Xiaowen L. Rudner

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.

Analysis of adhesion, piliation, protease production and ocular infectivity of several P. aeruginosa strains

The role of bacterial piliation and protease production in Pseudomonas aeruginosa adhesion to the injured corneal epithelial surface and subsequent infectivity was examined using several bacterial strains, including three that were hyperpiliated. To initiate this study, bacteria were examined by transmission EM to confirm their piliation characteristics. The PAK strain, like pseudomonas ATCC 19660, possessed about 1-4 polar pili. The mutant PAK/PR11 lacked pili while PAK/PR1, DB2, a mutant of PAO1, and PA1244, a wild-type clinical isolate, were hyperpiliated. Ocular infectivity of these bacterial strains and mutants was examined macroscopically and histopathologically in mice and these data compared to the well-characterized ocular disease response of a murine model of infection with pseudomonas ATCC 19660. The PAK strain was infective, but less virulent than strain 19660 by both macroscopic grading and histopathological analysis of infected eyes. Infectivity of the PR11 mutant was similar to the PAK parent strain, while PR1, DB2 and 1244, all hyperpiliated, were not infective. To explore the hypothesis that hyperpiliated bacteria bound less well to cornea and thus failed to induce corneal disease, in vitro quantitative studies of bacterial adhesion were done using an ocular organ culture model. The PR1 hyperpiliated mutant bound significantly less well to cornea than the PAK parent strain, PR11 mutant or pseudomonas 19660, while DB2 and 1244 binding did not differ significantly from 19660 or PAK. Examination of protease production, another factor which may influence adhesion, revealed that only 19660 and DB2 produced detectable protease. This study provides evidence that non-piliated, non-protease producing strains such as PAK/PR11 possess alternate virulence mechanisms to facilitate binding to and infectivity of corneal tissue.

B7/CD28 Costimulation Is Critical in Susceptibility to Pseudomonas aeruginosa Corneal Infection: A Comparative Study Using Monoclonal Antibody Blockade and CD28-Deficient Mice

Evidence suggests that Pseudomonas aeruginosa stromal keratitis and corneal perforation (susceptibility) is a CD4+ T cell-regulated inflammatory response following experimental P. aeruginosa infection. This study examined the role of Langerhans cells (LC) and the B7/CD28 costimulatory pathway in P. aeruginosa-infected cornea and the contribution of costimulatory signaling by this pathway to disease pathology. After bacterial challenge, the number of LC infiltrating the central cornea was compared in susceptible C57BL/6 (B6) vs resistant (cornea heals) BALB/c mice. LC were more numerous at 1 and 6 days postinfection (p.i.), but were similar at 4 days p.i., in susceptible vs resistant mice. Mature, B7 positive-stained LC in the cornea and pseudomonas Ag-associated LC in draining cervical lymph nodes also were increased significantly p.i. in susceptible mice. To test the relevance of these data, B6 mice were treated systemically and subconjunctivally with neutralizing B7 (B7-1/B7-2) mAbs. Treatment decreased corneal disease severity and reduced significantly the number of B7-positive cells as well as the recruitment and activation of CD4+ T cells in the cornea. IFN-γ mRNA levels also were decreased significantly in the cornea and in draining cervical lymph nodes of mAb-treated mice. When CD28−/− animals were tested, they exhibited a less severe disease response (no corneal perforation) than wild-type B6 mice and had a significantly lower delayed-type hypersensitivity response to heat-killed pseudomonas Ag. These results support a critical role for B7/CD28 costimulation in susceptibility to P. aeruginosa ocular infection.

Systemic and topical protection studies using Pseudomonas aeruginosa flagella in an ocular model of infection

Purified flagella from P. aeruginosa ATCC 19660 were used for active, passive, or topical immunization prior to corneal challenge with strain 19660. At 30 days post-infection, a significant number of mice actively or passively immunized with flagella and infected with the homologous bacterial strain were protected from ocular disease when compared to control animals. In topical immunization studies, premixing of 19660 flagella with the bacterial inoculum prior to ocular challenge with strain 19660, provided results similar to those of the active or passive immunization studies. A reduced lipopolysaccharide (LPS:1 E.U./mg) flagella preparation was also produced and used similarly. Again, significant protection was achieved in mice immunized by flagella regardless of the immunization route. An in vitro adherence assay also was performed to examine quantitatively the effect of exogenously applied flagella, or an antiflagella monoclonal antibody (MAb) on bacterial adhesion. Premixing of the bacterial inoculum with flagella or the MAb prior to applying it topically to corneas in organ culture all significantly inhibited bacterial binding. These results strongly suggest that significant ocular protection is achieved with either active or passive immunization, or premixing of the bacterial inoculum with flagella from strain 19660 prior to ocular challenge with the homologous bacterial strain. They also indicate that topical application of flagella or antiflagella MAb provide protection against ocular disease by decreasing bacterial adhesion to cornea.