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Extracellular matrix protein lumican promotes clearance and resolution of Pseudomonas aeruginosa keratitis in a mouse model.

ABSTRACT: Lumican is an extracellular protein that associates with CD14 on the surface of macrophages and neutrophils, and promotes CD14-TLR4 mediated response to bacterial lipopolysaccharides (LPS). Lumican-deficient (Lum(-/-)) mice and macrophages are impaired in TLR4 signals; raising the possibility that lumican may regulate host response to live bacterial infections. In a recent study we showed that invitro Lum(-/-) macrophages are impaired in phagocytosis of gram-negative bacteria and in a lung infection model the Lum(-/-) mice showed poor survival. The cornea is an immune privileged barrier tissue that relies primarily on innate immunity to protect against ocular infections. Lumican is a major component of the cornea, yet its role in counteracting live bacteria in the cornea remains poorly understood. Here we investigated Pseudomonas aeruginosa infections of the cornea in Lum(-/-) mice. By flow cytometry we found that 24 hours after infection macrophage and neutrophil counts were lower in the cornea of Lum(-/-) mice compared to wild types. Infected Lum(-/-) corneas showed lower levels of the leukocyte chemoattractant CXCL1 by 24-48 hours of infection, and increased bacterial counts up to 5 days after infection, compared to Lum(+/-) mice. The pro-inflammatory cytokine TNF-? was comparably low 24 hours after infection, but significantly higher in the Lum(-/-) compared to Lum(+/-) infected corneas by 2-5 days after infection. Taken together, the results indicate that lumican facilitates development of an innate immune response at the earlier stages of infection and lumican deficiency leads to poor bacterial clearance and resolution of corneal inflammation at a later stage.

SUBMITTER: Shao H

PROVIDER: S-EPMC3554612 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Extracellular matrix protein lumican promotes clearance and resolution of Pseudomonas aeruginosa keratitis in a mouse model.

Shao Hanjuan H Scott Sherri-Gae SG Nakata Chiaki C Hamad Abdel R AR Chakravarti Shukti S

PloS one 20130124 1

Lumican is an extracellular protein that associates with CD14 on the surface of macrophages and neutrophils, and promotes CD14-TLR4 mediated response to bacterial lipopolysaccharides (LPS). Lumican-deficient (Lum(-/-)) mice and macrophages are impaired in TLR4 signals; raising the possibility that lumican may regulate host response to live bacterial infections. In a recent study we showed that invitro Lum(-/-) macrophages are impaired in phagocytosis of gram-negative bacteria and in a lung infec ...[more]

PMID: 23358433

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Project description:Pseudomonas aeruginosa is an important pathogen that causes chronic infections that involve multicellular aggregates called biofilms. Within biofilms, bacteria are surrounded in a protective extracellular matrix of proteins, exopolysaccharides (EPS), and DNA. A key P. aeruginosa matrix protein is an extracellular adhesin called CdrA, which promotes aggregation by binding to the EPS Psl and via CdrA-CdrA interactions. We hypothesized that because of its ability to bind Psl, CdrA would be important only for strains that use Psl as the primary EPS (e.g., the laboratory strain PAO1). Thus, we predicted that cdrA might be dispensable for biofilm formation by strains that do not utilize Psl (e.g., the laboratory strain PA14). Instead, we observed that cdrA deletion strains exhibited biofilm defects, regardless of their EPS dependencies. We screened a panel of clinical and environmental P. aeruginosa isolates for the presence of the cdrA allele and production of CdrA protein. All isolates that we tested contained the cdrA allele, and these alleles had minimal sequence variation compared to the reference PAO1 cdrA gene. Additionally, all isolates except one produced detectable CdrA protein. We investigated the possible mechanisms of CdrA-promoted biofilm formation in these strains where Psl is not dominant, and we discovered that CdrA binds to Pel. Although Psl and Pel chemical structures are distinct, this appears to be a specific interaction, since previous work has shown that CdrA binds discriminately to other EPS. Our findings provide new understanding of biofilm formation across P. aeruginosa isolates and emphasize the versatility of CdrA.IMPORTANCE Depending upon the strain, Pseudomonas aeruginosa can use different exopolysaccharides (e.g., Psl, Pel, and alginate) to build its biofilm matrix. Previously, we demonstrated that the biofilm matrix protein CdrA binds to Psl, promoting biofilm formation and aggregate stability. As such, it was thought that CdrA might be important for biofilm assembly only in strains that rely upon Psl. However, past studies indicated that CdrA can interact with monosaccharides not present in Psl, including N-acetylglucosamine, a constituent of another EPS called Pel. We discovered that CdrA also binds to Pel and promotes biofilm formation by strains in which Psl is not dominant. Thus, our findings suggest that CdrA plays a common role as a biofilm matrix cross-linker across P. aeruginosa isolates with different EPS.

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.

Dataset Information

Extracellular matrix protein lumican promotes clearance and resolution of Pseudomonas aeruginosa keratitis in a mouse model.

Publications

Extracellular matrix protein lumican promotes clearance and resolution of Pseudomonas aeruginosa keratitis in a mouse model.

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