Access Type

Open Access Dissertation

Date of Award

January 2012

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Anatomy and Cell Biology

First Advisor

Linda Hazlett

Abstract

The purpose of the current dissertation was to examine how VIP regulates host immunity and corneal healing, specifically, its control of growth factors and TLR expression in the P. aeruginosa infected cornea. Firstly, VIP treatment increased growth factor expression (EGF, GHF, FGF and VEGF) in infected cornea. Notably, treatment with a mixture of EGF, FGF and HGF prevented corneal perforation, reduced pro-inflammatory cytokines and bacterial plate count, while increasing anti-inflammatory cytokines and antimicrobials such as murine beta-defensin2 and 3.

We also investigated the expression of TLR-signaling pathways in P. aeruginosa infected corneas with or without VIP treatment. PCR array and real-time RT-PCR data demonstrated that VIP treatment decreased pro-inflammatory, but increased anti-inflammatory TLRs. Immunohistochemistry, ELISA and western blot results further confirmed the PCR data. AC7 siRNA experiments indicated that VIP regulated TLR1, TRAF6 and ST2 in a cAMP dependent manner, but Chuk, IRAK1, 2, TLR4, 9 and SIGIRR were cAMP independent. In vitro, VIP down-regulated pro-inflammatory, but increased anti-inflamamtory TLRs in macrophages and Langerhans (XS52) cells. Exogenous VIP also decreased Langerhans cell number in the infected cornea.

Furthermore, we used VIP-/- mice to asses whether VIP is required for expression of growth factors and their receptors in normal and infected cornea. VIP-/- vs WT B6 mice showed earlier corneal perforation. PCR array showed growth factors are differentially changed between groups. Real time RT-PCR and immunostaining studies revealed that the infected cornea of VIP-/- vs WT mice expressed higher EGF and HGF, reduced FGF, EGFR and HGFR and similar FGFR; no significant difference between the two groups of mice were seen in normal cornea. VIP antagonist treatment decreased protein levels for growth factor receptors at 5 days p.i. in both B6 and BALB/c mice, with no significant changes in normal cornea.

In summary, these data provide evidence that VIP modulate growth factors, angiogenic molecules and beta defensins in the infected cornea; that it reduced pro-inflammatory, but increased anti-inflammatory TLRs after corneal infection; and that it is not required for growth factor production in the normal cornea but required in the infected cornea to delay perforation.

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