Targeting macrophage activation for the prevention and treatment of Staphylococcus aureus biofilm infections

Abstract

Biofilm infections often lead to significant morbidity due to their chronicity and recalcitrance to antibiotics. We have demonstrated that methicillin-resistant Staphylococcus aureus (MRSA) biofilms can evade macrophage (MΦ) antibacterial effector mechanisms by skewing MΦs toward an alternatively activated M2 phenotype. To overcome this immune evasion, we have used two complementary approaches. In the first, a proinflammatory milieu was elicited by local administration of classically activated M1 MΦs and in the second by treatment with the C5a receptor (CD88) agonist EP67, which invokes MΦ proinflammatory activity. Early administration of M1-activated MΦs or EP67 significantly attenuated biofilm formation in a mouse model of MRSA catheter-associated infection. Several proinflammatory mediators were significantly elevated in biofilm-infected tissues from MΦ- and EP67-treated animals, revealing effective reprogramming of the biofilm environment to a proinflammatory milieu. A requirement for MΦ proinflammatory activity was demonstrated by the fact that transfer of MyD88-deficient MΦs had minimal impact on biofilm growth. Likewise, neutrophil administration had no effect on biofilm formation. Treatment of established biofilm infections with M1-activated MΦs also significantly reduced catheter-associated biofilm burdens compared with antibiotic treatment. Collectively, these results demonstrate that targeting MΦ proinflammatory activity can overcome the local immune inhibitory environment created during biofilm infections and represents a novel therapeutic strategy.

Figures

Figure 1. M1 macrophage polarization enhances phagocytosis and killing of S. aureus biofilms

A) Non-activated macrophages (MΦs) and M1-activated MΦs (10 ng/ml IFN-γ or 100 ng/ml TNF-α + 10 µg/ml PGN) from C57BL/6 mice, as well as MyD88 knockout (KO) MΦs were labeled with CellTracker Blue (blue) and co-cultured with S. aureus-GFP (green) during biofilm or planktonic growth for 2 h and imaged to observe their phagocytic ability. B) After 24 h, biofilms were sonicated to quantitate bacterial burdens to evaluate the ability of the various MΦ populations to attenuate biofilm growth. Biofilms without MΦs were used as untreated controls. White arrows indicate phagocytic cells and significant differences are denoted by asterisks (**, p < 0.01; ***, p < 0.001). Results are representative of at least three independent experiments.

Figure 2. Activated macrophages, but not neutrophils, impair MRSA biofilm formation in vivo

C57BL/6 mice were infected with 103 colony forming units (cfu) of USA300 LAC in the lumen of surgically implanted catheters to establish biofilm infection. Animals were treated with vehicle, 106 neutrophils (PMN), 106 non-activated macrophages (MΦs), or 106 M1-activated MΦs at 12, 24, and 48 h post-infection, whereupon catheters (A) and surrounding tissues (B) were collected at 72 h to quantitate bacterial burdens. Results are expressed as the number of cfu per ml for catheters or cfu per mg tissue, to correct for differences in tissue sampling size. Significant differences are denoted by asterisks (*, p < 0.05). Results are presented from individual animals combined from at least two independent experiments.

Figure 3. Activated macrophages provide long-lasting defense from MRSA biofilm infections in vivo

C57BL/6 mice were infected with 103 colony forming units (cfu) of USA300 LAC in the lumen of surgically implanted catheters to establish biofilm infection. Animals were treated with vehicle, 106 non-activated or 106 M1-activated macrophages (MΦs) at 12, 24, and 48 h post-infection, whereupon catheters (A) and surrounding tissues (B) were recovered at day 14 to quantitate bacterial burdens. Results are expressed as the number of cfu per ml for catheters or cfu per mg tissue, to correct for differences in tissue sampling size. Significant differences in bacterial burdens between vehicle and MΦ-treated mice are denoted by asterisks (*, p < 0.05).

Figure 4. M1-polarized macrophages display superior efficacy at impairing MRSA biofilm formation

C57BL/6 mice were infected with 103 colony forming units (cfu) of USA300 LAC in the lumen of surgically implanted catheters to establish biofilm infection. Animals were treated with either vehicle or increasing numbers of non-activated or M1-activated macrophages (MΦs) at 12, 24, and 48 h post-infection, whereupon catheters (A) and surrounding tissues (B) were recovered at day 3 to quantitate bacterial burdens. Results are expressed as the number of cfu per ml for catheters or cfu per mg tissue, to correct for differences in tissue sampling size. Significant differences in bacterial burdens between vehicle and MΦ-treated mice are denoted by asterisks (*, p < 0.05).

Figure 5. The ability of M1-polarized macrophages to impair MRSA biofilm development is mediated by MyD88-dependent signals

C57BL/6 mice were infected with 103 colony forming units (cfu) of USA300 LAC in the lumen of surgically implanted catheters to establish biofilm infection. Animals were treated with vehicle or 106 M1-activated macrophages (MΦs) derived from wild type (WT) or MyD88 knockout (KO) mice at 12, 24, and 48 h post-infection, whereupon catheters (A) and surrounding tissues (B) were recovered at day 3 to quantitate bacterial burdens. Results are expressed as the number of cfu per ml for catheters or cfu per mg tissue, to correct for differences in tissue sampling size. Results are presented from individual animals combined from at least two independent experiments. Significant differences are denoted by asterisks (*, p < 0.05).

