Linda D. Hazlett

Corneal response to Pseudomonas aeruginosa infection

Pseudomonas aeruginosa (P. aeruginosa) is a common organism associated with bacterial keratitis, especially in those who use extended wear contact lenses. Recent advances in our understanding of host innate and adaptive immune responses to experimental infection have been made using a variety of animal models, including inbred murine models that are classed as resistant (cornea heals) vs. susceptible (cornea perforates). Evidence has been provided that sustained IL-12-driven IFN-gamma production in dominant Th1 responder strains such as C57BL/6 (B6) contributes to corneal destruction and perforation, while IL-18-driven production of IFN-gamma in the absence of IL-12 is associated with bacterial killing and less corneal destruction in dominant Th2 responder strains such as BALB/c. The critical role of IL-1 and chemotactic cytokines such as MIP-2 in PMN recruitment and the critical role of this cell in the innate immune response to bacterial infection is reviewed. Regulation of PMN persistence is also discussed and evidence provided that persistence of PMN in B6 cornea is regulated by CD4+ T cells, while macrophages regulate PMN number in the cornea of BALB/c mice. The studies provide a better understanding of the inflammatory mechanisms that are operative in the cornea after P. aeruginosa challenge and are consistent with long-term goals of providing targets for alternative or adjunctive treatment for this disease. Future studies will be aimed at better defining the role of Toll receptors, neuropeptides (as unconventional modulators of the immune response) and exploitation of disease control by new techniques, such as RNA silencing.

Defensins in innate immunity

The innate immune system is the first line of defense against many common microorganisms, which can initiate adaptive immune responses to provide increased protection against subsequent re-infection by the same pathogen. As a major family of antimicrobial peptides, defensins are widely expressed in a variety of epithelial cells and sometimes in leukocytes, playing an important role in the innate immune system due to their antimicrobial, chemotactic and regulatory activities. This review introduces their structure, classification, distribution, synthesis, and focuses on their biological activities and mechanisms, as well as clinical relevance. These studies of defensins in the innate immune system have implications for the prevention and treatment of a variety of infectious diseases, including bacterial ocular disease.

Prolonged Elevation of IL-1 in Pseudomonas aeruginosa Ocular Infection Regulates Macrophage-Inflammatory Protein-2 Production, Polymorphonuclear Neutrophil Persistence, and Corneal Perforation

The kinetics of IL-1 (α and β) production after Pseudomonas aeruginosa corneal infection was examined in susceptible (cornea perforates) C57BL/6J (B6) and resistant (cornea heals) BALB/cByJ (BALB/c) mice. IL-1α and -1β (mRNA and protein) were elevated in both mouse strains, and levels peaked at 1 day postinfection (p.i.). Significantly greater amounts of IL-1 protein were detected in B6 vs BALB/c mice at 1 and 3 days p.i. At 5 days p.i., IL-1α and -1β (mRNA and protein) remained elevated in B6, but began to decline in BALB/c mice. To test the significance of elevated IL-1 in B6 mice, a polyclonal neutralizing Ab against IL-1β was used to treat infected B6 mice. A combination of subconjunctival and i.p. administration of IL-1β polyclonal Ab significantly reduced corneal disease. The reduction in disease severity in infected B6 mice was accompanied by a reduction in corneal polymorphonuclear neutrophil number, bacterial load, and macrophage inflammatory protein-2 mRNA and protein levels. These data provide evidence that IL-1 is an important contributor to P. aeruginosa corneal infection. At least one mechanism by which prolonged and/or elevated IL-1 expression contributes to irreversible corneal tissue destruction appears to be by increasing macrophage inflammatory protein-2 production, resulting in a prolonged stimulation of polymorphonuclear neutrophil influx into cornea. In contrast, a timely down-regulation of IL-1 appears consistent with an inflammatory response that is sufficient to clear the bacterial infection with less corneal damage.

