MyD88-dependent signaling influences fibrosis and alternative macrophage activation during Staphylococcus aureus biofilm infection

Abstract

Bacterial biofilms represent a significant therapeutic challenge based on their ability to evade host immune and antibiotic-mediated clearance. Recent studies have implicated IL-1β in biofilm containment, whereas Toll-like receptors (TLRs) had no effect. This is intriguing, since both the IL-1 receptor (IL-1R) and most TLRs impinge on MyD88-dependent signaling pathways, yet the role of this key adaptor in modulating the host response to biofilm growth is unknown. Therefore, we examined the course of S. aureus catheter-associated biofilm infection in MyD88 knockout (KO) mice. MyD88 KO animals displayed significantly increased bacterial burdens on catheters and surrounding tissues during early infection, which coincided with enhanced dissemination to the heart and kidney compared to wild type (WT) mice. The expression of several proinflammatory mediators, including IL-6, IFN-γ, and CXCL1 was significantly reduced in MyD88 KO mice, primarily at the later stages of infection. Interestingly, immunofluorescence staining of biofilm-infected tissues revealed increased fibrosis in MyD88 KO mice concomitant with enhanced recruitment of alternatively activated M2 macrophages. Taken in the context of previous studies with IL-1β, TLR2, and TLR9 KO mice, the current report reveals that MyD88 signaling is a major effector pathway regulating fibrosis and macrophage polarization during biofilm formation. Together these findings represent a novel example of the divergence between TLR and MyD88 action in the context of S. aureus biofilm infection.

Figure 1. MyD88 regulates acute S. aureus biofilm development.

Biofilm infections were established in MyD88 knockout (KO) and wild type (WT) mice following the inoculation of 10³ CFU of USA300 LAC::lux into the lumen of subcutaneous implanted catheters. Animals were sacrificed at the indicated days following S. aureus 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 (*p<0.05).

Figure 2. MyD88 loss leads to early impairments in S. aureus containment at the site of biofilm infection.

Biofilm infections were established in MyD88 knockout (KO) and wild type (WT) mice following the inoculation of 10³ CFU of USA300 LAC::lux into the lumen of subcutaneous implanted catheters. Animals were sacrificed at the indicated days following S. aureus 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 (*p<0.05).

Figure 3. MyD88-dependent signals are important for cytokine and chemokine production during later stages of S. aureus biofilm infection.

Tissues surrounding S. aureus catheter-associated biofilm infections from MyD88 KO and WT mice were collected at the indicated time points post-infection to quantitate differences in IL-6 (A), IFN-γ (B), and CXCL1 (C) expression by MILLIPLEX analysis. Results are normalized to the amount of total protein recovered to correct for differences in tissue sampling size and represent the mean values from individual animals combined from two independent experiments (n = 6−10 per group). Significant differences between MyD88 KO and WT mice are denoted by asterisks (*p<0.05).

Figure 4. MyD88-dependent signals influence the host tissue response to S. aureus biofilms.

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 S. aureus 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 (*p<0.05).

Figure 5. Type I collagen deposition surrounding S. aureus biofilms is enhanced following MyD88 loss.

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 S. aureus 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 (*p<0.05).

Figure 6. Arginase-1 expression is increased in MyD88 KO mice during S. aureus biofilm infection.

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 S. aureus 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 (*p<0.05).

Figure 7. Macrophage recruitment into S. aureus biofilms is attenuated in MyD88 KO mice during early infection.

Biofilms were established in MyD88 knockout (KO) and wild type (WT) mice following the inoculation of 10³ CFU of USA300 LAC::lux into the lumen of subcutaneous implanted catheters. Animals were sacrificed at the indicated time points following S. aureus 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 (*p<0.05).

Figure 8. MyD88 loss during S. aureus biofilm infection augments alternatively activated M2 macrophage accumulation.

Biofilm infections were established in MyD88 knockout (KO) and wild type (WT) mice following the inoculation of 10³ CFU of USA300 LAC::lux into the lumen of subcutaneous implanted catheters. Animals were sacrificed at day 7 following S. aureus 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 (*p<0.05).

Figure 9. Loss of MyD88-dependent signaling augments the expression of genes associated with alternatively activated M2 macrophages following S. aureus biofilm exposure.

Bone marrow-derived macrophages from MyD88 KO or WT mice were co-cultured with S. aureus biofilms in vitro 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 S. aureus 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 (*p<0.05; **p<0.001).