Double-stranded RNA induces inflammation via the NF-κB pathway and inflammasome activation in the outer root sheath cells of hair follicles.

Alopecia areata (AA), a chronic, relapsing, hair-loss disorder, is considered to be a T cell-mediated autoimmune disease. It affects approximately 1.7% of the population, but its precise pathogenesis remains to be elucidated. Despite the recent attention focused on the roles of inflammasomes in the pathogenesis of autoinflammatory diseases, little is known about inflammasome activation in AA. Thus, in this study, we investigated the pattern of NLRP3 inflammasome activation in the outer root sheath (ORS) cells of hair follicles. We found that interleukin (IL)-1ß and caspase-1 expression was increased in hair follicle remnants and inflammatory cells of AA tissue specimens. After stimulation of ORS cells with the double-stranded (ds)RNA mimic polyinosinic:polycytidylic acid (poly[I:C]), the activation of caspase-1 and secretion of IL-1ß were enhanced. Moreover, NLRP3 knockdown decreased this poly(I:C)-induced IL-1ß production. Finally, we found that high-mobility group box 1 (HMGB1) translocated from the nucleus to the cytosol and was secreted into the extracellular space by inflammasome activation. Taken together, these findings suggest that ORS cells are important immunocompetent cells that induce NLRP3 inflammasomes. In addition, dsRNA-induced IL-1ß and HMGB1 secretion from ORS cells may contribute to clarifying the pathogenesis and therapeutic targets of AA.

Analysis of calcium-inducible genes in keratinocytes using suppression subtractive hybridization and cDNA microarray.

Terminal differentiation of skin keratinocytes is a vertically directed multistep process that is tightly controlled by the sequential expression of a variety of genes. To gain further insight into the molecular events involved in this process, we used suppression subtraction hybridization (SSH) and cDNA microarray analysis. Messenger RNAs were isolated from primary skin keratinocytes cultured in vitro after treatment with calcium and then SSH was performed. A total of 840 cDNA clones were obtained from subtracted libraries, and these cDNA clones were used to make the microarray slides. Time-course cDNA microarray analysis (1, 3, 7, and 14 days after calcium treatment) revealed the global gene expression profile during keratinocyte differentiation. Of the 840 genes tested, 290 showed a greater than twofold change in expression level at least once over four time points. The genes were clustered into six groups according to their expression pattern using self-organizing map analysis and showed the global feature of function-related regulation. The genes related to keratinocyte differentiation were markedly up-regulated by calcium treatment. In addition, a unique pattern of increase was seen in the expression of genes related to ribosomal proteins. On the other hand, transcripts involved in metabolism, DNA repair, transcription, and translation were generally down-regulated. These results demonstrate the complexity of the gene expression profile that contributes to the spatiotemporal regulation of keratinocyte differentiation.