After infection, in the absence of the neuropeptide, GFs and GFRs were disregluated when tested by PCR array, RT-PCR, and immunostaining, and the cornea perforated more rapidly (within 2 days p.i.) for the majority of the VIP
−/− versus WT mice. Furthermore, either the endogenous absence of VIP or antagonist treatment of B6 mice led to this disregulation. Specifically, VIP antagonist treatment of B6 mice reduced all three GFRs at the mRNA level (significantly, except for FGFR). When either B6 or BALB/c mice were treated with a VIP antagonist, GFR protein levels were decreased in both strains of mice when compared with controls. In this regard, others previously have shown interaction of VIP with the EGFR. In this regard, in vitro studies have shown that G
q protein-coupled receptor agonists such as VIP can directly activate (phosphorylate) the EGFR in T
84 colonic epithelial cells by signaling pathways involving cAMP and protein kinase A and thereby regulate Cl
− secretion.
18 Previous studies also have shown that VIP receptors are Gs (VPAC1/2)
19 and Gq coupled (VPAC2)
20 (PAC1).
21 cAMP, the downstream molecule of Gs coupled signaling, has been shown to increase nerve growth factor triggered signaling and differentiation in rat adrenal pheochromocytoma PC12 cells.
22 On the other hand, phospholipase C-ε, the downstream molecule of Gq signaling, augments EGF-dependent COS-7 cell growth by inhibiting EGFR downregulation.
23 Therefore, in the current study, we could not exclude the possibility that VIP binds to VPAC1/2 (Gs coupled) to activate cAMP, and/or binds PAC1 and VPAC2 (Gq coupled) to activate phospholipase C. The activated cAMP and/or phospholipase C, consequently, could regulate EGF and EGFR expression. In another study, it was reported that during infection of human alveolar epithelial cells with
Pseudomonas fluorescens, a gram-negative rod blocking the EGFR increased epithelial susceptibility to pathogen-induced epithelial cell death
24 consistent with, although not tested, in our keratitis model. Another growth factor, KGF/FGF-7 (FGF), and its receptor (FGFR) were both found to reduce
Pseudomonas infection in a lung model through reducing bacterial load because intratracheal FGF was found to increase clearance of
P. aeruginosa .
25 Another study using an experimental burn model in human keratinocytes, also found that when FGF was added to
P. aeruginosa in the presence of keratinocytes, bacterial growth was inhibited, and the same was observed when genetically modified keratinocytes were used.
26 In addition,
P. aeruginosa infection in a bioengineered skin model, in which FGF7 was expressed in diploid human keratinocytes, led to increased levels of antimicrobials β-defensin-2 and cathelicidin (LL-37) production and reduced bacterial load when compared with controls.
27 The current study essentially agreed with these past studies in that absence of VIP or treatment with a VIP antagonist that disregulates GF and GFR contributed to earlier corneal perforation and/or increased bacterial load (tested only in antagonist treated mice). Previous studies from this laboratory also found that treatment using a GF mixture composed of EGF, FGF, and HGF increases antimicrobial peptides including murine beta-defensin-2 (mBD-2) and mBD3 expression, as well as decreases bacterial plate counts, resulting in less disease severity in the infected cornea.
5 FGF also has been shown to promote wound healing in skin epithelium
28 and to participate in lung epithelial repair.
8 Because many cases of bacterial keratitis result directly or indirectly from disruption of the corneal epithelium,
29 it also is possible that earlier perforation observed in VIP
−/− mice may be due to reduced FGF levels with subsequent epithelial defect. HGFR mRNA and protein (immunostaining) also are decreased in the VIP
−/− mice. HGFR recognizes HGF, which has anti-inflammatory properties, in that it can target vascular endothelial cells and disrupt nuclear factor-kappa B (NF-κB) signaling in these cells,
30 critical to regulating inflammatory cytokines.