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Meredith S. Gregory-Ksander, Michelle Crane, William J. Vincent; TLR2 Mediated Upregulation Of Ciliary Body-derived CRAMP Is Critical In Defense Against S. Aureus Induced Endophthalmitis. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3649.
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Staphylococcus aureus (S. aureus) is a leading cause of destructive endophthalmitis associated with severe inflammation and loss of retinal function. Innate immunity is the first line of defense against pathogens and we demonstrated previously that the Toll-like receptor 2 (TLR2) was required for bacterial clearance. TLR2 is a signaling receptor of the innate immune system that recognizes S. aureus and induces; secretion of antimicrobial factors, phagocytosis of pathogens, and release of pro-inflammatory cytokines. The current experiments examine the mechanism of TLR2 mediated defense against S. aureus induced endophthalmitis.
TLR2-KO and C57BL/6 WT mice received intravitreal injections of 500 CFU S. aureus (RN6390). Clinical examinations and bacterial quantification were performed at 24, 48, and 72 hrs. H&E retinal sections and myeloperoxidase assay (MPO) were used to assess retinal damage and neutrophil infiltration respectively. Immunofluoresence and western blot analysis were used to determine the location and quantitate the expression of antimicrobial peptides (βdefensins 1-4 and cathelin related antimicrobial peptide (CRAMP)). C57BL/6-NFΚB-eGFP mice, in which activation of NF-ΚB causes the cells to fluoresce green, were used to identify the first cells activated following infection and establish the kinetics of the response.
C57BL/6 WT mice injected with 500 CFU S. aureus successfully cleared the infection as demonstrated by bacterial quantification. By contrast, TLR2-KO mice were unable to clear an identical inoculation resulting in extensive retinal damage. Unexpectedly, MPO analysis revealed no delay or decrease in the number of infiltrating neutrophils in TLR2-KO mice. However, immunofluorescence revealed a TLR2-dependent upregulation of CRAMP at 24 hrs post S. aureus infection in both ciliary body epithelium and neural retina. Western blot analysis revealed a significant increase in activated CRAMP in WT mice as early as 6 hrs post infection and this response was completely abrogated in TLR2-KO mice. Moreover, studies with NFΚB-eGFP mice indicated that upregulation of CRAMP expression coincided with NFΚB activation in the neural retina and ciliary body epithelial cells.
Together these data demonstrate that susceptibility to infection in TLR2-KO mice is not due to decreased or delayed neutrophil infiltration. Rather, our data suggests that an early TLR2-mediated activation of CRAMP produced by resident ocular tissue is critical in the defense against S. aureus induced endophthalmitis. Moreover, tracking NFΚB activation suggests that the ciliary body epithelium may be the primary source of activated CRAMP.
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