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

Histology of eyes 48 h.p.i.

<p>H and E staining of parasagittal sections through the eye showing the lens (Le), anterior chamber (Ac) and the cornea (Co) (A-E). Cellular inflammatory infiltrates (arrow head) were quantified by counting the nuclei using Image J from 3–5 animals per genotype (F) (additional details provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137129#pone.0137129.s002" target="_blank">S2 Fig</a>).</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

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

<i>Pglyrp</i> gene expressions regulated differentially after infection.

<p>Relative expressions of <i>Pglyrp-1</i>–<i>2</i>, <i>-3 and -4</i> compared to <i>Gapdh</i> were measured before and 48 h.p.i by qRTPCR on total RNA from six animals per genotype and shown here as mean ± SEM. Fold change was calculated as 2^-ΔΔCt. <i>Pglyrp</i>-1 and -4 expressions were slightly increased after infection but these were not significant.</p