A Search for Post-translationally-modified Trichohyalin Epitopes: Potential Triggers of Autoimmunity to Hair Follicles in Alopecia Areata

Abstract Text: Alopecia Areata (AA) is a non-scarring immune mediated hair loss disorder. Induction of ectopic immunocompetence in the lower hair-follicle (HF), followed by T-cell receptor recognition of exposed HF autoantigens by CD8+ T-cells is considered to play a central role. We propose that Trichohyalin (TCHH), a major structural protein of the inner root sheath produced in the anagen HF could be a potential autoantigen. TCHH undergoes significant anagen-mediated citrullination, a post-translational modification (PTM). Hence, we evaluate here TCHH’s putative autoantigenic role in AA and identify post-translationally-modified immunoreactive TCHH epitopes.
Ultrastructural TEM immuno-gold analysis of normal human anagen scalp HFs revealed that TCHH is expressed in the most external epithelial hair-bulb adjacent to the immunocompetent vasculature indicating that TCHH epitopes are accessible to peri-follicular sentinel immune cells. We extracted TCHH from human scalp anagen HFs and mapped for the immunologically-sensitive PTM-citrullination. 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 were synthesized, along with their native forms, and subjected to immunoassays. Cell-mediated autoimmune reactivity against putative epitope peptides was assessed using PBMCs of AA patients & healthy-controls by ELISpot. Humoral autoimmune reactivity was assessed using serum samples by ELISA.
In summary, we demonstrate that earliest TCHH epitopes occurs in the most peripheral anagen hair bulb, the site of immune cell attack in AA. This site harbors immunologically-relevant citrullinated TCHH residues that could render TCHH a major autoantigen in AA.