Toll-like receptors mediate induction of peptidoglycan recognition proteins in human corneal epithelial cells.
Human peptidoglycan recognition proteins (PGLYRPs) are a novel family of pattern recognition receptors, and also act as anti-bacterial proteins. This study was to explore the toll-like receptor (TLR)-mediated regulation of PGLYRPs in human corneal epithelial cells (HCECs). Fresh human donor corneoscleral tissues were used to prepare cryosections. Primary HCECs, established from limbal explants, were treated with microbial ligands to TLRs 1-9 for 4-48 h, with or without pretreatment of TLR antibodies, NFkB inhibitor, or siRNA transfection. The mRNA of PGLYRPs was evaluated by RT and real-time PCR, and their proteins and NFkB activation were determined by immunostaining and Western blot. The nuclear IRF3 activity was quantified using an ELISA-based TransAM kit. PGLYRP-2, -3 and -4 were found to be expressed by human corneal epithelium while PGLYRP-1 was not detected. In primary HCEC cultures, PGLYRP-3 and -4 were constitutively expressed while PGLYRP-2 was largely inducible. PGLYRP-2 was induced by bacterial components, Pam3CSK4, PGN, flagellin and FSL-1, ligands for TLR2/1, 2, 5 and 2/6, respectively. Interestingly, PGLYRP-2 was strongest stimulated by polyI:C representing viral dsRNA. TLR3 antibody or NFkB inhibitor blocked IRF3 and NFkB p65 activation as well as polyI:C-stimulated PGLYRP-2. RNA interference indicates that the polyI:C-induced PGLYRP-2 was dramatically blocked in the cells transfected with siRNA-TRIF but neither siRNA-MyD88 nor the negative control siRNA-F. These findings suggest that human corneal epithelium may response to viral or bacterial infection by producing PGLYRPs through TLRs, and the induction of PGLYRP-2 by dsRNA was through TLR3-TRIF-IRF3-NFkB signaling pathways.
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun-Yat Sen University, Guangzhou, China.
Ping
P
Ma
Zhichong
Z
Wang
Stephen C
SC
Pflugfelder
De-Quan
DQ
Li
D000328:Adult; D000368:Aged; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D008024:Ligands; D008875:Middle Aged; D016328:NF-kappa B / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051193:Toll-Like Receptors / Q000502:physiology*; D055815:Young Adult
D002352:Carrier Proteins; D008024:Ligands; D016328:NF-kappa B; D012333:RNA, Messenger; D051193:Toll-Like Receptors; C118897:peptidoglycan recognition protein