Suppressive effects of azithromycin on zymosan-induced production of proinflammatory mediators by human corneal epithelial cells.
OBJECTIVE
In addition to its antibiotic effects, azithromycin has been noted to have anti-inflammatory activity, particularly in the context of microbial infections. This study was conducted to explore the suppressive effects of azithromycin on the production of proinflammatory mediators by human corneal epithelial cells (HCECs) stimulated by a fungal component, zymosan.


METHODS
Primary HCECs were cultured from donor corneal limbal explants and grown to subconfluence. The cells were treated with toll-like receptor (TLR) 2 agonist zymosan (1-50 μg/mL) for 4 to 48 hours, with or without preincubation with azithromycin (1-50 μg/mL), TLR2 antibody, or NF-κB activation inhibitor quinazoline (NF-κB-I). The cells were subjected to total RNA extraction, reverse transcription (RT), and real-time PCR using gene expression assays. Cells treated for 48 hours were used for immunofluorescence staining and Western blot analysis, and their medium supernatants were collected for protein quantitation by immunobead assays.


RESULTS
The mRNA expression and protein production of proinflammatory cytokines (TNF-α and IL-1β), chemokines (IL-8 and RANTES), and matrix metalloproteinases (MMP-1, -3, and -9) by HCECs were stimulated by zymosan in a concentration-dependent manner, with peak levels noted at 4 hours. These stimulated levels of proinflammatory mediators by zymosan were significantly inhibited by TLR2 antibody, NF-κB-I, or azithromycin, which blocked zymosan-induced NF-κB activation as determined by p65 protein nuclear translocation.


CONCLUSIONS
These findings demonstrated that the fungal component zymosan induces proinflammatory responses through TLR2 and NF-κB signaling pathways, whereas azithromycin suppresses its stimulation by blocking NF-κB activation in HCECs, suggesting the potential efficacy of this antibiotic for treating ocular surface inflammatory disorders.
Department of Ophthalmology, Baylor College of Medicine, Ocular Surface Center, Cullen Eye Institute, Houston, Texas 77030, USA.
De-Quan
DQ
Li
Nan
N
Zhou
Lili
L
Zhang
Ping
P
Ma
Stephen C
SC
Pflugfelder
D000328:Adult; D000368:Aged; D017963:Azithromycin / Q000494:pharmacology*; D015153:Blotting, Western; D002478:Cells, Cultured; D018925:Chemokines / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019573:Epithelium, Corneal / Q000187:drug effects* / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D018836:Inflammation Mediators / Q000378:metabolism*; D053583:Interleukin-1beta / Q000378:metabolism; D020782:Matrix Metalloproteinases / Q000378:metabolism; D008875:Middle Aged; D016328:NF-kappa B / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae; D051195:Toll-Like Receptor 2 / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D055815:Young Adult; D015054:Zymosan / Q000494:pharmacology*
D018925:Chemokines; D007166:Immunosuppressive Agents; D018836:Inflammation Mediators; D053583:Interleukin-1beta; D016328:NF-kappa B; D012333:RNA, Messenger; C495342:TLR2 protein, human; D051195:Toll-Like Receptor 2; D014409:Tumor Necrosis Factor-alpha; D017963:Azithromycin; D015054:Zymosan; D020782:Matrix Metalloproteinases