Figure 1.
T cell infiltration and loss of melanocytes in lesional and leading edge vitiligo skin.
(A) Hematoxylin and eosin staining of non-lesional, leading edge and depigmented vitiligo skin does not reveal any abnormality, but significant immune cell infiltrations were found in the dermal regions of leading edge vitiligo biopsies. Bar = 100 mm. (B) Immunohistochemical staining of Melan-A on non-lesional, leading edge and depigmented vitiligo skin. The staining pattern of Melan-A is consistent with the degree of depigmentation in our vitiligo biopsies. Bar = 100 mm. Bar charts to the right of (B) is cell count of Melan-A positive cells of non-lesional, depigmented lesional and leading edge skin in biopsies of vitiligo patients. Non-lesional vitiligo skin has significantly higher number of melanocytes than lesional (p<0.0027) and leading edge skin (p<0.0082). Leading edge has significantly higher numbers of Melan-A positive cells than lesional vitiligo skin (C) Immunohistochemical staining of CD3+ T cells at non-lesional, leading edge and depigmented lesional vitiligo skin. (D) Immunohistochemical staining of CD8+ T cells at non-lesional, leading edge and depigmented lesional vitiligo skin. Bar = 100 mm, applies to both C&D. T cell infiltrates are predominantly found at the dermal-epidermal junctions in the leading edge vitiligo skin. Bar charts on the right to C&D provide the numbers from quantitative cell counting of CD3+ and CD8+ T cells, respectively.
Figure 2.
Characterization of Langerhans cells, dermal dendritic cells subpopulations in matched non-lesional, leading edge and depigmented lesional skin.
Quantification of each cell population per mm of skin appears on the right side of the micrograph image panels. Representative immunohistochemical staining on non-lesional, leading edge and depigmented lesional skin of (A) CD207/Langerin: Langerhans cell marker; (B) CD11c: myeloid dermal dendritic cell marker; (C) CD1c/BDCA-1: resident dermal dendritic cell marker; (D) HLA-DR: Activated Langerhans cell and mature dermal DC marker. (E) DC-LAMP: mature DC marker. (F) Mouse IgG1, IgG2a, and IgG2b were applied at the same concentrations as the matching primaries antibodies on leading edge vitiligo skin. Bar = 100 mm, applies to 2A, B, C, D&E.
Figure 3.
NALP-1 positive Langerhans cells are found in lesional vitiligo skin.
Immunohistochemical staining of NALP-1 on vitiligo biopsies and double staining of NALP-1 with CD207 and HLA-DR on lesional skin. (A) Immunohistochemical staining shows higher numbers of NALP-1 positive inflammasomes in leading edge vitiligo skin biopsies when compared to normal skin from healthy patients. (B) Quantification of NALP-1 positive cells per mm of skin (p<0.05) (C) NALP-1 colocalized with Langerin/CD207, a Langerhans cell marker, in lesional vitiligo skin. (D) NALP-1 colocalized with HLA-DR, a marker for activated dendritic cells, in lesional vitiligo skin. In 3C&D, single-stained controls are above the merged image, white line denotes dermal-epidermal junction. Fluorescent signals of NALP-1 appear as red, whereas Langerin and HLA-DR signals appear as green. Areas of colocalization appear as orange. Bar = 100 mm, applies to both A, C&D.
Figure 4.
qPCR analysis of Th1, Th2 and Th17 cytokines in non-lesional, leading edge and lesional skin.
Gene expression levels of key cytokines were adjusted against hARP across matching non-lesional, leading edge and lesional vitiligo skin pairs. (A) IFN-g and (B) IL-4 are hallmark cytokines for Th1 and Th2 polarization, respectively. (C) IL-17A and (D) IL-1ß serve as hallmark cytokines for Th17 polarization; Two way paired t-tests are performed for each data of gene expression data between non-lesional v.s. lesional, non-lesional v.s. leading-edge vitiligo skin. *p<0.05; **, p<0.01.
Figure 5.
IL-17A and IL-17RA are found on vitiligo skin biopsies.
Immunohistochemistry and immunofluorescence staining of IL-17A and IL-17 receptor A on vitiligo skin biopsies. (A)&(B) In immunohistochemistry, IL-17A and IL-17RA showed strong staining on the upper dermis of leading edge vitiligo skin when compared to non-lesional/pigmented vitiligo skin. (C) Double immunofluorescence staining of IL-17A and IL-17RA, areas of orange shows receptor bound IL-17A molecules. 50–60% of IL-17RA positive cells are also IL-17A positive in leading edge vitiligo biopsies. In comparison, only 20–30% of IL-17RA positive cells are IL-17A positive in non-lesional and lesional skin. Bar = 100 mm, applies to 5A, B&C.
Figure 6.
Detection of IL-17A producing T cells in vitiligo skin biopsies.
Double immunofluorescent staining of IL-17A/IL-17RA with CD3 on leading edge vitiligo skin biopsies. (A) Co-staining of CD3 and IL-17A reveals IL-17A+ T cells in vitiligo skin biopsies, but overlapping signals were only found on a small number of CD3+ cells. IL-17A signals on CD3 negative cells may reflect IL-17A molecules that are receptor bound. (B) Co-staining of CD3 and IL-17RA does not show any overlapping signals across the three groups of vitiligo biopsies, which indicates that receptor bound IL-17A are not likely to colocalize with CD3+ T cells. Bar = 100 mm, applies to 6A&B.