Figure 1.
Increased clinical score in P.aeruginosa infected Lum −/− mice.
Mice were infected with 2×104 CFU P.aeruginosa ATCC19660 in one eye and scored in a blinded manner for 6 days. A representative image of infected eyes 1 and 6 days post infection (d.p.i) shown for each genotype indicates relatively similar severity on day 1 but increased opacity and damage in the Lum−/− cornea on 6 d.p.i (A). Daily disease scores (B) of individual animals shows increased average scores for infected Lum−/− corneas.* p≤0.05.
Figure 2.
Poor clearance of P.aeruginosa in infected Lum −/− corneas.
Viable bacterial CFU were quantified from infected Lum +/− and Lum−/− corneas. CFU were higher in ocular surfaces sampled with filter lifts (A) and whole eye homogenates (B) during the infection. Both methods yielded significantly higher CFU counts from Lum −/− corneas as shown at day 4 (cornea surface lift) and day 2 (whole eye homogenate). *p≤0.05.
Figure 3.
Flow cytometry of 24 hrs infected corneas shows reduced inflammatory cell percentages in Lum−/− corneas.
Total cells from control and infected corneas per genotype (n = 5) were subjected to FACS analysis. Cells were immunostained with F4/80 (macrophage or MΦ marker), Gr-1 (neutrophil or PMN marker) (A) and with APC-Annexin V for detecting apoptotic cells (B). The percentage of MΦs and PMNs with respect to total cells was reduced in the Lum−/− pool to 3.45% and 70.29% respectively, compared to the Lum +/+ and Lum+/− controls. There was a decrease in the percentage of AnnexinV+cells in the Lum−/− PMN population.
Figure 4.
Increased lumican expression in Lum +/− corneas 6 hr post infection.
Lumican transcript relative to18S RNA was significantly increased in Lum+/− corneas 6 hrs after infection as determined by qRT-PCR (n = 4). * p≤0.05.
Figure 5.
Proinflammatory cytokines measured by ELISA in P.aeruginosa infected corneas.
TNF-α level was low and comparable in Lum+/− and Lum−/− corneal protein extracts 3 hrs after infection (A) but significantly higher in Lum−/− infected corneas 2 days after infection (B). IL-1β was induced comparably in Lum+/− and Lum−/− infected corneas for up to 5 days after infection (C) and IL-12 (D) was detected at very low levels in infected corneas of both genotypes. * p≤0.05.
Figure 6.
Differential regulation of CXCL1 in infected Lum +/− and Lum −/− corneas.
CXCL1 measured by ELISA increased 24 hrs after infection in both genotypes, with its level being consistently lower in the Lum−/− corneal extracts - the difference being statistically significant at the 2 d.p.i time point (A). Three hours after infection CXCL1 levels were comparably low in both genotypes (n = 3) (B). The Cxcl1 transcript measured by qRT-PCR in quadruplicates/animal was increased over baseline 6 hrs post infection; by 24–48 hrs post infection Lum −/− corneas showed higher levels of Cxcl1 compared to Lum +/− (C). * p≤0.05.
Figure 7.
Increased tissue damage in infected corneas of Lum +/− and Lum −/− mice.
Paraffin-embedded sections of eyes 24 hrs (A) and 48 hrs (B) after infection were stained with H and E. To examine tissue damage in mice with comparable disease all infected animals used for histology had an initial disease score of 2 to 3 and showed PMN infiltrations in the cornea and anterior chamber. The Lum −/− infected corneas showed large areas of epithelial ulcerations (arrow) and stromal damage (arrowhead). Scale bar, 100 µm.