Intranasal sensitization model of alopecia areata using pertussis toxin as adjuvant

Abstract

Background: Nasopharyngeal Bordetella pertussis (BP) colonization is common, with about 5% of individuals having PCR evidence of subclinical BP infection on nasal swab, even in countries with high vaccination rates. BP secretes pertussis toxin (PTx). PTx is an adjuvant commonly used to induce autoimmunity in multiple animal models of human disease. Colocalization of PTx and myelin from myelinated nerves in the nasopharynx may lead to host sensitization to myelin with subsequent autoimmune pathology.

Methods: C57BL/6J female adult mice were given varied doses and schedules of intranasal PTx, MOG_35-55 antigen, or controls to test whether intranasal administration of PTx and myelin oligodendrocyte peptide (MOG_35-55) could induce experimental autoimmune encephalomyelitis (EAE) in mice. While we observed systemic cell-mediated immunity against MOG_35-55, we did not observe EAE. Unexpectedly, many mice developed alopecia. We systematically investigated this finding.

Results: Patchy alopecia developed in 36.4% of mice with the optimized protocol. Pathology consistent with alopecia areata was confirmed histologically by documenting concomitant reduced anagen phase and increased telogen phase hair follicles (HFs) in biopsies from patches of hair loss in mice with alopecia. We also found reduced CD200 staining and increased CD3⁺T cells surrounding the HFs of mice with alopecia compared to the mice without alopecia, indicating HF Immune Privilege (HFIP) collapse. Systemic immune responses were also found, with increased proportions of activated T cells and B cells, as well as MHCII⁺ dendritic cells in peripheral blood and/or splenocytes. Finally, in mice initially exposed to intranasal MOG_35-55 and PTx in combination, but not to either agent alone, splenocytes were shown to proliferate after in vitro stimulation by MOG_35-55. Consistent with prior investigations, PTx exhibited a dose-response effect on immune cell induction and phenotype, with the lowest PTx dose failing to induce autoimmunity, the highest PTx dose suppressing autoimmunity, and intermediate doses optimizing autoimmunity.

Conclusions: We propose that this is the first report of an autoimmune disease in an animal model triggered by colocalization of intranasal PTx and autoantigen. This model parallels a natural exposure and potential intranasal sensitization-to-pathology paradigm and supports the plausibility that nasopharyngeal subclinical BP colonization is a cause of alopecia areata.

Keywords: Bordetella pertussis; autoantigen; autoimmunity; immunization; myelin oligodendrocyte peptide; nasopharyngeal colonization.

Figure 1

Development of autoimmune alopecia in mice after repeated intranasal administration of PTx and MOG_35-55. (A) Experimental protocol for inducing alopecia. (B) The onset of alopecia and % developing alopecia during the observation period. Mouse numbers in each group are as follows: MOG_35-55/PTx, intranasal, 5 ng × 4, n = 55; MOG_35-55/PTx, intranasal, 25 ng × 4, n = 14; MOG_35-55/PTx, intranasal, 100 ng × 4, n = 10; PTx, intranasal, 5 ng × 4, n=17; PTx, intranasal, 25 ng × 4, n = 5; MOG_35-55, intranasal, 100 μg × 4, n = 17. Each dot represents several mice with or without alopecia on the observed indicated dates. (C) Representative images are shown for mice with alopecia areata.

Figure 2

Histological evaluation of autoimmune alopecia with mouse skin biopsies. (A) Representative H&E-stained images. Images in the left panel are from mice with no alopecia, the arrows indicate anagens. Images in the right panel are from mice with alopecia; the arrows indicate telogens. 100 × magnification. (B) % of anagen and % of telogen follicles in mice with alopecia compared to those without alopecia. Significant p values indicated in the figure.

Figure 3

Skin biopsies: CD3 and CD200 expression. (A, B) Images show increased CD3⁺ T lymphocytes around the hair follicles (HFs, Red Circles) in mice with alopecia (B) compared to normal skin biopsies (A). (C-E) Images show CD200 is highly expressed hair follicle epithelium and dermis around HFs in normal mice without alopecia (C) but diminished in those with alopecia (D). (E) % of # of HFs with CD200⁺stained epithelium in mice with alopecia compared to those without alopecia. 200 × magnification. Significant p values indicated in the figure.

Figure 4

Systemic immune activation. (A) The percentage of GL7⁺CD3⁺ T cells (indicating activated T cells) among lymphocytes in the spleen and blood of mice with different intranasal exposures. (B) XTT spectrophotometric absorbance, representing cell proliferation after splenocytes were stimulated in vitro with 10 μM MOG_35-55 for 72h. Splenic lymphocytes isolated from each mouse were studied in triplicate from n = 5-15 mice per group. Only groups with significant differences are shown; a-d indicates the significance of difference with group comparisons: a, groups vs. MOG_35-55/PTx, intranasal, 25 ng × 4; b, groups vs. MOG_35-55/PTx, intranasal, 100 ng × 4; c, groups vs. MOG_35-55/PTx, intranasal, 200 ng × 2; and d, groups vs. MOG_35-55/PTx, intranasal, 400 ng × 2. (C) The percentage of MHCII-expressing cells among CD11b⁺CD11c⁺ antigen-presenting cells in the spleen and blood of mice with different intranasal treatment protocols. (D) The percentage of GL7⁺CD19⁺ B cells (indicating activated B cells) from the lymphocyte population in the spleen and blood of mice with different intranasal treatment protocols. N = 5-15 mice per group, *p < 0.05, **p < 0.01, ***p < 0.001, ****p<0.0001, and ns, no significant difference.