Purpose Peptidoglycan recognition proteins (PGRPs), a novel family of pattern recognition receptors (PRRs) in innate immunity conserved from insects to mammals, recognize bacterial cell wall peptidoglycan (PG) and act as antibacterial proteins. In humans, four PGRPs (PGRP-S, -L, -Iα, and -Iβ) have been cloned and all bind PG. In this study, we investigated the expression and regulation of PGRPs in human corneal epithelium in response to microbial ligands.
Methods Fresh human corneoscleral tissues from normal donors were used to prepare cryosections for immunostaining. Primary human corneal epithelial cells were established from limbal explants. The confluent cell cultures were treated with 11 microbial components, ligands to toll-like receptors (TLRs) 1-9, for a different time periods (4-48 hours) with or without TLR antibodies or NFkB inhibitors. The mRNA expression of PGRPs was evaluated by RT and real time PCR using TaqMan primers and probes, with GAPDH as an internal control. Their proteins and NF-kB signaling pathway activation were determined by immunofluorescent staining and Western blot.
Results PGRP-L, -Iα, and -Iβ were found to be expressed by human corneal epithelium and primary cultured human corneal epithelial cells. Their mRNA levels peaked at 16 hour in response to the TLR ligands. PGRP-Iα was the most inducible with its mRNA markedly stimulated by multiple microbial components including Pam3csk4, PG, poly I:C (dsRNA), flagellin and FSL-1, ligands for TLRs 1, 2, 3, 5, and 6. PGRP-L and -Iβ were inducible mainly by poly I:C and flagellin, although they were constitutively expressed at higher levels than PGRP-Iα in human corneal epithelial cells. The induction of PGRPs was confirmed at the protein levels by immunostaining and Western blot. TLR antibodies, IKK inhibitor II (wedelolactone) or NF-kB activation inhibitor (quinazoline) blocked NF-kB p65 nuclear translocation and phosphorylation, and also suppressed PGRP-Iα and PGRP-Iβ expression stimulated by Poly I:C or flagellin.
Conclusion These findings demonstrate that human corneal epithelial cells produce the innate immunity molecules, PGRP-L, PGRP-Iα and PGRP-Iβ, which were induced through TLRs and NF-kB pathways in response to certain microbial components. In addition to antibacterial effects, PGRPs may participate in the immune response to viral infection on the ocular surface.
Support National Natural Science Fundation of China (Grant No. 81100643) and Promotive Research Fund for excellent young and middle-aged Scientisits of Shandong Province (Grant No. BS2010YY052). |
