vasoactive-intestinal-peptide and Keratitis

Vasoactive intestinal peptide (VIP) has been shown to regulate corneal inflammation. Formyl peptide receptor 2 (FPR2) is a transmembrane protein belonging to the GPCR family. Ligands include pro-resolving lipids, lipoxin A4 (LXA4) and resolvin D1 (RvD1). The current study focuses on the effect of VIP regarding the FPR2 receptor axis in improving disease outcome in a mouse model of bacterial keratitis. Infection was induced in C57BL/6 (B6) mice using P. aeruginosa (PA) ATCC 19660. Mice received topical treatment (VIP or PBS) 3× daily after infection. Mean clinical scores, bacterial plate counts, Griess and myeloperoxidase (MPO) assays indicate that topical VIP effectively abrogates the disease response. Findings also reveal that VIP influences FPR2 pathway activation independent of archetypal VIP receptors. Exploring the immunoresolving role of FPR2, its ligand RvD1 and related enzymes (5-LOX, 12/15-LOX), our results suggest a mechanism by which VIP treatment influences the disease response in bacterial keratitis, which could offer a therapeutic point of intervention for enhancing this pro-resolving circuit.

Topics: Animals; Disease Models, Animal; Female; Homeodomain Proteins; Inflammation Mediators; Keratitis; Mice; Mice, Inbred C57BL; Pseudomonas aeruginosa; Receptors, Formyl Peptide; Vasoactive Intestinal Peptide

Previous studies have demonstrated the efficacy of vasoactive intestinal peptide (VIP) treatment in regulating inflammation following bacterial keratitis induced by the P. aeruginosa strain 19660. However, in the current study we assessed whether disease outcome is specific to 19660 or if VIP treatment is effective against multiple P. aeruginosa strains.. B6 mice received daily IP injections of VIP from -1 through 5 days post injection (p.i.). Control mice were similarly injected with PBS. Corneal infection was induced using PA 19660, PAO1 or KEI 1025. Disease response was documented and bacterial plate counts and myeloperoxidase assays were performed. Expression of select inflammatory mediators as well as enzymes associated with lipid mediator production was assessed after VIP treatment. KEI 1025 was characterized by cytotoxicity and invasion assays and then confirmed for ExoS/ExoU expression.. VIP treatment converted the susceptible response to resistant for the three P. aeruginosa strains tested. Disease response was significantly reduced with no corneal perforation. Anti-inflammatory mediators were enhanced after VIP treatment, while pro-inflammatory molecules were reduced compared to controls. Furthermore, VIP reduced inflammatory cell persistence in the cornea after infection with each of the P. aeruginosa strains.. VIP treatment is effective at ameliorating disease pathogenesis for multiple P. aeruginosa strains, both cytotoxic and invasive. This study is also the first to indicate a possible role for VIP regarding lipid mediator expression in the eye. In addition, the clinical isolate, KEI 1025, was characterized as an invasive strain. Overall, this study strengthens the preclinical development of VIP as a therapeutic agent for ocular infectious disease.

Topics: Animals; Enzyme-Linked Immunosorbent Assay; Eye Infections, Bacterial; Female; Intravitreal Injections; Keratitis; Mice; Mice, Inbred C57BL; Pseudomonas aeruginosa; Pseudomonas Infections; Real-Time Polymerase Chain Reaction; Vasoactive Intestinal Peptide

