CXCL10 is critical for the progression and maintenance of depigmentation in a mouse model of vitiligo

Abstract

Vitiligo is an autoimmune disease of the skin that results in disfiguring white spots. There are no U.S. Food and Drug Administration-approved treatments for vitiligo, and most off-label treatments yield unsatisfactory results. Vitiligo patients have increased numbers of autoreactive, melanocyte-specific CD8(+) T cells in the skin and blood, which are directly responsible for melanocyte destruction. We report that gene expression in lesional skin from vitiligo patients revealed an interferon-γ (IFN-γ)-specific signature, including the chemokine CXCL10. CXCL10 was elevated in both vitiligo patient skin and serum, and CXCR3, its receptor, was expressed on pathogenic T cells. To address the function of CXCL10 in vitiligo, we used a mouse model of disease that also exhibited an IFN-γ-specific gene signature, expression of CXCL10 in the skin, and up-regulation of CXCR3 on antigen-specific T cells. Mice that received Cxcr3(-/-) T cells developed minimal depigmentation, as did mice lacking Cxcl10 or treated with CXCL10-neutralizing antibody. CXCL9 promoted autoreactive T cell global recruitment to the skin but not effector function, whereas CXCL10 was required for effector function and localization within the skin. Surprisingly, CXCL10 neutralization in mice with established, widespread depigmentation induces reversal of disease, evidenced by repigmentation. These data identify a critical role for CXCL10 in both the progression and maintenance of vitiligo and thereby support inhibiting CXCL10 as a targeted treatment strategy.

Conflict of interest statement

Competing interests: The authors declare they have no competing interests.

Figures

Fig. 1. Vitiligo patients express IFNγ-dependent chemokines in skin and blood

(A) We analyzed the gene expression profiles within skin biopsies from vitiligo patients compared to age- and site-matched controls using quantile normalization (n=5 per group). Melanocyte-specific transcripts were significantly reduced, consistent with melanocyte destruction in vitiligo. Chemokine expression reflected a TH1 profile, and the expression of specific gene targets of either Type I or Type II IFNs revealed an IFNγ-dominant response. The expression of genes capable of T cell recruitment to the skin reflected expression of chemokines, rather than adhesion molecules. (B–D) Serum samples from vitiligo donors or healthy controls were analyzed by ELISA to determine levels of CXCL9 (B), CXCL10 (C), and CXCL11 (D). CXCL10 was significantly elevated, whereas CXCL9 and CXCL11 were not (n=16–32 donors assayed in duplicate; two-tailed Mann-Whitney U test, *p=0.0148).

Fig. 2. CXCR3 is expressed on antigen-specific cells in the blood and CD8+ T cells in the skin lesions of vitiligo patients

(A) Expression of CXCR3 on melanocyte antigen-specific pentamer+ CD8+ T cells in the blood of HLA-A2+ patients with vitiligo, pre-gated on CD8+ T cells. Flow plots representative of 5 vitiligo patients and 5 healthy controls are shown; gp100-specific pentamer (gp100), tyrosinase-specific pentamer (Tyr), and control, no pentamer (None). (B) The ratio of CXCR3+:CXCR3− antigen-specific T cells is significantly higher in vitiligo patients than in healthy controls (n=5 donors with 2 pentamers each; two-tailed unpaired t test, **p=0.0098). (C) Immunohistochemical staining of CD3, CD8, and CXCR3 in the skin of 4 patients with active vitiligo, identified from a FFPE clinical tissue bank. All showed positive staining for CD3, CD8, and CXCR3.

Fig. 3. Expression of CXCL9, CXCL10, and CXCR3 in a mouse model of vitiligo

(A) Gene expression in fresh ear skin from mice 5 weeks after vitiligo induction and controls were analyzed via microarray using quantile normalization (n=3 per group). As in human disease, mice displayed TH1-specific response in the skin, and expressed the IFNγ-dependent chemokines CXCL9, CXCL10, and CCL5. (B) CXCR3 was expressed on melanocyte-specific CD8+ T cells (GFP+ PMEL) in the blood, spleen, and skin-draining lymph nodes from mice with vitiligo, gated on CD8+ T cells (representative flow plots, n=4 mice from 3 experiments).

