Activated Mesenchymal Stem Cells Interact with Antibiotics and Host Innate Immune Responses to Control Chronic Bacterial Infections

Abstract

Chronic bacterial infections associated with biofilm formation are often difficult to resolve without extended courses of antibiotic therapy. Mesenchymal stem cells (MSC) exert antibacterial activity in vitro and in acute bacterial infection models, but their activity in chronic infection with biofilm models has not been previously investigated. Therefore, we studied the effects of MSC administration in mouse and dog models of chronic infections associated with biofilms. Mice with chronic Staphylococcus aureus implant infections were treated by i.v. administration of activated or non-activated MSC, with or without antibiotic therapy. The most effective treatment protocol was identified as activated MSC co-administered with antibiotic therapy. Activated MSC were found to accumulate in the wound margins several days after i.v. administration. Macrophages in infected tissues assumed an M2 phenotype, compared to untreated infections which contained predominately M1 macrophages. Bacterial killing by MSC was found to be mediated in part by secretion of cathelicidin and was significantly increased by antibiotics. Studies in pet dogs with spontaneous chronic multi drug-resistant wound infections demonstrated clearance of bacteria and wound healing following repeated i.v. administration of activated allogeneic canine MSC. Thus, systemic therapy with activated MSC may be an effective new, non-antimicrobial approach to treatment of chronic, drug-resistant infections.

Figure 1

Effects of MSC and antibiotic administration on bacterial infection in mouse chronic wound implant infection model. CD-1 mice (n = 5 per group) were implanted with surgical mesh on which biofilms has been established with a luciferase-expressing strain (Xen36) of S. aureus, as described in Methods. On day 2 after implant placement, mice were randomly assigned to the following treatment groups: untreated controls; treatment with antibiotic (amoxicillin-clavulanic acid, continuous treatment in drinking water) only; treatment by i.v. administration of untreated MSC; treatment by i.v. administration of activated MSC; treatment by administration of untreated MSC plus antibiotics; and treatment with activated MSC plus antibiotics. Mice were imaged by IVIS bioluminescence imaging every 2–3 days following mesh implantation to assess the effects of treatment on bacterial burden. In (A), representative IVIS images of wounds in one mouse of each treatment group (n = 5 animals per group) are depicted. Quantitative mean photon intensity from each group of treated animals over time is depicted in (B). Similar results were obtained in 2 additional experiments. In (C), the mean bacterial burden in wounds were determined on day 14 of infection by quantitative counting, as described in Methods, and compared statistically by ANOVA and Tukey multiple means comparison. *Denotes p < 0.05.

Figure 2

Effects of activated MSC administration on wound healing in mouse chronic infection model. Mice with S. aureus infected mesh implants were treated as described in Fig. 1. At the completion of the study (day 14), mice were euthanized and skin was dissected to reveal the implant site. In (A), representative photograph of a wound treated with antibiotics only, and corresponding representative photomicrographs of H&E sections (B,C) revealing suppurative inflammation at the wound site. In (D) representative photograph of a wound in a mouse treated with activated MSC plus antibiotics, with implanted mesh visible in wound bed. In (E,F) representative photomicrographs of (H&E) sections from a mouse treated with activated MSC plus antibiotics, revealing mild monocytic inflammation.

Figure 3

Migration of labeled MSC to infected wound sites following i.v. administration. In (A–C) representative wound tissues from animals receiving no MSC (A), resting MSC (B) and activated MSC (C). MSC were labeled with the fluorescent membrane dye DiD and detected via fluorescent microscopy (DiD⁺ cells light blue in these images). Animals were injected with labeled MSC 24 hours after implantation of infected mesh and received a second injection 3 days later. Animals were sacrificed 2 days after the second injection and tissues were evaluated histologically. Quantitative counts of DiD⁺ cells revealed significantly more cells present in tissues from animals receiving activated MSC (D). All animals in this study received antibiotics. Counts were compared using ANOVA with *depicting p < 0.05. In (E,F), wound tissues from a control animal and an animal injected i.v. with activated GFP-transgenic MSC to allow detection in wound tissues. Cells were administered 2 separate injections 3 days apart, and 48 h later, the wound tissues were collected and evaluated by fluorescence microscopy for detection of GFP + cells (F). In other studies, MSC (activated or non-activated) were labeled with the fluorescent dye DiR, and live mice were imaged using an IVIS imager (G–I). In the first 24 h after injection, DiR + cells were detected in the lung and spleen. By 48 h post-injection, DiR + cells were apparent in the region of the infected wound. The cells accumulated at the wound sites and images were taken 3 days after the third MSC injection (H,I). There were also more activated DiR + cells localized in the region of the wound than in mice injected with non-activated cells. Similar results were obtained in one additional animal study.

