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Exogenous vasoactive intestinal peptide (VIP) down-regulates pro-inflammatory but up-regulates anti-inflammatory cytokines, growth factors (GFs) and Toll-like receptors promoting healing in experimental Pseudomonas aeruginosa (P. aeruginosa) keratitis. Whether VIP is required for GF or GF receptor (R) expression in normal and infected corneas is unknown and is the purpose of this study.VIP knockout ((-/-)) and wild-type (WT) C57BL/6 (B6) mice were infected and tested using PCR array, real-time RT-PCR, ELISA, and immunostaining. VIP antagonist treatment studies also were done using B6 and BALB/c mice.Infected corneas of VIP(-/-) versus WT B6 mice perforated earlier (2 vs. 5 days postinfection [p.i.]), and array data showed that GFs were differentially changed between groups. RT-PCR revealed that the infected cornea of VIP(-/-) versus WT mice expressed higher mRNA levels of epidermal growth factor (EGF) and hepatocyte growth factor (HGF), reduced FGF, EGFR, and HGFR, with no difference in FGFR; differences between groups were not seen in normal cornea. Immunostaining for GF and GFR in the normal cornea of VIP(-/-) versus WT mice was similar. However, at 1 day p.i., VIP(-/-) versus WT mice had more intense EGF and HGF, similar FGFR, and reduced FGF, EGFR, and HGFR staining. VIP antagonist treatment decreased protein levels for GFR at 5 days p.i. in both B6 and BALB/c mice, with no significant changes in normal cornea.The data showed that endogenous VIP is not requisite for GF or GFR expression in the normal cornea but, after infection, its absence or reduction is critical for their regulation.

An opportunistic, gram-negative pathogen, Pseudomonas aeruginosa is one of the most virulent organisms associated with microbial keratitis and is often associated with contact lens usage.1 Both bacterial (e.g., lipopolysaccharide) and host factors released from infiltrating cells during infection are thought to contribute to a rapidly progressing liquefactive stromal necrosis.2–5 Experimental murine models of the disease have been established: Th1 responder mouse strains (e.g., C57BL/6, B6) are susceptible (cornea perforates), whereas Th2 responder strains (e.g., BALB/c) are resistant (cornea heals).6 In previous studies using these murine models, we provided evidence that an anti-inflammatory neuropeptide, vasoactive intestinal peptide (VIP) given exogenously, promotes resistance against P. aeruginosa corneal infection by regulation of cytokine production and subsequent alteration of the host inflammatory cell response.7 Neuropeptides, such as VIP are small protein-like molecules used by neurons to bidirectionally communicate with the cells of the immune system.8 In the eye, VIP can be detected in corneal nerves and the aqueous humor in both mice and humans.9 Others also have shown previously that VIP reduces pro-inflammatory cytokine production, limits cell-mediated immunity, and inhibits macrophage and T cell proliferation9; and by downregulation of IL-6 and TNF-α, the neuropeptide showed a protective effect in models of endotoxemia.10 Despite these anti-inflammatory effects which have been reported in diverse systems and which clearly modulate disease outcome, the role of VIP in regulation of corneal healing, specifically, control of growth factor production, has not been examined. Classically, growth factors, which also may be considered as cytokines, bind to their receptors and regulate cell growth, proliferation, migration, and differentiation.11 Thus, the current studies investigated the potential regulation by VIP of growth factors in P. aeruginosa-infected corneas in B6 (susceptible) mice that exhibit lower levels of VIP when compared with resistant (BALB/c) mice.7 Exogenous VIP treatment was given to B6 mice to determine whether VIP modulates growth factor production and thereby, favors resolution of disease. Data from these studies provide evidence that VIP treatment leads to upregulation of growth factor production in the cornea after P. aeruginosa infection, and that both epithelial and stromal cells are participatory. Furthermore, evidence is provided to show that treatment with a mixture of growth factors, given topically after infection, prevents perforation of the cornea. Mechanistically this is achieved by downregulation of proinflammatory cytokines, upregulation of anti-inflammatory cytokines, antimicrobials β-defensins 2 and 3, and decreased bacterial plate counts. The data suggest that VIP modulates growth factor production in the infected cornea and that this in turn, contributes to better disease outcome.

