MyD88 loss during <i>S. aureus</i> biofilm infection augments alternatively activated M2 macrophage accumulation.

<p>Biofilm infections were established in MyD88 knockout (KO) and wild type (WT) mice following the inoculation of 10³ CFU of USA300 LAC::<i>lux</i> into the lumen of subcutaneous implanted catheters. Animals were sacrificed at day 7 following <i>S. aureus</i> infection, whereupon tissues surrounding infected catheters were collected to quantitate M2 (A) and M1 (B) macrophage infiltrates by FACS on the basis of CD206 and IRF-5 staining, respectively. Results are expressed as the percent macrophages (F4/80⁺) that were also positive for CD206 or IRF-5 after correction for isotype control staining and represent the mean ± SEM of three independent experiments. Significant differences between MyD88 KO and WT infiltrates are denoted by asterisks (*<i>p</i><0.05).</p

Loss of MyD88-dependent signaling augments the expression of genes associated with alternatively activated M2 macrophages following <i>S. aureus</i> biofilm exposure.

<p>Bone marrow-derived macrophages from MyD88 KO or WT mice were co-cultured with <i>S. aureus</i> biofilms <i>in vitro</i> for 2 h, whereupon viable macrophages were purified by FACS and RNA immediately isolated for qRT-PCR analysis. The expression levels of M1 (A), M2 (B), and extracellular matrix (C) genes in macrophages exposed to <i>S. aureus</i> biofilms were calculated after normalizing signals against GAPDH and are presented as fold-change relative to unstimulated macrophages. Results represent the mean ± SEM of at least two independent experiments (*<i>p</i><0.05; **<i>p</i><0.001).</p

Type I collagen deposition surrounding <i>S. aureus</i> biofilms is enhanced following MyD88 loss.

<p>Biofilm-infected tissues were recovered from MyD88 knockout (KO) and wild type (WT) mice at the indicated time points following infection, whereupon sections were processed by immunofluorescence staining for type I collagen (red; A). Tissues were stained with DAPI (blue) to demarcate nuclei and asterisks denote the original location of infected catheters that were non-adherent to glass slides. (B) Quantitation of type I collagen fluorescence surrounding <i>S. aureus</i> biofilms of MyD88 KO and WT mice. Results are representative of tissues collected from five individual animals per group for each time point where significant differences are indicated with an asterisk (*<i>p</i><0.05).</p

MyD88-dependent signals influence the host tissue response to <i>S. aureus</i> biofilms.

<p>Biofilm-infected tissues were recovered from MyD88 knockout (KO) and wild type (WT) mice at the indicated time points following infection, whereupon sections were processed by hematoxylin and eosin (H&E) staining to demonstrate changes in tissue architecture (A). The deposition of host material surrounding infected catheters is denoted by arrows. (B) Quantitation of the fibrotic thickness surrounding <i>S. aureus</i> biofilms of MyD88 KO and WT mice. Results represent measurements taken from at least seven individual animals per group for each time point where significant differences are indicated with asterisks (*<i>p</i><0.05).</p

Macrophage recruitment into <i>S. aureus</i> biofilms is attenuated in MyD88 KO mice during early infection.

<p>Biofilms were established in MyD88 knockout (KO) and wild type (WT) mice following the inoculation of 10³ CFU of USA300 LAC::<i>lux</i> into the lumen of subcutaneous implanted catheters. Animals were sacrificed at the indicated time points following <i>S. aureus</i> infection, whereupon tissues surrounding infected catheters were collected to quantitate macrophage infiltrates by FACS. Results are expressed as the absolute number of F4/80⁺ macrophages after normalization to adjust for the recovery of different cell numbers from MyD88 KO and WT mice and represent the mean ± SEM of three independent experiments. Significant differences in macrophage infiltrates are denoted by an asterisk (*<i>p</i><0.05).</p

MyD88 loss leads to early impairments in <i>S. aureus</i> containment at the site of biofilm infection.

<p>Biofilm infections were established in MyD88 knockout (KO) and wild type (WT) mice following the inoculation of 10³ CFU of USA300 LAC::<i>lux</i> into the lumen of subcutaneous implanted catheters. Animals were sacrificed at the indicated days following <i>S. aureus</i> biofilm infection, whereupon the heart and kidneys were removed to quantitate bacterial burdens with results expressed as CFU per mg tissue. Results are presented from individual animals in each group combined from a total of at least 2 independent experiments with bars representing the mean of each group. Significant differences in bacterial burdens between MyD88 KO and WT mice are denoted by asterisks (*<i>p</i><0.05).</p

MyD88 regulates acute <i>S. aureus</i> biofilm development.

<p>Biofilm infections were established in MyD88 knockout (KO) and wild type (WT) mice following the inoculation of 10³ CFU of USA300 LAC::<i>lux</i> into the lumen of subcutaneous implanted catheters. Animals were sacrificed at the indicated days following <i>S. aureus</i> infection, whereupon catheters and surrounding host tissues were removed to quantitate bacterial burdens. Results are expressed as the number of CFU per ml of fluid used for sonication (for catheters) or CFU per mg host tissue to correct for differences in tissue sampling size. Results are presented from individual animals in each group combined from a total of 3 independent experiments with bars representing the mean of each group. Significant differences in bacterial burdens between MyD88 KO and WT mice are denoted by asterisks (*<i>p</i><0.05).</p

Arginase-1 expression is increased in MyD88 KO mice during <i>S. aureus</i> biofilm infection.

<p>Biofilm-infected tissues were recovered from MyD88 knockout (KO) and wild type (WT) mice at the indicated time points following infection, whereupon sections were processed by immunofluorescence staining for arginase-1 (green; A). Tissues were stained with DAPI (blue) to demarcate nuclei and asterisks denote the original location of infected catheters that were non-adherent to glass slides. (B) Quantitation of arginase-1 fluorescence surrounding <i>S. aureus</i> biofilms of MyD88 KO and WT mice. Results are representative of tissues collected from five individual animals per group for each time point where significant differences are indicated with an asterisk (*<i>p</i><0.05).</p

CNS catheter-associated biofilm infection is associated with attenuated inflammation compared to parenchymal abscesses.

<p>The tissues encompassing the brain abscess and the tissues surrounding the infected catheters were homogenized and the resulting supernatants analyzed for levels of the pro-inflammatory mediators CXCL1 (A, B) and IL-17 (C, D). Raw cytokine/chemokine levels are reported (A, C) as well as correction for disparate bacterial burdens (B, D) * = p<0.05 (n = 12–18 mice/group/time point).</p

<i>sarA</i> influences peripheral immune cell influx during CNS catheter-associated infection.

<p>Catheter associated cells were recovered from mice at days 3, 7 and 14-infection, whereupon pooled tissue from 4–5 mice per group (wild type ACH1719– <b>WT</b> vs <i>ACH1719</i>Δ<i>sarA</i> – <b>Δ</b><b><i>sarA</i></b>) was analyzed for neutrophil (A), macrophage (B) and T cell (C) infiltrates, with three independent replicates. Data was corrected for bacterial burdens at each time point. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084089#pone.0084089.s002" target="_blank">Figure S2</a> for representative histograms.</p