Glycyrrhizin Interacts with TLR4 and TLR9 to Resolve P. aeruginosa Keratitis

This study tests the mechanism(s) of glycyrrhizin (GLY) protection against P. aeruginosa keratitis. Female C57BL/6 (B6), TLR4 knockout (TLR4KO), myeloid specific TLR4KO (mTLR4KO), their wildtype (WT) littermates, and TLR9 knockout (TLR9KO) mice were infected with P. aeruginosa KEI 1025 and treated with GLY or PBS onto the cornea after infection. Clinical scores, photography with a slit lamp, RT-PCR and ELISA were used. GLY effects on macrophages (Mϕ) and polymorphonuclear neutrophils (PMN) isolated from WT and mTLR4KO and challenged with KEI 1025 were also tested. Comparing B6 and TLR4KO, GLY treatment reduced clinical scores and improved disease outcome after infection and decreased mRNA expression levels in cornea for TLR4, HMGB1, and RAGE in B6 mice. TLR9 mRNA expression was significantly reduced by GLY in both mouse strains after infection. GLY also significantly reduced HMGB1 (B6 only) and TLR9 protein (both B6 and TLR4KO). In TLR9KO mice, GLY did not significantly reduce clinical scores and only slightly improved disease outcome after infection. In these mice, GLY significantly reduced TLR4, but not HMGB1 or RAGE mRNA expression levels after infection. In contrast, in the mTLR4KO and their WT littermates, GLY significantly reduced corneal disease, TLR4, TLR9, HMGB1, and RAGE corneal mRNA expression after infection. GLY also significantly reduced TLR9 and HMGB1 corneal protein levels in both WT and mTLR4KO mice. In vitro, GLY significantly lowered mRNA expression levels for TLR9 in both Mϕ and PMN isolated from mTLR4KO or WT mice after incubation with KEI 1025. In conclusion, we provide evidence to show that GLY mediates its effects by blocking TLR4 and TLR9 signaling pathways and both are required to protect against disease.