Figure 6. M1-activated macrophage therapy augments the local proinflammatory milieu during MRSA biofilm infection

Tissues surrounding S. aureus biofilms of vehicle, non-activated macrophage (NA MΦ), and M1-activated MΦ (A MΦ) treated mice were collected at day 3 (early treatment) or day 10 (established biofilm treatment) post-infection and homogenized to quantitate CXCL9 (A and E), CCL5 (B), IFN-γ (C), IL-10 (D), IL-17 (F), CXCL2 (G) and IL-6 (H) expression by MILLIPLEX analysis. Results were normalized to the amount of total protein recovered to correct for differences in tissue sampling size. Significant differences are denoted by asterisks (*, p < 0.05) and are representative of 5–8 mice/group (N.D. = not detected).

Figure 7. M1-activated macrophages attenuate established MRSA biofilm infection

C57BL/6 mice were infected with 103 colony forming units (cfu) of USA300 LAC in the lumen of surgically implanted catheters to establish biofilm infection. On days 7 and 9 post-infection, animals received injections of vehicle, antibiotic (rifampicin + daptomycin), 106 non-activated macrophages (MΦs), or 106 M1-activated macrophages, whereupon catheters (A) and surrounding tissues (B) were recovered at day 10 to quantitate bacterial burdens. Significant differences between groups are denoted by asterisks (*, p < 0.05) and represent animals from two independent experiments.

Figure 8. Administration of M1-activated macrophages attenuates arginase-1 expression in established biofilms

Mice received injections of vehicle (A & D), 106 non-activated macrophages (B & E) or 106 M1-activated macrophages (C & F) beginning at days 7 and 9 post-infection, whereupon tissues surrounding infected catheters were collected at day 10 and subjected to immunofluorescence staining with Iba-1 to identify MΦs (red), arginase-1 (green), and nuclear staining with DAPI (blue). Asterisks represent the original location of the catheter, which is non-adherent to glass slides. (G & H) Quantitation of arginase-1 and Iba-1 immunofluorescence staining associated with S. aureus biofilms of vehicle-, non-activated-, or M1 MΦ-treated animals. Significant differences are indicated with asterisks (*, p < 0.05; **, p < 0.01; ***, p < 0.001) and are representative of two independent experiments with 8 mice per group.

Figure 9. M1-activated macrophages remain localized at the site of biofilm infection and maintain a M1 phenotype

Mice received one dose of 107 Quantum Dot-labeled M1-activated macrophages (MΦs; red) either at the time of S. aureus challenge (A) or at day 7 following infection (B), representing early and established therapies, respectively. The same cohort of animals was subjected to daily IVIS imaging to visualize MΦ persistence. Results are representative of 10 individual animals per group.

Figure 10. The macrophage activating peptide EP67 attenuates S. aureus biofilm growth in vivo

Mice were infected with 103 cfu of USA300 LAC in the lumen of surgically implanted catheters to establish biofilms. Animals were treated with vehicle, EP67, or a biologically inactive scrambled derivative peptide (sEP67), beginning at the time of infection and again at 24 and 48 h, whereupon catheters (A and C) and surrounding tissues (B and D) were recovered to quantitate bacterial burdens at days 3 or 14 after infection. Data are expressed as the number of cfu per ml for catheters or cfu per mg host tissue for normalization. Results are presented from individual animals from at least two independent experiments. Significant are denoted by asterisks (*, p < 0.05).

Figure 11. EP67 augments proinflammatory mediator expression in biofilm infected tissues

Mice were infected with 103 CFU of USA300 LAC in the lumen of surgically implanted catheters to establish biofilms. Animals were treated with vehicle or EP67 beginning at the time of infection and again at 24 and 48 h, whereupon tissues were collected at day 3 to quantitate the effects of EP67 treatment on IL-12p40 (A), CCL5 (B), IL-17 (C), IL-1α (D) and IFN-γ (E) expression by MILLIPLEX analysis. Results were normalized to the amount of total protein recovered to correct for differences in tissue sampling size. Results are presented from individual animals combined from two independent experiments (n = 14 per group). Significant differences between EP67- vs. vehicle-treated tissues are denoted by asterisks (*, p < 0.05).

Figure 12. EP67 augments macrophage infiltration into MRSA biofilms

(A & B) Mice were infected with 103 CFU of USA300 LAC in the lumen of surgically implanted catheters to establish biofilms. Animals were treated with vehicle or EP67 beginning at the time of infection and again at 24 and 48 h, whereupon tissues surrounding infected catheters were collected at day 3 and subjected to immunofluorescence staining with Iba-1 to identify macrophages (MΦs; red) and nuclear staining with DAPI (blue). Asterisks represent the original location of the catheter, which is non-adherent to glass slides. (C) Macrophage (F4/80+) infiltrates in tissues surrounding infected catheters from vehicle- or EP67-treated animals were quantitated by FACS. Results are expressed as the percentage of cells after correction for isotype control staining and are representative of three independent experiments with 8 mice per group (*, p < 0.05; ***, p < 0.001).