Macrophage Inflammatory Protein-2 Is a Mediator of Polymorphonuclear Neutrophil Influx in Ocular Bacterial Infection

Polymorphonuclear neutrophils (PMN) in Pseudomonas aeruginosa-infected cornea are required to clear bacteria from affected tissue, yet their persistence may contribute to irreversible tissue destruction. This study examined the role of C-X-C chemokines in PMN infiltration into P. aeruginosa-infected cornea and the contribution of these mediators to disease pathology. After P. aeruginosa challenge, corneal PMN number and macrophage inflammatory protein-2 (MIP-2) and KC levels were compared in mice that are susceptible (cornea perforates) or resistant (cornea heals) to P. aeruginosa infection. While corneal PMN myeloperoxidase activity (indicator of PMN number) was similar in both groups of mice at 1 and 3 days postinfection, by 5–7 days postinfection corneas of susceptible mice contained a significantly greater number of inflammatory cells. Corneal MIP-2, but not KC, levels correlated with persistence of PMN in the cornea of susceptible mice. To test the biological relevance of these data, resistant mice were treated systemically with rMIP-2. This treatment resulted in increased corneal PMN number and significantly exacerbated corneal disease. Conversely, administration of neutralizing MIP-2 pAb to susceptible mice reduced both PMN infiltration and corneal destruction. Collectively, these findings support an important role for MIP-2 in recruitment of PMN to P. aeruginosa-infected cornea. These data also strongly suggest that a timely down-regulation of the host inflammatory response is critical for resolution of infection.

IL-33 Shifts Macrophage Polarization, Promoting Resistance against Pseudomonas aeruginosa Keratitis

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 (Mvarphi) 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-1beta, MIP-2, and TNF-alpha. Th2-type cytokines (IL-4, -5, -10) also were significantly upregulated, and protein levels for TNF-alpha and IL-10 confirmed the mRNA data. To further investigate IL-33 in corneal inflammation, it was overexpressed in Mvarphi (RAW264.7 cells). This significantly increased IL-5 and IL-10, while it decreased IFN-gamma and other pro-inflammatory cytokines. The role of the Mvarphi was further tested in infected rmIL-33 compared with PBS-injected mice. Immunostaining showed that rmIL-33 injection shifted Mvarphi 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 Mvarphi production of anti-inflammatory mediators in the cornea.

SIGIRR Promotes Resistance against Pseudomonas aeruginosa Keratitis by Down-Regulating Type-1 Immunity and IL-1R1 and TLR4 Signaling

Pseudomonas aeruginosa keratitis destroys the cornea in susceptible Th1 responder C57BL/6 (B6), but not resistant Th2 responder (BALB/c) mice. To determine whether single Ig IL-1R-related molecule (SIGIRR) played a role in resistance, mRNA and protein expression levels were tested. Both were constitutively expressed in the cornea of the two mouse groups. A disparate mRNA and protein expression pattern was detected in the cornea of BALB/c vs B6 mice after infection. SIGIRR protein decreased significantly in BALB/c over B6 mice at 1 day postinfection. Thus, BALB/c mice were injected with an anti-SIGIRR Ab or IgG control. Anti-SIGIRR Ab over control-treated mice showed increased corneal opacity, stromal damage, and bacterial load. Corneal mRNA levels for IL-1β, MIP-2, IL-1R1, TLR4, IL-18, and IFN-γ and protein levels for IL-1β and MIP-2 also were significantly up-regulated in anti-SIGIRR Ab over control mice, while no changes in polymorphonuclear cell number, IL-4, or IL-10 mRNA expression were detected. To further define the role of SIGIRR, RAW264.7 macrophage-like cells were transiently transfected with SIGIRR and stimulated with heat-killed P. aeruginosa or LPS. SIGIRR transfection significantly decreased mRNA levels for IL-1R1, TLR4, and type 1 immune response-associated cytokines (IL-12, IL-18, and IFN-γ) as well as proinflammatory cytokines IL-1β and MIP-2 protein expression. SIGIRR also negatively regulated IL-1 and LPS, but not poly(I:C)-mediated signaling and NF-κB activation. These data provide evidence that SIGIRR is critical in resistance to P. aeruginosa corneal infection by down-regulating type 1 immunity, and that it negatively regulates IL-1 and TLR4 signaling.

Macrophages Restrict Pseudomonas aeruginosa Growth, Regulate Polymorphonuclear Neutrophil Influx, and Balance Pro- and Anti-Inflammatory Cytokines in BALB/c Mice

