Human Limbal Basal Epithelium-Derived GDNF Suppresses The Stress And Inflammation-Induced Th17 Cytokines Through NF-kB Signaling Pathway

Purpose Corneal epithelial stem cells are well known for their self-renewal and regenerative capacity. However their immune defensive privilege is not well elucidated. Th17 is a newly recognized T helper cell subset distinct from Th1,Th2 and Treg cells. 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.Methods Cryosections were prepared from fresh human corneoscleral tissues for Immunostaining. Primary corneal epithelial cultures established from limbal explants were treated with hyperosmolar media (350–500 mOsM) , proinflammatory cytokines (TNF-αor IL-1β) ,or IL-17A with or without GDNF or NF-kB inhibitors for 4 and 24 hours. The mRNA expression was determined by Affymetrix microarray with whole human genome U133 Plus 2.0 chips and by real time PCR with Taqman primers and probes. NF-kB p65 nuclear translocation and phosphorylation was detected by immunostaining and a cellular activation of signaling ELISA kit.Results The immunofluorescent staining and laser scanning confocal microscopy on human corneal cryosections identified that GDNF and its receptor GFRα-1 were exclusively localized in the basal layer of limbal epithelium where stem cells reside. Affymetrix microarray revealed the epithelium-derived Th17 family cytokines,IL-17C (IL-21),IL-17D (IL-22),and receptors,IL-17RC and IL-17RD,which expressed at lower levels in limbal progenitor cells than the differentiated cells. Hyper-osmolar (400–500 mOsM) stress and inflammatory cytokines TNF-αand IL-1βsignificantly stimulated the transcript expression 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 induce IL-17F expression in human corneal epithelial cells. The hyperosmotic stress or TNF-αactivated NF-kB nuclear translocation and NF-kB Ser536 phosphorylation. Addition with GDNF blocked the NF-kB protein nuclear translocation and phosphorylation,further suppressed these four Th17 inducers and IL-17F stimulated by hyperosmolar media and TNF-α. Exogenously added IL-17A promoted the expression of inflammatory cytokines (TNF-αand IL-1β) ,chemokine IL-8,and extracellular metalloproteinases MMP-9 and -3 by corneal epithelial cells. IL-17A also stimulated their inducers IL-6,IL-23 and IL-15. Interestingly,addition with GDNF partially suppressed these inflammatory responses except TNF-α.Conclusion These findings demonstrated that limbal basal epithelium-expressed GDNF suppresses the stress and inflammation-induced Th17 cytokines through inhibition of NF-kB signaling pathway. GDNF also partially suppressed the IL-17 induced inflammatory response,which protects corneal epithelial stem cells against inflammation. GDNF may have therapeutic potential for the treatment of Th17 mediated ocular surface diseases.