120 Identification of post-translationally modified trichohyalin epitopes responsible for triggering autoimmunity in alopecia areata

Trichohyalin (TCHH) is a major structural protein of the anagen-associated hair follicle (HF) inner root sheath and is our long-standing potential autoantigen that may trigger autoinflammatory hair loss in alopecia areata (AA) in several mammalian species including humans. The precise TCHH epitopes responsible for triggering the anti-HF immune-mediated attack in AA are unknown. TCHH undergoes significant anagen-mediated citrullination. Post-translational modification (PTM) citrullination is increasingly implicated in a host of autoimmune disorders. Hence, in this study, we aim to assess TCHH’s putative role as an HF autoantigen in AA and identify post-translationally modified immunoreactive TCHH epitopes. First, ultrastructural TEM immuno-gold analysis of normal human anagen scalp HFs revealed TCHH epitopes located in the most external epithelial hair bulb adjacent to the immunocompetent vasculature, indicating accessibility of TCHH epitopes to peri-follicular sentinel immune cells. Second, TCHH protein was extracted from human anagen HFs and mapped for the immunologically-sensitive PTM citrullination, with follow-on in-silico analysis identifying potential immunoreactive epitopes. Mass-spectrometric and in-silico analyses revealed that TCHH harbors several citrullinated residues; several of which exist within sequences predicted to be immunologically relevant. A total of 6 putative antigenic peptides harboring citrullinated residues along with their native forms were synthesized and subjected to immunoassays. Cell-mediated autoreactivity against putative epitope peptides was assessed using peripheral blood mononuclear cells of AA patients & controls by ELISpot assay and humoral autoreactivity was assessed through ELISA assay using serum samples. In summary, we demonstrate that the earliest TCHH epitopes are expressed in the most peripheral region of the anagen hair bulb, the site of immune cell attack in AA and that this site harbors immunologically-relevant citrullinated residues that could render TCHH, a major autoantigen in AA.