The role of macrophages in Pseudomonas aeruginosa corneal infection in susceptible (cornea perforates), C57BL/6 (B6) vs resistant (cornea heals), BALB/c mice was tested by depleting macrophages using subconjunctival injections of clodronate-containing liposomes before corneal infection. Both groups of inbred mice treated with clodronate-liposomes compared with PBS-liposomes (controls) exhibited more severe disease. In B6 mice, the cornea perforated and the eye became extremely shrunken, whereas in BALB/c mice, the cornea perforated rather than healed. The myeloperoxidase assay detected significantly more PMN in the cornea of both groups of mice treated with clodronate-liposomes vs PBS-liposomes. In independent experiments, ELISA analysis showed that protein levels for IL-1β, macrophage-inflammatory protein 2, and macrophage-inflammatory protein 1α, all regulators of PMN chemotaxis, also were elevated in both groups of mice treated with clodronate-liposomes. Bacterial plate counts in B6 mice treated with clodronate-liposomes were unchanged at 3 days and were higher in control-treated mice at 5 days postinfection (p.i.), whereas in BALB/c mice, bacterial load was significantly elevated in the cornea of mice treated with clodronate-liposomes at both 3 and 5 days p.i. mRNA expression levels for pro (IFN-γ and TNF-α)- and anti (IL-4 and IL-10)-inflammatory cytokines also were determined in BALB/c mice treated with clodronate-liposomes vs control-treated mice. Expression levels for IFN-γ were significantly elevated in mice treated with clodronate-liposomes at 3 and 5 days p.i., while IL-10 levels (mRNA and protein) were reduced. These data provide evidence that macrophages control resistance to P. aeruginosa corneal infection through regulation of PMN number, bacterial killing and balancing pro- and anti-inflammatory cytokine levels.

Flagellin Suppresses the Inflammatory Response and Enhances Bacterial Clearance in a Murine Model of Pseudomonas aeruginosa Keratitis

ABSTRACT Pseudomonas aeruginosa is a common organism associated with bacterial keratitis, especially in extended-wear contact lens users. In the present study, we determined that pretreatment of cultured human corneal epithelial cells with flagellin isolated from the P. aeruginosa PAO1 strain attenuated cytokine production when the cells were challenged with a cytotoxic strain (ATCC 19660), suggesting a potential use of bacterial flagellin to downregulate infection-associated inflammation in vivo. Administration of flagellin via the subconjunctival and intraperitoneal routes 24 h prior to Pseudomonas inoculation significantly improved the disease outcome, preserved structural integrity and transparency, and thus maintained vision in otherwise perforated corneas of C57BL/6 (B6) mice. The flagellin pretreatment resulted in suppression of polymorphonuclear leukocyte infiltration at a late stage of infection but not at an early stage of infection, decreased the expression of proinflammatory cytokine genes (genes encoding interleukin-1β [IL-1β], macrophage inflammatory protein 2, IL-12, and gamma interferon), and greatly enhanced bacterial clearance in the corneas of B6 mice probably through induced expression of the cathelicidin-related antimicrobial peptide and inducible nitric oxide synthase. This is the first report that describes the protective mechanisms induced by a Toll-like receptor agonist that not only curbs the host inflammatory response but also eliminates invading bacteria in the B6 mouse cornea.

β-Defensin-2 Promotes Resistance against Infection with P. aeruginosa

Corneal infection with Pseudomonas aeruginosa results in corneal perforation in susceptible C57BL/6 (B6) mice, but not in resistant BALB/c mice. To explore the role of two important defensins, murine β-defensin-1 (mBD1) and mBD2, in the ocular immune defense system, their mRNA and protein expression levels were tested by real-time RT-PCR and Western blot, respectively. mRNA, protein, and immunostaining data demonstrated that both mBD1 and mBD2 were constitutively expressed in normal BALB/c and B6 corneas, and they were disparately up-regulated in BALB/c (more) vs B6 (less) corneas after infection. To determine whether either defensin played a role in host resistance, BALB/c mice were treated with either mBD1 or mBD2 small interfering RNA by subconjunctival injection together with topical application. Increased corneal opacity and worsened disease were displayed after knockdown of mBD2 but not of mBD1. mBD2 silencing also increased bacterial counts and polymorphonuclear neutrophil infiltration in BALB/c corneas. Real-time RT-PCR data further demonstrated that mBD2, not mBD1, differentially modulated mRNA expression of proinflammatory cytokines/molecules such as IFN-γ, MIP-2, IL-1β, TNF-α, IL-6, and inducible NO synthase; TLR signaling molecules, including TLR2, TLR4, TLR9, and MyD88; and the transcription factor NF-κB. Additionally, in vivo studies indicated that mBD2 silencing enhanced corneal nitrite levels and NF-κB activation. Collectively, the data provide evidence that mBD2, but not mBD1, is required for host resistance against P. aeruginosa-induced corneal infection.

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.