TLRs recognize microbial pathogens and trigger an immune response, but their regulation by neuropeptides, such as vasoactive intestinal peptide (VIP), during Pseudomonas aeruginosa corneal infection remains unexplored. Therefore, C57BL/6 (B6) mice were injected i.p. with VIP, and mRNA, protein, and immunostaining assays were performed. After VIP treatment, PCR array and real-time RT-PCR demonstrated that proinflammatory TLRs (conserved helix-loop-helix ubiquitous kinase, IRAK1, TLR1, TLR4, TLR6, TLR8, TLR9, and TNFR-associated factor 6) were downregulated, whereas anti-inflammatory TLRs (single Ig IL-1-related receptor [SIGIRR] and ST2) were upregulated. ELISA showed that VIP modestly downregulated phosphorylated inhibitor of NF-κB kinase subunit α but upregulated ST2 ~2-fold. SIGIRR was also upregulated, whereas TLR4 immunostaining was reduced in cornea; all confirmed the mRNA data. To determine whether VIP effects were cAMP dependent, mice were injected with small interfering RNA for type 7 adenylate cyclase (AC7), with or without VIP treatment. After silencing AC7, changes in mRNA levels of TLR1, TNFR-associated factor 6, and ST2 were seen and unchanged with addition of VIP, indicating that their regulation was cAMP dependent. In contrast, changes were seen in mRNA levels of conserved helix-loop-helix ubiquitous kinase, IRAK1, 2, TLR4, 9 and SIGIRR following AC7 silencing alone; these were modified by VIP addition, indicating their cAMP independence. In vitro studies assessed the effects of VIP on TLR regulation in macrophages and Langerhans cells. VIP downregulated mRNA expression of proinflammatory TLRs while upregulating anti-inflammatory TLRs in both cell types. Collectively, the data provide evidence that VIP downregulates proinflammatory TLRs and upregulates anti-inflammatory TLRs and that this regulation is both cAMP dependent and independent and involves immune cell types found in the infected cornea.

Topics: Animals; Cells, Cultured; Down-Regulation; Female; Inflammation Mediators; Keratitis; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Pseudomonas aeruginosa; Pseudomonas Infections; Toll-Like Receptors; Up-Regulation; Vasoactive Intestinal Peptide

Studies from our laboratory have demonstrated that vasoactive intestinal peptide (VIP) directly converts the normally susceptible C57BL/6J (B6) mouse to resistant after ocular infection through modulation of the inflammatory response. This study examines mechanisms by which VIP influences the healing phase following infection--specifically reconstitution of the extracellular matrix (ECM).. B6 mice received daily intraperitoneal (IP) injections of VIP, while control mice were similarly injected with sterile phosphate buffered saline (PBS). Real-time RT-PCR, ELISA, and immunofluorescent staining were used to assess the effects of VIP treatment on ECM molecule expression after Pseudomonas aeruginosa-induced keratitis. We also compared the effect of VIP treatment on lipopolysaccharide (LPS)-stimulated B6- and BALB/c-derived fibroblasts.. In vivo analyses revealed that VIP treatment of P. aeruginosa-infected B6 corneas led to a significant increase in ECM molecules associated with healing/homeostasis, while those associated with ECM degradation were significantly down-regulated when compared to wild-type (WT) controls. In vitro studies revealed that VIP treatment of lipopolysaccharide-stimulated fibroblasts derived from susceptible B6 and resistant BALB/c mice expressed distinct differences in ECM molecule expression, whereby the latter expressed higher levels of ECM molecules aimed at reconstitution. Furthermore, differential expression of VIP receptor-1/VIP receptor-2 (VIPR1/VIPR2) was observed between B6 and BALB/c after VIP treatment of LPS-stimulated fibroblasts.. VIP treatment functions to enhance ECM reconstitution, which appears to be carried out in large part by fibroblasts via VIPR2. Overall, the data from this study suggest that VIP not only regulates disease pathogenesis, but also functions to restore integrity of the corneal stroma.

Topics: Animals; Cornea; Disease Models, Animal; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix; Eye Infections, Bacterial; Female; Homeostasis; Keratitis; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neuroprotective Agents; Pseudomonas aeruginosa; Pseudomonas Infections; Receptors, Vasoactive Intestinal Peptide, Type II; Receptors, Vasoactive Intestinal Polypeptide, Type I; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Vasoactive Intestinal Peptide

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.

Topics: Animals; Colony Count, Microbial; Cornea; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Eye Infections, Bacterial; Female; Gene Expression Regulation; Keratitis; Mice; Mice, Inbred BALB C; Neuroprotective Agents; Pseudomonas aeruginosa; Pseudomonas Infections; Receptors, Growth Factor; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Toll-Like Receptors; Vasoactive Intestinal Peptide