Shahrzad Lighvani

Substance P regulates natural killer cell interferon‐γ production and resistance to Pseudomonas aeruginosa infection

Studies have shown that after Pseudomonas aeruginosa (P. aeruginosa) corneal infection, BALB/c mice that are capable of resolving the disease, locally produce IFN‐γ. As T cells are not detected in the infected cornea of these mice, antibody depletion was used to test whether NK cells produce the cytokine. After depletion, decreased corneal IFN‐γ mRNA and increased disease severity, bacterial load, and PMN infiltrate resulted. Further work determined if substance P (SP), a pro‐inflammatory neuropeptide, participated in regulation of this response. To this end, mice were treated with the SP antagonist, spantide I that blocks SP interaction with neurokinin‐1, its major receptor. The treatment significantly decreased corneal IFN‐γ and IL‐18 protein levels and corneal perforation resulted. In vitro experiments using isolated splenic NK cells confirmed their ability to respond to IL‐18 and SP and to secrete IFN‐γ protein. We conclude: that for development of the BALB/c resistance response, NK cells are required to produce IFN‐γ; that the cells express the neurokinin‐1 receptor; and that SP directly regulates IFN‐γ production through this receptor. The data suggest a unique link between the nervous system and development of innate immunity in the cornea.

Vasoactive Intestinal Peptide Balances Pro- and Anti-Inflammatory Cytokines in the Pseudomonas aeruginosa-Infected Cornea and Protects against Corneal Perforation

Corneal infection with Pseudomonas aeruginosa perforates the cornea in susceptible C57BL/6 (B6), but not resistant BALB/c, mice. To determine whether vasoactive intestinal peptide (VIP) played a role in development of the resistant response, protein expression levels were tested by immunocytochemistry and enzyme immunoassay in BALB/c and B6 corneas. Both mouse strains showed constitutive expression of corneal VIP protein and nerve fiber distribution. However, disparate expression patterns were detected in the cornea after infection. VIP protein was elevated significantly in BALB/c over B6 mice at 5 and 7 days postinfection. Therefore, B6 mice were injected with rVIP and subsequently demonstrated decreased corneal opacity and resistance to corneal perforation compared with PBS controls. rVIP- vs PBS-treated B6 mice also demonstrated down-regulation of corneal mRNA and/or protein levels for proinflammatory cytokines/chemokines: IFN-γ, IL-1β, MIP-2, and TNF-α, whereas anti-inflammatory mediators, IL-10 and TGF-β1, were up-regulated. Treatment with rVIP decreased NO levels and polymorphonuclear neutrophil (PMN) number. To further define the role of VIP, peritoneal macrophages (Mφ) and PMN from BALB/c and B6 mice were stimulated with LPS and treated with rVIP. Treatment of LPS-stimulated Mφ from both mouse strains resulted in decreased IL-1β and MIP-2 protein levels; PMN responded similarly. Both cell types also displayed a strain-dependent differential response to rVIP, whereby B6 Mφ/PMN responded only to a higher concentration of VIP compared with cells from BALB/c mice. These data provide evidence that neuroimmune regulation of the cytokine network and host inflammatory cells functions to promote resistance against P. aeruginosa corneal infection.

IFN-γ: Regulation of nitric oxide in the P. aeruginosa-infected cornea

Purpose: BALB/c mice are resistant to Pseudomonas aeruginosa (P. aeruginosa) keratitis and bacterial killing/stasis requires nitric oxide (NO). NO regulation in the cornea is unknown and was tested in this model. Methods: Nitrite detection, IFN-γ-knockout mice, TNF-α neutralization, ELISA, aminoguanidine (AG) treatment, MPO, and plate counts were done. Results: Evidence shows (i) without IFN-γ, nitrite levels are elevated, (ii) neutralization of TNF-α does not change nitrite levels, and (iii) absence of IFN-γ and reduced NO synergistically increases disease progress and upregulates pro-inflammatory cytokines, PMN number, and bacterial load. Conclusion: IFN-γ regulates NO levels, and synergistic interaction between the two regulates disease outcome in resistant mice.