Autophagy Suppresses Toll-Like Receptor 3-Mediated Inflammatory Reaction in Human Epidermal Keratinocytes

Figure 3

(a) Keratinocytes were transduced with recombinant adenovirus expressing green fluorescent protein-light chain 3 (GFP-LC3) overnight. After changing the medium, the cells were further incubated with fresh growth medium for 1 day. The cells were then treated with 1 μg/mL of poly (I:C) and rapamycin (1 μM). Autophagic LC3 puncta were observed under fluorescent microscopy. Cotreatment with rapamycin enhanced autophagy induction. (b) Cells were cotreated with poly (I:C) and rapamycin for 2 h. The messenger RNA (mRNA) level for inflammatory cytokines were determined by real-time polymerase chain reaction. Rapamycin cotreatment inhibited poly (I:C)-induced gene expression of inflammatory cytokines. (c) The cells were cotreated with poly (I:C) and rapamycin for 24 h. The conditioned media were collected, and secreted inflammatory cytokines were determined by enzyme-linked immunosorbent assay. Rapamycin cotreatment inhibited poly (I:C)-induced secretion of inflammatory cytokines. (d) Effect of rapamycin on inflammasome-related molecules.

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