TGF-β1 plays a critical role in epithelial migration and corneal scar development. Immunofluorescence staining of the corneal epithelium of WT mice revealed an increase in the TGF-β1 expression in the injured corneas on days 5 and 7 after injury, as compared with that in normal corneas as well as in the injured ones on days 1 and 3 after injury (
Fig. 7A). These observations were further confirmed by the progressive increase in the TGF-β1 mRNA (
Fig. 7B) and protein expressions (
Figs. 7C, D) in the injured corneas of WT mice. In subsequent in vitro experiments, the IL-1β recombinant factor–stimulated human corneal epithelial cells demonstrated an upregulated TGF-β1 mRNA and protein level, as compared with that in the control group; in fact, the TGF-β1 expression increased along with the increase in the dose of IL-1β factor treatment (
Figs. 7E–G). Furthermore, the experiments demonstrated that the fluorescence intensity as well as the protein expression level of TGF-β1 was significantly decreased in the corneas of
NLRP3−/− mice and
NLRP3 Lyz-KO mice, as compared with those in
NLRP3loxp mice after injury (
Figs. 8A–C). To ascertain the effect of NLRP3 activation in macrophages in vitro, peritoneal macrophages were extracted from WT and
NLRP3−/− mice. Coincidentally, LPS treatment followed by Nig stimulation significantly increased the protein expression of NLRP3 in macrophages derived from WT mice. However, macrophages derived from
NLRP3−/− mice hardly expressed NLRP3 under similar stimulus (
Fig. 8D). Additionally, the NLRP3-induced IL-1β secretion was predominantly decreased in the cell supernatants derived from
NLRP3−/− mice (
Fig. 8E). To examine whether the NLRP3-induced IL-1β secretion by the mouse macrophages has any effect on TGF-β1 expression in the corneal epithelium, the pMCECs derived from the WT mice were preincubated with mouse macrophage supernatants for 24 hours. Even though significantly high TGF-β1 levels were observed in the culture supernatants of LPS-treated macrophages derived from WT mice, this effect was significantly diminished in case of the pMCECs cocultured with supernatants obtained from
NLRP3−/− macrophages (
Fig. 8F). Interestingly, IL-1β–blocking antibodies reversed the IL-1β-induced TGF-β1 upregulation in macrophages from WT mice (
Fig. 8G). The macrophage NLRP3-mediated IL-1β production stimulated TGF-β1 expression in the corneal epithelium, thereby causing myofibroblast differentiation.