Purpose.: To determine the role of IL-33 in resistance to Pseudomonas aeruginosa keratitis. Methods.: Corneal IL-33 mRNA and protein levels were tested in susceptible C57BL/6 (B6) and resistant BALB/c mice. B6 mice were injected with recombinant mouse IL-33 (rmIL-33) and disease severity, bacterial load, polymorphonuclear neutrophils (PMN) infiltrate, gene expression of inflammatory, and T-helper (Th)1/Th2 cytokines were tested by RT-PCR. IL-33 signaling and macrophage (Mφ) polarization also were examined. Results.: IL-33 mRNA and protein were expressed constitutively in the normal corneas of both groups and were significantly elevated at 1 to 5 days after infection in BALB/c over B6 mice. rmIL-33–treated B6 mice showed less severe disease than did PBS controls and exhibited decreased bacterial load, PMN infiltrate, and corneal mRNA levels for IL-1β, MIP-2, and TNF-α. Th2-type cytokines (IL-4, -5, -10) also were significantly upregulated, and protein levels for TNF-α and IL-10 confirmed the mRNA data. To further investigate IL-33 in corneal inflammation, it was overexpressed in Mφ (RAW264.7 cells). This significantly increased IL-5 and IL-10, while it decreased IFN-γ and other pro-inflammatory cytokines. The role of the Mφ was further tested in infected rmIL-33 compared with PBS-injected mice. Immunostaining showed that rmIL-33 injection shifted Mφ polarization from NO synthase 2 to arginase production. Furthermore, peritoneally elicited cells (B6 mice) treated with lipopolysaccharide and rmIL-33 exhibited elevated ST2 levels and a shift from IL-12 to IL-10 mRNA production. Conclusions.: These data provide evidence that IL-33 promotes a Th2-type immune response and reduces inflammation by polarizing the Mφ production of anti-inflammatory mediators in the cornea.

Hypoxia-inducible factor (HIF)-1α, is a transcription factor that controls energy metabolism and angiogenesis under hypoxic conditions, and a potent regulator of innate immunity. The studies described herein examined the role of HIF-1α in disease resolution in BALB/c (resistant, cornea heals) mice after ocular infection with Pseudomonas (P.) aeruginosa. Furthermore, the current studies focused on the neutrophil (PMN), the predominant cell infiltrate in keratitis. Using both siRNA and an antagonist (17-DMAG), the role of HIF-1α was assessed in P. aeruginosa-infected BALB/c mice. Clinical score and slit lamp photography indicated HIF-1α inhibition exacerbated disease and corneal destruction. Real time RT-PCR, immunohistochemistry, ELISA, Greiss and MPO assays, bacterial load, intracellular killing, phagocytosis and apoptosis assays further tested the regulatory role of HIF-1α. Despite increased pro-inflammatory cytokine expression and increased MPO levels after knocking down HIF-1α expression, in vivo studies revealed a decrease in NO production and higher bacterial load. In vitro studies using PMN provided evidence that although inhibition of HIF-1α did not affect phagocytosis, both bacterial killing and apoptosis were significantly affected, as was production of antimicrobial peptides. Overall, data provide evidence that inhibition of HIF-1α converts a normally resistant disease response to susceptible (corneal thinning and perforation) after induction of bacterial keratitis. Although this inhibition does not appear to affect PMN transmigration or phagocytosis, both in vivo and in vitro approaches indicate that the transcriptional factor is essential for effective bacterial killing, apoptosis and antimicrobial peptide production. Infections of the eye, especially the cornea, often result in vision loss and may require corneal transplantation. Pseudomonas aeruginosa is a common, opportunistic bacterium that can infect the cornea, especially in extended wear contact lens users. There are more than 30 million such individuals in the US alone and they account for over 40% of corneal bacterial infection (keratitis) cases. The cornea itself is a specialized tissue that must effectively combat infections, while preserving intact visual acuity. Using various techniques to mimic in an animal model, what can occur in the human eye, we test how cells and molecules of the immune response react to the bacteria, leading to either its eradication or blindness. By better understanding the functions of the immune system during infection, rational design of more effective treatments for this disease, which preserve a patient's eyesight, are feasible. In addition, what we have learned about the different cells and molecules of the immune response in the eye may be applicable to other infectious diseases, as well.

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.

Using a multiprobe RNase protection assay, we examined cytokine and chemokine mRNAs that were expressed after corneal infection withPseudomonas aeruginosain mice. Cytokines that were upregulated included interleukin-1α (IL-1α) and -1β, IL-1 receptor antagonist, IL-6, IL-11, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, stem cell factor, lymphotoxin β, transforming growth factor β1, and tumor necrosis factor alpha. Chemokine transcripts that were upregulated included Eotaxin; gamma-interferon-inducible protein 10; monocyte chemoattractant protein 1; macrophage inflammatory proteins 1α, 1β, and 2; and RANTES. Peak expression of these cytokines and chemokines was observed between 1 and 3 days after infection. These responses returned to or approached baseline preinfection levels by 7 days after ocular challenge. Identification of the various cytokines and chemokines upregulated during corneal infection provides important information relevant to unraveling the pathogenesis induced by this bacterium and provides hope that specific molecules can be targeted for therapy.