Gsdma3 Mutation Causes Bulge Stem Cell Depletion And Alopecia Mediated By Skin Inflammation

Cicatricial (scarring) alopecias are a group of hair loss disorders resulted from immune-mediated irreversible damage to bulge stem cells in the hair follicle. In primary cicatricial alopecia (PCA), the hair follicle is the key target of an inflammatory process. Inflammation infiltrate in cicatricial alopecia is predominantly focused on the non-cycling region of the hair follicle which contains the bulge stem cells and this leads to permanent hair loss. PCA is uncommon, but often results in major disfigurement, discomfort and psychological distress, so it is clinically important. However, the pathogenesis of PCA remains unclear.In this study, we describe a new dominant alopecia mutant strain, Alopecia and Excoriation (AE), generated from ENU mutagenesis. Mutation mapping and sequencing revealed that AE mice harbored a point mutation in gasdermin A3 (Gsdma3) on mouse chromosome 11. By substitution of nucleotide 1112 (T to C) in Gsdma3 gene, the AE mutation changed tyrosine 344 of Gsdma3 protein to histidine (TAC to CAC). AE mice began to show thickened and wrinkled skin at P7 and could be differentiated from wild-type mice. When the hair follicles had grown out of the skin surface, AE mice showed rough hair coat. The onset of hair loss in mutants began on the head and around the eyelid at P22. From then on, mutant hairs became more and more sparse compared with wild-type. AE mice progressed to nearly complete hair loss in both males and females by 6 months of age. Histological analysis indicated that in the first postnatal catagen, mutant hair follicles started to show catagen delay. AE strain is a novel Gsdma3 mutant, which share similar alopecia phenotype to other previously reported Gsdma3 mutant mice.Immunofluorescence and immunohistochemistry revealed that the expression of K10 and Involucrin are both increased in AE epidermis compared with that in wild-type, whereas the expression of Lef-1, AE13 and AE15 are all reduced to a more proximal region in mutant hair follicles compared with that in controls. These results indicate that AE mice exhibit hyperplasia in the epidermis but hypoplasia in the hair follicles. Next, Oil Red 0 staining found that the differentiation of sebaceous glands has defects in AE skin. In addition, BrdU incorporation assay revealed hyperproliferation in upper region of AE hair follicles from the first postnatal catagen and TUNEL assay indicated normal apoptosis status in AE skin. These data suggest that catagen delay in AE mice may be associated with increased proliferation rather than down-regulated apoptosis.To investigate the reason for hair loss in AE mice, we examined expression levels of some differentiation markers in bulge stem cells. Surprisingly, we found the expression of stem cell markers (CD34, K15 and Sox9) and the number of LRCs in AE mice were all significantly decreased compared with wild-type mice. These results suggest that AE mice undergo gradual depletion of bulge stem cells and loss of stem cells could be an important pathological mechanism of alopecia.We further discovered that AE mice displayed severe skin inflammation. H&E staining indicated that inflammatory cell infiltrate had appeared in the dermis of AE skin by P7. Results of quantitative real-time PCR showed that the mRNA levels of TNF-αand MCP-1 were elevated in AE skin at P7. And immunofluorescence of F4/80 and CD11c revealed increasing of macrophages and dendritic cells in AE skin. Besides, low levels of bulge immune privilege markers, MHC class I and II, were detected in wild-type skin, but the levels of these markers were up-regulated around and in the bulge region of AE skin. This demonstrates that AE mice display collapse of immune privilege in bulge stem cells. The above results suggest that inflammatory attack in AE skin results in damage of bulge stem cells and eventually leads to hair loss. Consistently, after treatment with an immunosuppressive agent cyclosporine A, bulge immune privilege collapse, stem cell destruction and alopecia phenotype of AE mice were all remarkably rescued. Collectively, our data demonstrate that immune-mediated destruction of bulge stem cells plays a crucial role in the pathogenesis of alopecia in AE mice and this strain might be an interesting model for PCAs, especially for lichen planopilaris.