Immune Activities in Choroids of Visually Impaired Smyth Chickens With Autoimmune Vitiligo

Abstract

Vitiligo is a common dermatological disorder affecting 1-2% of the world's population. It is characterized by postnatal, autoimmune destructions of melanocytes in the skin, resulting in patches of depigmentation. Autoimmunity in vitiligo may also affect melanocytes in non-integumental tissues, including the eyes where choroidal melanocytes are the target of the autoimmune response. The Smyth line (SL) of chicken is the only animal model that spontaneously and predictably develops all clinical and biological manifestations of autoimmune vitiligo. In SL vitiligo (SLV), destruction of epidermal melanocytes in growing feathers (GFs) involves a melanocyte-specific, Th1-mediated cellular immune response. Smyth chickens may also exhibit uveitis and vision impairment. Previous studies established a strong association between SLV and vision impairment, including similar pathology in affected eyes and GFs. To determine the presence, types, and activities of choroid infiltrating mononuclear cells, we collected eyes before, near onset, and during active SLV from sighted, partially blind, and blind SL chickens. All SL chickens with vision impairment had SLV. Immunohistochemistry and quantitative reverse transcriptase-PCR analyses revealed mononuclear cell and cytokine expression profiles in the autoimmune destruction of melanocytes in choroids that are identical to those described in GF, demonstrating the systemic nature of autoimmunity against melanocytes in SLV. In addition, we observed aberrant melanogenesis in SL eyes. The immunopathogenesis in SL vision impairment resembles human vitiligo-associated ocular diseases, especially Vogt-Koyanagi-Harada syndrome and sympathetic ophthalmia. Hence, the Smyth chicken autoimmune vitiligo model provides the opportunity to expand our understanding of spontaneous autoimmune pigmentation disorders and to develop effective treatment strategies.

Keywords: Smyth chicken vitiligo model; autoimmune vitiligo; choroid melanocytes; uveitis; vitiligo-associated diseases.

FIGURE 1

Mononuclear leukocyte presence and MHC class II expression in choroids of healthy controls and Smyth line (SL) chickens with and without vitiligo and visual impairment. (A) Representative CD4 or CD8 IHC-stained cells in frozen sections (7 μm) of eyes from 12-week-old chickens. BL-NV: Normally sighted (NV) parental Brown line (BL) control without vitiligo (no-vit); SL-NV: normally sighted, vitiligo-prone Smyth line (SL) chicken without vitiligo; SL-IV: vision-impaired SL chickens [partially sighted (P) or blind], all SL-IV chickens had vitiligo. RPE (retinal pigmented epithelium) is destroyed in blind SL-IV chickens. (B) The appearance of eyes in an SL-NV chicken without vitiligo (top) and a vitiliginous, blind SL-IV chicken (bottom). (C) Presence (% area) of T cell subsets (γδ, CD4, and CD8), B cells, macrophages, and MHC class II+ cells in choroids of controls, SL-NV, and SL-IV (includes P and blind). At each age, mononuclear cells were identified by indirect immunohistochemistry (IHC) using chicken marker-specific mouse monoclonal antibodies, biotinylated horse-antimouse IgG secondary antibody, and Vekta-stain Elite ABC reagents (streptavidin and biotin-conjugated peroxidase). Size-bar = 100 μm. Data are mean ± SEM; per age group, n = 6 and 3 for control and SL-NV, respectively; SL-IV: n = 0 at 1 week, n = 1 at 4 weeks, and n = 10 at 12 weeks. (A–C) Within an age group and across sample categories (control-NV, SL-NV, and SL-IV), mean levels of individual cell types without a common letter are different (P ≤ 0.05).

FIGURE 2

Expression of cytokine and melanogenesis-related genes in eye tissue from healthy controls and Smyth line (SL) chickens with and without vitiligo and visual impairment. (A) Relative mRNA levels of interleukin-1 (IL-1), IL-6, IL-8 (CXCL8), IL-10, IL-21, and interferon-γ (IFN-γ); (B) relative mRNA levels of melanocortin 1-receptor (MC1R), melanocyte-inducing transcription factor (MITF), tyrosinase (TYR), tyrosinase-related protein-1 (TRP1), and TRP2 (dopachrome tautomerase) in eyes from 1-, 4-, and 12-week-old controls, SL with normal (NV) and impaired vision (SL-IV; partial vision and blind). Relative mRNA expression of cytokines and melanogenesis-related genes in collected eyes was determined using qPCR. Data are mean ± SEM; for each age-group, n = 6 and 3 for control-NV and SL-NV, respectively; SL-IV: n = 0 at 1 week, n = 1 at 4 weeks, and n = 10 at 12 weeks. (A,B) Within an age group and across sample categories (control-NV, SL-NV, and SL-IV), mean mRNA expression levels of individual target genes without a common letter are different (P ≤ 0.05).