Background: C3H/HeJ mouse models progress gradually in hair loss from acute to chronic phase and reflect the symptoms of patients with alopecia areata (AA). However, the underlying pathological characteristics alteration associated with disease progression and autoantigens remain unclear. Objective: We aimed at elucidating the pathological differences between acute and chronic-AA in the C3H/HeJ mouse model. Methods: We analyzed populations of PBMCs, skin-draining lymph node (SDLN) cells, and cutaneous cells of AA mice using flow cytometry. The cytokine and chemokine expressions in the serum and skin were determined using multiplex assay and qPCR. The antibody serum levels were determined using ELISA and the antigen-specific T cells were detected using the MHC class I tetramer. Results: The CD8 + NKG2D + T and CD8 + T EM cell percentage in the chronic-AA SDLNs or among the unaffected and acute-AA mice PBMCs increased. The Th1 and CD4 + T EM cell percentage in the SDLNs and among PBMCs increased in the unaffected and AA mice. The percentage of CD8 + T EM /T RM cells and MHC class I expression increased in the lesions of acute-AA or the non-lesions and lesions of chronic-AA. The Th1 cells, dendritic cell-related cytokines, CD11c + cells and MHC class II expression increased in the skin of AA mice. The antibody levels and TYRP2 and tyrosinase-specific CD8 + T cell percentages were upregulated in AA mice. Conclusion:These results suggest that the CD8 + and CD4 + T cell subpopulations, cytokine and chemokine expressions differ between the disease phases. Moreover, TYRP2 and tyrosinase are potential autoreactive targets in the AA mouse model.

Background: Alopecia areata (AA) is an autoimmune disease resulting in non-scarring hair loss. Animal models are useful means to identify candidates for therapeutic agents. The C3H/HeJ mouse AA model induced via transferring cultured lymphoid cells isolated from AA-affected mice is widely used for AA research. However, this conventional method requires the continuous breeding of AA mice. Objective: We aimed to establish a new method to generate AA model using the transfer of cryopreserved cells, which allows the rapid induction of a large number of AA mice when needed. Methods: We cryopreserved lymph node cells soon after isolation from AA-affected mice and injected thawed-cultured cells into recipient mice. H&E staining, immunohistochemical staining, quantitative real-time PCR and ELISA were conducted to identify pathological characteristics. Flow cytometry was performed to reveal the profile of transferred cells. Results: More than 90 % of recipient mice developed AA-like hair loss and showed inflammatory cell infiltration around anagen hair follicles, markedly increased mRNA expressions of interferon-g, CXCL11, and granzyme B, and elevated interferon-α protein levels in the skin compared with naïve mice. Higher percentages of effector memory T cells and dendritic cells in transferred cells resulted in a higher incidence of AA. Conclusion: This is the first report to establish a method for generating AA mice using cryopreserved lymphocytes. These AA mice have similar pathological characteristics to AA mice generated with the conventional method and AA patients. This convenient and reproducible method is expected to be valuable for AA study.

Alopecia areata (AA) is an autoimmune non‐scarring hair loss disease. Recently, several reports have suggested that innate immune systems such as interferon‐α (IFN‐α)‐producing plasmacytoid dendritic cells and NOD‐like receptor family pyrin domain‐containing protein 3 (NLRP3) inflammasomes play a role in the pathogenesis of AA. However, critical studies about their involvement in the initiation of AA have not yet been reported. Therefore, we investigated the expression of innate immune cytokines in serum and skin, and examined the effect of a selective NLRP3 inhibitor, MCC950, on AA in C3H/HeJ mice, induced by transferring cultured skin‐draining lymph node cells. IFN‐α production was upregulated in lesions of AA‐affected mice, and interleukin‐1β in serum and skin was highly expressed before onset as well as postonset. Furthermore, MCC950 treatment prevented AA development and promoted hair growth in AA mouse models by reducing NLRP3 signalling and Th1/Tc1 chemokines and cytokines in the skin. These results suggest that NLRP3 inflammasome contributes to AA onset and chronicity, and NLRP3 inhibitor may be a potential therapeutic agent for AA.