Misbalanced CXCL12 and CCL5 Chemotactic Signals in Vitiligo Onset and Progression  Ahmed F. Rezk, Daria Marley Kemp, Moetaz El-Domyati, Wael Hosam El-Din, Jason B. Lee, Jouni Uitto, Olga Igoucheva, Vitali Alexeev  Journal of Investigative Dermatology  Volume 137, Issue 5, Pages 1126-1134 (May 2017) DOI: 10.1016/j.jid.2016.12.028 Copyright © 2017 The Authors Terms and Conditions

Figure 1 Chemokine expression profiles in vitiligo and normal human melanocytes. (a) Representative microarrays showing expression of chemotaxis-related molecules in normal (1801c) and vitiligo (PIG3V) cells. Deregulated genes are circled in black; red and green circles represent up- and down-regulated genes, respectively. Clustergram on the right side shows differentially expressed genes in normal (N) and vitiligo (V) cells. Cell lines are shown on the top. E6E7 are immortalized cells. (b) Representative reverse transcription–PCR assessment of CXCL12 and CCL5 expression in representative normal (1801c) and vitiligo (PIG3V, HM208, Ma9308) cells. Amplified genes are indicated above the panel. (c) Representative antibody array analysis of chemokine secretion from vitiligo (red), normal light-pigmented (green), and dark-pigmented (blue) melanocytes. Images were pseudo-colored and superimposed. Differently secreted chemokines are labeled on the panel. (d) Reverse transcription–PCR and Western blot (WB) analyses of CCL5 and CXCL12 expression and secretion after exposure to thapsigargin for various times (indicated above the panel). Detected chemokines are shown to the left of the panel. (e) ELISA-based assessment of CCL5 and CXCL12 secretion from light- (1604c) and dark-pigmented (1256b) melanocytes treated with thapsigargin. Duration of exposure shown under the columns. Data are presented as a mean of three independent measurements ± standard deviation. avg, average; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; h, hours; max, maximum; min, minimum; N, normal; V, vitiligo; WB, Western blot. Journal of Investigative Dermatology 2017 137, 1126-1134DOI: (10.1016/j.jid.2016.12.028) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Analyses of vitiligo skin infiltration and chemokine expression. Top row: H&E staining of control and early and advanced vitiligo-affected skin sections showing increased infiltration of the vitiliginous skin. Other rows: indirect immunofluorescent detection of antigens on skin sections (as indicated in corresponding colors to the left of the panels). Orange arrows point to CXCL12+S100A1+ melanocytes, red arrows point to CCL5+S100A1+ melanocytes, and yellow arrows point to CCL5+ intradermal blood vessels Blue indicates DAPI nuclear staining. Scale bar = 100 μm. H&E, hematoxylin and eosin. Journal of Investigative Dermatology 2017 137, 1126-1134DOI: (10.1016/j.jid.2016.12.028) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Immunofluorescent analysis of vitiliginous skin infiltration. (a) Analysis of CXCL12+ infiltrating APCs in vitiliginous skin. Detected antigens shown to the right of the panels in corresponding colors. Magnified insert (top row) shows CXCL12+ melanocyte-like cells in the epidermis with CXCR4+ cells. White arrows point to CXCL12+ melanocytes, orange arrows point to CXCL12+CXCR4+ infiltrating cells, yellow arrows point to the representative CXCR4+ and CXCL12+ APCs, and light blue arrows point to representative CD11c+CD86+ APCs. (b) Analysis of T-cell infiltrate in vitiligo skin. Detected antigens in corresponding colors are shown on the sides of the panels. White arrowheads point to representative CCR4+ T cells, green arrows point to CCR7+CD8+ T cells, red arrows point to CCR3+CD3+ T cells, and yellow arrows point to melanocyte-interacting T cells. Magnified insert depicts cytotoxic T lymphocyte-melanocyte interaction. Blue indicates DAPI nuclear staining. Long scale bars = 100 μm; short scale bars = 10 μm. APC, antigen presenting cell. Journal of Investigative Dermatology 2017 137, 1126-1134DOI: (10.1016/j.jid.2016.12.028) Copyright © 2017 The Authors Terms and Conditions

Figure 4 CCL5- and CXCL12-mediated recruitment of leukocytes to the skin and induction of autoimmune vitiligo in mice. (a, b) Analysis of skin inflammation in mice injected with recombinant CCL5 and CXCL12 are shown on the top of the panels; infiltrating/detected leukocytes are shown on the bottom. (a) Full-thickness H&E-stained skin sections. (b) Representative photographs of H&E staining and indirect immunofluorescent detection of lymphocytes and APCs in chemokine-treated skin. Scale bar = 100 μm. (c) H&E staining and indirect immunofluorescent detection of cells in mouse skin transplanted with parental (control) and CXCL5+CXCL12+ Melan-a cells (indicated to the right of the panels). Detected antigens in corresponding colors are shown on the top of the panels. TRP2-specific antibodies were used to outline transplanted cells. Scale bar = 10 μm. (d) Representative photographs of K14-SCF mice, mouse skin, and DOPA-stained cryosections showing loss of melanocytes in the hair follicles and the skin of depigmented lesions. Scale bar = 100 μm. (e) Indirect immunofluorescent detection of the CD8+ cytotoxic T cells in control, depigmented lesional, and perilesional mouse skin. Green indicates CD8+ T cells; blue indicates DAPI nuclear staining. Scale bar = 100 μm. APC, antigen presenting cell; DOPA, dihydroxyphenylalanine; H&E, hematoxylin and eosin. Journal of Investigative Dermatology 2017 137, 1126-1134DOI: (10.1016/j.jid.2016.12.028) Copyright © 2017 The Authors Terms and Conditions