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Pseudomonas aeruginosa keratitis: passive immunotherapy with antibodies raised against divalent flagellin.

ABSTRACT: Objectives:Pseudomonas aeruginosa infections such as keratitis are considered among the major health problems worldwide due to the complexity of pathogenesis and antibiotic resistance crisis, thus, finding new effective approaches for prevention and treatment of the infections seem to be still vital. In this report, we aimed to investigate the therapeutic effects of topical administration of the antibodies against type a and b-flagellin (FLA and FLB) in Pseudomonas keratitis model of infection in mice. Materials and Methods:Scratched corneas of mice were treated with approximately 107 CFUs/eye of PAK and/or PAO1 strains of P. aeruginosa. Specific IgG to FLA, FLB or divalent flagellin were topically applied to the infected corneas for 20 min, 24, and 36 hr post-infection. The bacterial burden and myeloperoxidase activity (as a marker for polymorphonuclears (PMNs) infiltration) were determined in the corneas. The biological activity of the anti-FLA and FLB IgG was evaluated in vitro by opsonophagocytosis test. Results:Compared to other treated corneas, divalent anti-flagellin IgG treatment showed a significant decrease in the bacterial CFUs and myeloperoxidase activity in the infected corneas (P<0.05). Results of opsonophagocytosis revealed that the specific antibodies raised against FLA and FLB had more potent opsonic killing activity on their homologous strains as compared with control group (P<0.05). Conclusion:It appears that in P. aeruginosa keratitis, topical administration of the combined antibodies likely via decreasing the bacterial load, and PMNs infiltration as well as increasing opsonophagocytosis could lead to dramatic improvement of the infected corneas.

SUBMITTER: Mahin Samadi P

PROVIDER: S-EPMC6437458 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Project description:Pseudomonas aeruginosa (PA) is the leading cause of bacterial keratitis, especially in those who wear contact lens and who are immunocompromised. Once the invading pathogens are recognized by pattern recognition receptors expressed on the innate immune cells, the innate immune response is stimulated to exert host defense function, which is the first line to fight against PA infection. As a converging point of cytosolic DNA sense signaling, stimulator of interferon genes (STING) was reported to participate in host-pathogen interaction. However, the role of STING in regulating PA-induced corneal inflammation and bacterial clearance remains unknown. Our data demonstrated that STING was activated in murine model of PA keratitis and in in vitro-cultured macrophages, indicated by Western blot, immunostaining, and flow cytometry. To explore the role of STING in PA keratitis, we used siRNA to silence STING and 2',3'-cGAMP to activate STING in vivo and in vitro, and the in vivo data found out that STING promoted host resistance against PA infection. To investigate the reason why STING played a protective role in PA keratitis, the inflammatory cytokine secretion and bacterial load were measured by using real-time PCR and bacterial plate count, respectively. Our data demonstrated that STING suppressed the production of inflammatory cytokines and enhanced bacterial elimination in murine model of PA keratitis and in PA-infected macrophages. To further investigate the mechanism beneath, the phosphorylation of mitogen-activated protein kinase, the nuclear translocation of nuclear factor-κB (NF-κB) and the bactericidal mechanism were measured by western-blot, immunofluorescence, and real-time PCR, respectively. Our data indicated that STING suppressed inflammatory cytokine expressing via restraining NF-κB activity and enhanced inducible NO synthase expression, an oxygen-dependent bactericidal mechanism. In conclusion, this study demonstrated that STING promoted host resistance against PA keratitis and played a protective role in PA-infected corneal disease, via inhibiting corneal inflammation and enhancing bacterial killing.

Project description:A worrisome trend in the study and treatment of infectious disease noted in recent years is the increase in multidrug resistant strains of bacteria concurrent with a scarcity of new antimicrobial agents to counteract this rise. This is particularly true amongst bacteria within the Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species (ESKAPE) designation. P. aeruginosa is one of the most common causes of bacterial keratitis. Therefore, it is of vital importance to characterize new antimicrobial agents with anti-Pseudomonal activity for use with the ocular surface. MEDI3902 is a multifunctional antibody that targets the P. aeruginosa persistence factor Psl exopolysaccharide, and the type 3 secretion protein PcrV. We initially assessed this antibody for ocular surface toxicity. The antimicrobial activity of the antibody was then tested by treating mice with established P. aeruginosa keratitis with both topical and intravenous treatment modalities. MEDI3902, was shown to be non-toxic to the ocular surface of mice when given topically. It was also effective compared to the control antibody at preventing P. aeruginosa keratitis with a one-time treatment at the time of infection. Both topical and intravenous administration of MEDI3902 has been proved significant in treating established keratitis infections as well, speeding the resolution of infection significantly more than that of the control IgG. We report the first use of a topical immunotherapeutic multifunctional agent targeting Psl and type 3 secretion on the ocular surface as an antimicrobial agent. While MEDI3902 has been shown to prevent Pseudomonas biofilm formation in keratitis models when given prophylactically intravitally, we show that MEDI3902 has the capability to also treat an active infection when given intravenously to mice with Pseudomonas keratitis. Our data indicate antibodies are well tolerated and nontoxic on the ocular surface. They reduce infection in mice treated concurrently at inoculation and reduced the signs of cornea pathology in mice with established infection. Taken together, these data indicate treatment with monoclonal antibodies directed against Psl and PcrV may be clinically effective in the treatment of P. aeruginosa keratitis and suggest that the design of further antibodies to be an additional tool in the treatment of bacterial keratitis.

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Pseudomonas aeruginosa keratitis: passive immunotherapy with antibodies raised against divalent flagellin.

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