TLR4 is required for host resistance in Pseudomonas aeruginosa keratitis.
OBJECTIVE
To determine the role of Toll-like receptor 4 (TLR4) in Pseudomonas aeruginosa (P. aeruginosa) keratitis in resistant (cornea-healing) BALB/c mice.


METHODS
Corneal TLR4 mRNA levels were tested by real-time PCR in BALB/c mice before and after infection. Clinical score, slit lamp, histopathology, bacterial counts, and polymorphonuclear neutrophil (PMN) quantitation were performed in the infected cornea of TLR4-deficient (TLR4(lps-d)) and wild-type BALB/c mice. mRNA for IL-1beta, MIP-2, IFN-gamma, IL-18, inducible nitric oxide synthase (iNOS), and beta-defensin-2 levels were measured by real-time PCR. Protein levels for IL-1beta, MIP-2, and IFN-gamma were tested by ELISA.


RESULTS
In resistant BALB/c mice, TLR4 mRNA expression was significantly upregulated in the cornea after P. aeruginosa infection. In contrast, TLR4-deficient mice were susceptible to infection with P. aeruginosa and showed increased corneal opacity, PMN infiltration, bacterial counts, and perforated infected corneas. After infection, TLR4-deficient mice also showed increased mRNA expression of proinflammatory cytokines (IL-1beta and MIP-2) and type-1-associated cytokines (IFN-gamma and IL-18) when compared with wild-type BALB/c mice. ELISA analyses showed that IL-1beta, MIP-2, and IFN-gamma protein levels also were significantly upregulated in the cornea of TLR4-deficient versus wild-type mice. In contrast, levels of iNOs and beta-defensin-2 were significantly decreased in TLR4-deficient compared with wild-type mice.


CONCLUSIONS
TLR4 is critical in host resistance to P. aeruginosa, as its deficiency results in increased PMN infiltration and proinflammatory cytokine production, decreased iNOs and beta-defensin-2 production, impaired bacterial killing, and a susceptible phenotype.
Huang
Xi
X
Department of Anatomy and Cell Biology, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201, USA. xhuang@med.wayne.edu
Du
Wenjin
W
McClellan
Sharon A
SA
Barrett
Ronald P
RP
Hazlett
Linda D
LD
D000818:Animals; D015169:Colony Count, Microbial; D016207:Cytokines / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D015818:Eye Infections, Bacterial / Q000276:immunology* / Q000382:microbiology; D005260:Female; D007113:Immunity, Innate / Q000276:immunology*; D007634:Keratitis / Q000276:immunology* / Q000382:microbiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D018345:Mice, Knockout; D009504:Neutrophils / Q000502:physiology; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D009195:Peroxidase / Q000378:metabolism; D011552:Pseudomonas Infections / Q000276:immunology* / Q000382:microbiology; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051197:Toll-Like Receptor 4 / Q000502:physiology*; D015854:Up-Regulation; D023083:beta-Defensins / Q000378:metabolism
D016207:Cytokines; D012333:RNA, Messenger; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D023083:beta-Defensins; D009195:Peroxidase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse