Extracellular Matrix Protein Lumican Promotes Clearance and Resolution of <em>Pseudomonas aeruginosa</em> Keratitis in a Mouse Model

<div><p>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 (<em>Lum</em>^−/−) 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 <em>in</em><em>vitro Lum</em>^−/− macrophages are impaired in phagocytosis of gram-negative bacteria and in a lung infection model the <em>Lum</em>^−/− 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 <em>Lum</em>^−/− mice. By flow cytometry we found that 24 hours after infection macrophage and neutrophil counts were lower in the cornea of <em>Lum</em>^−/− mice compared to wild types. Infected <em>Lum</em>^−/− 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 <em>Lum^+/−</em> mice. The pro-inflammatory cytokine TNF-α was comparably low 24 hours after infection, but significantly higher in the <em>Lum</em>^−/− compared to <em>Lum</em>^+/− 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.</p> </div

Differential regulation of CXCL1 in infected <i>Lum ^+/−</i> and <i>Lum ^−/−</i> corneas.

<p>CXCL1 measured by ELISA increased 24 hrs after infection in both genotypes, with its level being consistently lower in the <i>Lum</i>^−/− 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 <i>Cxcl1</i> transcript measured by qRT-PCR in quadruplicates/animal was increased over baseline 6 hrs post infection; by 24–48 hrs post infection <i>Lum </i>^−/− corneas showed higher levels of <i>Cxcl</i>1 compared to <i>Lum </i>^+/− (C). * <i>p</i>≤0.05.</p

Poor clearance of <i>P.aeruginosa</i> in infected <i>Lum ^−/−</i> corneas.

<p>Viable bacterial CFU were quantified from infected <i>Lum ^+/−</i> and <i>Lum</i>^−/− 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 <i>Lum ^−/−</i> corneas as shown at day 4 (cornea surface lift) and day 2 (whole eye homogenate). *<i>p</i>≤0.05.</p

Proinflammatory cytokines measured by ELISA in <i>P.aeruginosa</i> infected corneas.

<p>TNF-α level was low and comparable in <i>Lum</i>^+/− and <i>Lum</i>^−/− corneal protein extracts 3 hrs after infection (A) but significantly higher in <i>Lum</i>^−/− infected corneas 2 days after infection (B). IL-1β was induced comparably in <i>Lum</i>^+/− and <i>Lum</i>^−/− 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. * <i>p</i>≤0.05.</p

Increased lumican expression in <i>Lum ^+/−</i> corneas 6 hr post infection.

<p>Lumican transcript relative to18S RNA was significantly increased in <i>Lum</i>^+/− corneas 6 hrs after infection as determined by qRT-PCR (n = 4). * <i>p</i>≤0.05.</p

Increased clinical score in <i>P.aeruginosa</i> infected <i>Lum</i>^−/− mice.

<p>Mice were infected with 2×10⁴ CFU <i>P.aeruginosa</i> 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 <i>Lum</i>^−/− cornea on 6 d.p.i (A). Daily disease scores (B) of individual animals shows increased average scores for infected <i>Lum</i>^−/− corneas.* <i>p</i>≤0.05.</p

Increased tissue damage in infected corneas of <i>Lum</i>^+/− and <i>Lum ^−/−</i> mice.

<p>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 <i>Lum ^−/−</i> infected corneas showed large areas of epithelial ulcerations (arrow) and stromal damage (arrowhead). Scale bar, 100 µm.</p

Clinical score frequencies.

<p>Clinical disease scores were given in a blinded manner (A) 24 and (B) 48-hours post infection (h p.i.). Results are shown as percent of animals examined in a total of 5 trials per genotype with each trial containing 6–9 animals per genotype. The <i>Pglyrp</i>-2^-/- mice showed clinical scores comparable to WT in the 24 h.p.i group; the <i>Pglyrp</i>-3^-/- and -4^-/- mice had a few animals with maximal scores by 24 h.p.i suggesting escalated disease. By 48 hours the <i>Pglyrp</i>-2^-/- mice showed reduced clinical scores.</p

Functions of Peptidoglycan Recognition Proteins (Pglyrps) at the Ocular Surface: Bacterial Keratitis in Gene-Targeted Mice Deficient in <i>Pglyrp</i>-2, -3 and -4

<div><p>Purpose</p><p>Functions of antimicrobial peptidoglycan recognition proteins (Pglyrp1-4) at the ocular surface are poorly understood. Earlier, we reported an antibacterial role for <i>Pglyrp</i>-1 in <i>Pseudomonas aeruginosa</i> keratitis. Here we investigated functions of three other related genes <i>Pglyrp</i>-2, -3 and -4 in a mouse model of <i>P</i>. <i>aeruginosa</i> keratitis.</p><p>Methods</p><p>Wild type (WT) and each of the <i>Pglyrp</i>-null genotypes were challenged with <i>P</i>. <i>aeruginosa</i> keratitis. The eyes were scored in a blinded manner 24 and 48h post infection. Viable bacterial counts and inflammatory factors (IL-12, TNF-α, IFN-γ, CCL2, IL-6 and IL-10) were measured in whole eye homogenates using cytometric bead arrays. Expressions of <i>Pglyrp-1-4</i>, mouse beta defensins (<i>mBD)-2</i>,<i>-3</i>, cathelicidin-related antimicrobial peptide (CRAMP) were determined by qRTPCR in total RNA extracts of uninfected and infected eyes of WT and each of the <i>Pglyrp</i>-null mouse types.</p><p>Results</p><p>The <i>Pglyrp-2</i>^<i>-/-</i> mice showed reduced disease and lower induction of pro-inflammatory TNF-α (<i>p</i> = 0.02) than WT or the other <i>Pglyrp</i> null mice. Viable bacterial yield was significantly lower in the <i>Pglyrp</i>-2^-/- (<i>p</i> = 0.0007) and the <i>Pglyrp</i>-4^-/- (<i>p</i> = 0.098) mice. With regards to expression of these antimicrobial genes, <i>Pglyrp</i>-2 expression was induced after infection in WT mice. <i>Pglyrp</i>-3 expression was low before and after infection in WT mice, while <i>Pglyrp</i>-4 expression was slightly elevated after infection in WT, <i>Pglyrp</i>-2 and -3 null mice. <i>Pglyrp</i>-1 expression was slightly elevated after infection in all genotypes without statistical significance. Transcripts for antimicrobial peptides mBD2, mBD3 and CRAMP were elevated in infected <i>Pglyrp-2</i>^-/- males without statistical significance.</p><p>Conclusions</p><p>Efficient resolution of keratitis in the <i>Pglyrp-2</i>^<i>-/-</i> mice may be due to a reduced pro-inflammatory microenvironment and synergistic antibacterial activities of defensins, CRAMP and Pglyrp-1. Therefore, in ocular infections the pro-inflammatory functions of <i>Pglyrp</i>-2 must be regulated to benefit the host.</p></div

Improved bacterial clearance in the <i>Pglyrp</i>-2^-/- mice.

<p>Dilutions of whole eye homogenates from mice harvested at 48.h.p.i were plated to obtain colony forming unit counts (CFU) per eye. Uninfected eyes did not yield viable bacteria (not shown). The <i>Pglyrp</i>-2^-/- mouse eyes showed significantly lower viable CFU (t = -3.45, p-value = 0.0007). Bacterial yield in <i>Pglyrp</i>-4^-/- mice were also lower than WT (t = -1.66, p-value = 0.098). Bacterial yields together with their corresponding clinical scores are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137129#pone.0137129.s001" target="_blank">S1 Fig</a>.</p