TLR4 and TLR5 on Corneal Macrophages Regulate Pseudomonas aeruginosa Keratitis by Signaling through MyD88-Dependent and -Independent Pathways

Abstract

Pseudomonas aeruginosa is a major cause of blindness and visual impairment in the United States and worldwide. Using a murine model of keratitis in which abraded corneas are infected with P. aeruginosa parent and ΔfliC (aflagellar) strains 19660 and PAO1, we found that F4/80+ macrophages were the predominant cell type in the cornea expressing TLR2, TLR4, and TLR5. Depletion of macrophages and dendritic cells using transgenic Mafia mice, in which Fas ligand is selectively activated in these cells, resulted in diminished cytokine production and cellular infiltration to the corneal stroma and unimpaired bacterial growth. TLR4−/− mice showed a similar phenotype postinfection with ΔfliC strains, whereas TLR4/5−/− mice were susceptible to corneal infection with parent strains. Bone marrow-derived macrophages stimulated with ΔfliC bacteria induced Toll/IL-1R intracellular domain (TIR)-containing adaptor inducing IFN-β (TRIF)-dependent phosphorylation of IFN regulatory factor 3 in addition to TIR-containing adaptor protein/MyD88-dependent phosphorylation of IκB and nuclear translocation of the p65 subunit of NFκB. Furthermore, TRIF−/− mice showed a similar phenotype as TLR4−/− mice in regulating only ΔfliC bacteria, whereas MyD88−/− mice were unable to clear parent or ΔfliC bacteria. Finally, IL-1R1−/− and IL-1α/β−/− mice were highly susceptible to infection. Taken together, these findings indicate that P. aeruginosa activates TLR4/5 on resident corneal macrophages, which signal through TRIF and TIR-containing adaptor protein/MyD88 pathways, leading to NF-κB translocation to the nucleus, transcription of CXCL1 and other CXC chemokines, recruitment of neutrophils to the corneal stroma, and subsequent bacterial killing and tissue damage. IL-1α and IL-1β are also produced, which activate an IL-1R1/MyD88-positive feedback loop in macrophages and IL-1R on other resident cells in the cornea.

Acanthamoeba Activates Macrophages Predominantly through Toll-Like Receptor 4- and MyD88-Dependent Mechanisms To Induce Interleukin-12 (IL-12) and IL-6

“…Furthermore, these macrophages have been found to be important in a murine model of Pseudomonas aeruginosa infection. Thus, F4/80 ϩ cells present in the corneal stroma expressing TLR4, TLR5, and TLR2 were found to respond to Pseudomonas by releasing cytokines in a TLR4/TIRAP/MyD88 and TLR4/TRIF-NF-B translocation-dependent manner (41). Thus, the studies described herein could also have relevance to AK.…”

Interferon-γ-induced MD-2 Protein Expression and Lipopolysaccharide (LPS) Responsiveness in Corneal Epithelial Cells Is Mediated by Janus Tyrosine Kinase-2 Activation and Direct Binding of STAT1 Protein to the MD-2 Promoter

“…For corneal inflammation, 20 g of UltaPure TLR4-specific Escherichia coli LPS (strain K12; Invivogen) in 2 l of sterile endotoxin-free water were placed on the ocular surface as described (9,18,19). After 24 h, mice were euthanized, corneal haze was measured by in vivo confocal microscopy using the Nidek Confoscan TM , and neutrophil recruitment to the cornea was examined by immunohistochemistry using rat anti-mouse neutrophil antibody (NIMP-R14; Abcam, Cambridge, MA).…”

Unexpected Roles for Toll-Like Receptor 4 and TRIF in Intraocular Infection with Gram-Positive Bacteria

“…However, in the EIU model, some of TRIFdependent cytokines were found to be proinflammatory (46), while other TRIF-dependent cytokines were anti-inflammatory (43,47). In Pseudomonas aeruginosa keratitis models, MyD88-and TRIF-mediated signaling following LPS recognition by TLR4 has been reported (48)(49)(50). MyD88 has also been reported to be an important contributor in other retinal diseases, such as diabetic retinopathy (51) and age-related macular degeneration (AMD) (52).…”