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F. Bian, H. Qi, S. C. Pflugfelder, D.-Q. Li; Human Limbal Basal Epithelium-Derived GDNF Suppresses the Stress and Inflammation-Induced Th17 Cytokines Through NF-kB Signaling Pathway. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4520.
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The immune defensive privilege of corneal epithelial stem cells is not clear. This study was to explore the immune defensive role of glial cell-derived neurotrophic factor (GDNF) in protecting human limbal stem cells against stress and inflammation-stimulated Th17 pathway.
Fresh human corneoscleral cryosections were used for immunostaining. Primary human corneal epithelial cultures were treated with hyperosmolar media, TNF-α, IL-1β or IL-17A, with or without GDNF or NF-kB inhibitors for 2-24 hours. The mRNA expression was determined by Affymetrix microarray and real time PCR with TaqMan primers and probes. NF-kB activation was detected by immunostaining, Western blot and cell-bases activation ELISA.
GDNF and its receptor GFRα-1 were found to be exclusively localized in the basal layer of limbal epithelium where stem cells reside. Affymetrix microarray revealed the Th17 family cytokines IL-17C, IL-17D (but not IL-17A, B, and F) and their receptors, IL-17RA, IL-17RB, IL-17RC and IL-17RD, were expressed by normal corneal epithelia. IL-17RA was immuno-localized in most corneal and limbal epithelial layers, but was negative in the limbal basal cells. The hyperosmotic (400-500 mOsM) stress and inflammatory cytokines TNF-α and IL-1β significantly increased mRNA levels of IL-6 and TGF-β2 (inducers for TH17 initiation) as well as IL-23 and IL-15 (inducers for Th17 survival and expansion), and further induced IL-17F expression in human corneal epithelial cells. Hyperosmotic stress or TNF-α activated NF-kB p65 nuclear translocation and Ser536 phosphorylation. GDNF, NF-kB inhibitor (quinazoline) or IKK inhibitor II (Wedelolactone) blocked the NF-kB activation, and suppressed stimulated production of these Th17 inducers and IL-17F by hyperosmolarity and TNF-α. Exogenous IL-17A promoted the expression of TNF-α, IL-1β, IL-8, MMP-9 and MMP-3. IL-17A also stimulated Th17 inducers IL-6, IL-23 and IL-15. Interestingly, GDNF partially suppressed these inflammatory responses to Th17 cytokine.
These findings demonstrate that limbal basal epithelial-expressed GDNF suppresses stress and inflammation-induced Th17 cytokines by inhibiting the NF-kB signaling pathway. GDNF also partially suppressed the IL-17 induced inflammatory response, which protects corneal epithelial stem cells against inflammatory insults.
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