Infection with Pseudomonas aeruginosa is often associated with keratitis, especially in users of extended-wear contact lenses,1–3 with disease outcome largely determined by the host inflammatory response. In fact, we propose that although an intense response may be needed to eradicate the bacteria, if left uncontrolled, it is often detrimental, favoring bystander tissue damage and in turn providing nutrients for bacterial growth and replication. Thus, tight regulation of inflammation and a balance between proinflammatory and antiinflammatory factors is requisite for disease resolution, including restoration of tissue homeostasis.4 Whether cells undergo necrosis and when they undergo apoptosis also may contribute to disease outcome. In this regard, previous studies5 have shown that apoptosis of polymorphonuclear leukocytes (PMNs) is delayed until 3 days p.i. in the corneas of susceptible C57BL/6 (B6) mice, whereas in resistant BALB/c mice, cells undergo apoptosis at 1 day p.i. The importance of earlier apoptosis in disease resolution was further confirmed in B6 mice by blocking interaction of the antiapoptotic neuropeptide substance P with its receptor (NK-1R), which resulted in earlier apoptosis and improved disease outcome. Apoptosis can be initiated by a variety of interactions, including the interaction between Fas and Fas Ligand (FasL), the well-documented death ligand and its receptor,6,7 leading to the subsequent activation of downstream caspases and the induction of cell lysis. It has been suggested that the Fas-FasL system may play an important role in the pathophysiology of infectious diseases.8 Evidence shows that P. aeruginosa infection induces lung epithelial cell apoptosis through the activation of endogenous Fas and FasL in vitro and in vivo, leading to improved disease outcomes in C3H mice.8 Others have reported that ExoS of P. aeruginosa triggers apoptosis in various cultured cell lines through clustering membrane Fas and activating the Fas-FasL pathway.9 Evidence has also suggested a direct regulatory role of the Fas-FasL system on local cytokine/chemokine production.10 For example, it was reported that C3H/HeJgld mice, which bear a nonfunctional mutation in FasL, showed significantly reduced Borrelia burgdorferi–specific cytokine response and reduced severity of arthritis.10 However, whether Fas-FasL interaction functions in P. aeruginosa–induced keratitis and has a similar impact on disease outcome remain to be determined. Our studies provide evidence that compared with WT mice, FasL−/− mice have a delayed onset of apoptosis and an exacerbated production of proinflammatory cytokines/chemokines and nitric oxide (NO) after P. aeruginosa corneal challenge, subsequently leading to worsening of disease. We also document in vitro that LPS-stimulated Mφ from FasL−/− mice compared with WT BALB/c mice showed decreased apoptosis, increased production of TNF-α, MIP-2, and IL-1β, and decreased production of IL-10, consistent with our in vivo findings.

Purpose Studies have shown that blocking substance P (SP) binding to neurokinin 1 receptor with spantide I prevents Pseudomonas aeruginosa-induced corneal perforation in susceptible C57BL/6 mice. This study tested the effect of SP injection on the resistance response (cornea heals) of BALB/c mice. Methods The day before infection, mice were injected intraperitoneally with SP or PBS. Disease was graded by clinical score, slit lamp, plate count, real-time RT-PCR, and ELISA assays, and polymorphonuclear neutrophils (PMNs) were quantitated using a myeloperoxidase assay. In additional experiments, BALB/c mice were injected intraperitoneally with vasoactive intestinal peptide (VIP) antagonist and similarly analyzed. Results Mice injected with SP exhibited worsened disease on days 1 to 7 after infection compared with controls. SP injection resulted in elevated PMN levels and viable bacterial counts in the cornea 3 and 5 days after infection. mRNA expression for NFkappaB and type 1 cytokines (e.g., IFN-gamma), as well as for TNF-alpha, MIP-2, IL-18, IL-6, and IL-1beta, were significantly elevated, whereas VIP and cytokines TGF-beta and IL-10 were significantly reduced. Differences in mRNA expression were selectively confirmed at the protein level by ELISA for NFkappaB, IL-1beta, and IL-10. VIP antagonist treatment also resulted in exacerbated disease scores, elevated proinflammatory mediators, and reduced anti-inflammatory mediators. Conclusions These data provide evidence that the neuropeptide SP, among its broad systemic effects, is a potent neuroimmunoregulator that promotes susceptibility in the resistant BALB/c mouse by overcoming the anti-inflammatory effects of VIP and IL-10 and that a balance between SP and VIP levels may be critical in disease resolution.

Purpose The purpose of this study was to determine the role of matrix metalloproteinases (MMP) in Pseudomonas aeruginosa keratitis. Methods Gene array and selective real-time PCR examined MMP expression in the cornea of susceptible (C57BL/6, B6) versus resistant (BALB/c) mice before and after infection; zymography tested enzyme activity for MMP-2 and -9. Clinical score, Langerhans cell (LC), and Neutrophil (PMN) quantitation were done in recombinant (r) MMP-9, antibody neutralized, and MMP-9(-/-) mice. The chemotactic potential of MMP-9 was tested in a Boyden chamber assay; light and transmission microscopy and immunostaining for collagen IV and MMP-9 were used to examine the effects and the source of MMP-9 after infection. ELISA was used to assess IL-1beta and MIP-2 levels. Results Gene array (confirmed by PCR) revealed sixfold more MMP-9, and zymography showed greater enzyme activity in the infected cornea of B6 over BALB/c mice. rMMP-9 injection of BALB/c mice enhanced, whereas MMP-9 antibody neutralization in B6 mice and its absence in MMP-9(-/-) mice decreased corneal disease. MMP-9(-/-) and antibody neutralized mice had fewer LCs in cornea; rMMP-9-treated mice had more. A myeloperoxidase (MPO) assay showed a similar pattern for PMN. MMP-9 was not chemotactic for LC or PMN. The basement membrane was more intact in MMP-9(-/-) over wild-type infected mice and correlated with staining for collagen IV; PMN was a source of MMP-9. IL-1beta and MIP-2 were increased in rMMP-9 but decreased in MMP-9 antibody neutralized and MMP-9(-/-) over control groups. Conclusions MMP-9 regulates immune function in cornea by proteolysis, potentiating P. aeruginosa keratitis by degrading collagen IV and upregulating chemotactic cytokines/chemokines IL-1beta and MIP-2.