myofibroblast phenotype, and extracellular matrix production (ECM) formation and remodeling in fibrosis and NGF/p75NTR may have a role to play in early fibrosis [16,17]. Ultimately, total ocular surface failure results from extreme dry eye, often complicated by recurrent infection, and leading to surface keratinization.

More objective biomarkers are required to identify fibrosis signaling pathway and associated inflammatory cascade activity. As longitudinal conjunctival tissue biopsy is not feasible and not considered acceptable to patients, the main objective for this pilot study was to optimize a and test the feasibility of a relatively non-invasive conjunctival swab sampling technique used in combination with the Nanostring platform to phenotype the global conjunctival transcriptome comprising several hundred genes in healthy subjects and OcMMP patients. Interrogation of the differential gene expression of the conjunctival transcriptome may provide insight into new targets for anti-fibrotic therapy, and guide clinical decision-making for initiation, maintenance, and withdrawal of immunosuppressive, and when available, anti-scarring therapies.