Abstract
Purpose: :
We have identified that polyinosinic-polycytidylic Acid (poly I:C), a synthetic double-stranded RNA (dsRNA) molecule designed to mimic RNA virus infection, strongly induced the production of thymic stromal lymphopoietin (TSLP), a novel pro-allergic cytokine, in human corneal epithelium. This study was to investigate the signaling pathways mediated in this TSLP induction.
Methods: :
Fresh human corneoscleral tissues were used to prepare cryosections for immunostaining. Primary human corneal epithelial cells established from limbal explants were treated with different concentrations of poly I:C (1-50 µg) for 4-48 hours with or without TLR3 antibody, or different inhibitors for endosomal acidification, dsRNA-activated protein kinase (PKR) or NFkB activation, respectively. The mRNA expression was determined by RT and real time PCR with Taqman primers and its protein levels were measured by ELISA. IkB- and NF-kB activation was determined by Western blot and immunostaining. Interferon regulatory factor (IRF) 3 activity in cell nuclear extracts was measured by a TransAM IRF3 kit.
Results: :
TSLP was largely induced by a synthetic dsRNA poly I:C at both mRNA and protein levels (up to 60-fold) in human corneal epithelium and its primary cultured cells. Poly I:C activated IkB- with its dramatic degradation in 1-2 hours, and activated NFkB p65 protein nuclear translocation in 4 hours. Furthermore, poly I:C stimulated transcription factor IRF3 activity up to 5-10 fold in the cell nuclear extracts. A rabbit TLR3 antibody, endosomal acidification inhibitor chloroquin, PKR inhibitor 2-aminopurine, IKK inhibitor II wedelolactone, or NF-kB activation inhibitor quinazoline blocked the activation of IkB- and NF-kB, inhibited the nuclear IRF3 activity, and further suppressed TSLP expression and production, partially or completely.
Keywords: cornea: epithelium • inflammation • immunomodulation/immunoregulation