Fig. 4. CXCR3 expression on T cells is required for the development of vitiligo in mice

(A) Vitiligo was induced through adoptive transfer of WT or Cxcr3−/− PMELs. Hosts receiving Cxcr3−/− PMELs developed less depigmentation than those that received WT PMELs (representative images of tail depigmentation shown). (B) The degree of depigmentation was scored blindly by one observer 5 weeks after vitiligo induction. Disease scores were significantly lower in mice that received Cxcr3−/− PMELs compared to those that received WT PMELs (one way ANOVA with Tukey’s post-tests: control vs. WT p<0.0001, WT vs. Cxcr3−/− p<0.0001). (C) There was impaired accumulation of Cxcr3−/− PMELs in the ear skin 5–6 weeks after vitiligo induction (two-tailed unpaired t test p<0.0001), however there was (D) no significant difference in the skin-draining lymph nodes. ((A–D) data pooled from 3 independent experiments, n=30 mice per group). (E) WT CFP+ PMELs and WT or Cxcr3−/− GFP+ PMELs were co-transferred in equal proportions into hosts to induce vitiligo. The ratio of WT:WT or WT:Cxcr3−/− PMELs (frequency ratio) in the skin and skin-draining lymph nodes was analyzed by flow cytometry. The frequency ratio for WT:Cxcr3−/− was significantly higher than WT:WT in the skin, but equal frequency ratios were found skin-draining lymph nodes, indicating impaired skin homing of Cxcr3−/− PMELs (n=4–5 mice per group, 3 experiments; representative experiment shown; two-tailed unpaired t test, skin p=0.0303).

Fig. 5. CXCL9 and CXCL10 play non-redundant roles in vitiligo: CXCL9 induces global recruitment, while CXCL10 promotes epidermal positioning and activation status

Vitiligo was induced in WT, Cxcl9−/− or Cxcl10−/− hosts. (A) Representative pictures of tail depigmentation. (B) The degree of depigmentation was scored blindly 5–6 weeks after vitiligo induction. Cxcl10−/− mice had significantly lower disease scores than WT mice (n=27 control, 80 WT, 44 Cxcl9−/−, 59 Cxcl10−/− mice from 9 separate experiments; one-way ANOVA p<0.0001 with Tukey’s post-tests: control vs. WT p=0.0003, control vs. Cxcl9−/− p=0.0002, WT vs. Cxcl10−/− p=0.0009, Cxcl9−/− vs. Cxcl10−/− p=0.0007). PMEL accumulation in the skin was analyzed by flow cytometry of the dermis and epidermis. (C) Reduced number of PMELs in the dermis of Cxcl9−/−, but not Cxcl10−/− mice (one-way ANOVA p=0.0219 with Tukey’s post-tests: WT vs. Cxcl9−/− p=0.0304). (D) Reduced number of PMELs in the epidermis of Cxcl9−/−, and a trend toward reduced PMELs in Cxcl10−/− mice compared to WT mice (one-way ANOVA p=0.022 with Tukey’s post-tests: WT vs. Cxcl9−/− p=0.0223). ((C–D) data pooled from 2 separate experiments, n=11 WT, 11 Cxcl9−/−, and 8 Cxcl10−/− age-matched male mice). (E) The ratio of antigen-experienced (CD69+) CD44lo to CD44hi PMELs was significantly higher in the epidermis of Cxcl10-deficient mice compared to WT mice (n=9 WT, 7 Cxcl9−/−, and 7 Cxcl10−/− pooled from 2 separate experiments, zero ratios not reported; one way ANOVA with Tukey’s post-tests: WT vs. Cxcl10−/− p=0.0478). (F) However this ratio was unchanged in skin-draining lymph nodes, indicating an epidermis-specific defect.

Fig. 6. CXCL10 neutralizing antibody both prevents and reverses vitiligo

(A) Vitiligo was induced and mice were treated with either no treatment or PBS (No Tx, n=21), CXCL9 neutralizing antibody (CXCL9 Ab, n=15), or CXCL10 neutralizing antibody (CXCL10 Ab, n=15). Antibodies were administered 3x weekly beginning 2 weeks after vitiligo induction. The degree of depigmentation was scored blindly by one observer 5 weeks after vitiligo induction (after 3 weeks of treatment). CXCL10 Ab-treated mice had significantly lower disease scores than control mice (pooled from 3 independent experiments; one-way ANOVA p=0.0012, with Tukey’s post-tests: control vs. no treatment p=0.0011, no tx vs. CXCL10 Ab p=0.0273). (B–C) Mice with extensive vitiligo (>50% depigmentation on the tail) were treated with CXCL10 neutralizing antibody (CXCL10 Ab, n=7) or vehicle control (PBS, n=3) for 8 weeks. (B) Degree of repigmentation in the tails. Photographs of mouse tails before and 8 weeks after treatment were analyzed using ImageJ software to quantify the extent of repigmentation. Fold change over baseline reflects the total pigmentation following treatment divided by total pigmentation prior to treatment, therefore a score of 1.0 reflects no change. CXCL10 Ab treatment resulted in repigmentation of the tails, whereas control mice experienced disease progression (one-tailed unpaired t test, **p=0.0019). (C) Repigmentation in a mouse with vitiligo following 8 weeks of treatment with CXCL10 Ab (representative example shown).