Figure 4

Bacterial killing by MSC mediated in part by the antimicrobial peptide Cramp. The ability of MSC to kill S. aureus was assessed by co-culture of bacteria directly with MSC (A) or with MSC CM (B). MSC (5 × 10⁵ cells/well) were co-cultured with bacteria (MOI = 2) in triplicate wells of 24-well plates for 3 h, then bacteria were collected from supernatants resuspended by gentle pipetting, and CFU were determined by serial dilution and manual counting. Bacteria were incubated directly in MSC CM for 3 h in (B). In (C) synergistic killing of S. aureus by MSC CM and cefazolin. Cefazolin was added at a dose of 50 ng/ml and bacteria added at 1 × 10⁶ per well to 1 ml of MSC-CM or media alone in a 24 well plate and incubated for 3 hours at 37 °C, *denotes p < 0.05 as assessed by ANOVA and Tukey multiple means post-test. Synergy assessed via two way ANOVA for detection of significant interaction, as described previously. Immunocytology was used to assess intracellular expression of the antimicrobial peptide Cramp by MSC in (D), as described in Methods. Cells were immunostained with an irrelevant antibody (isotype panel insert) or with an anti-Cramp antibody (D) and cells were evaluated by fluorescence microscopy. Synergistic killing between antimicrobial peptide LL-37 and beta lactam antibiotic demonstrated in (E). Human LL-37 at 30 ug/ml was incubated with 50 ng/ml Cefazolin for 3 hours with 1 × 10⁶ CFU/ml of S. aureus. In (F), MSC were co-cultured with bacteria and the effects of Cramp neutralization on bacterial killing were assessed Synergistic killing of bacteria was demonstrated between MSC and cefazolin (C) and between LL-37 and cefazolin (E). *Denotes p < 0.05 as assessed by ANOVA and Tukey multiple means post-test. Similar results were obtained in two additional experiments.

Figure 5

Incubation of neutrophils with MSC-CM increases phagocytosis of S. aureus. The effect of MSC secreted factors on neutrophil phagocytosis was assessed by first incubating mouse neutrophils (derived from peritoneal lavage as noted in Methods), then assessing neutrophil phagocytosis of S. aureus. Following incubation neutrophils were infected with S. aureus at an MOI of 1 for 30 minutes and extracellular bacteria was killed by the addition of gentamycin. Neutrophils were stained with CD11b (green) and S. aureus stained with anti-staph antibody (red). Neutrophils incubated with medium alone (A), resting MSC-CM (B) or activated MSC-CM (C). Image analysis was done using Image J software (NIH). Statistical analysis was performed using way ANOVA and Newman-Keul post test *indicates p < 0.05. Similar results were obtained in two additional experiments.

Figure 6

Mesenchymal stem cells increase monocyte migration and change the phenotype of macrophages in tissues. Production of CCL2 by MSC was measured in CM from resting MSC and pIC activated MSC (A). The effects of MSC CM on monocyte migration was assessed using Boyden chambers and monocytes isolated from the peritoneal cavity of thioglycollate-treated mice, as described in Methods. Cell migration data was quantitated using Cell Sens software, Olympus). MSC-CM significantly stimulated monocyte migration compared to medium alone, and CM from activated MSC triggered significantly more migration than CM from non-activated MSC (B). In mice treated with MSC in vivo, monocyte recruitment to infected wounds was assessed using CCR2-GFP reporter mice. Monocytes were present in relatively small numbers in tissues of untreated mice (D), whereas the numbers of infiltrating monocytes were significantly higher in mice treated with MSC and further increased in mice treated with activated MSC. These results were verified using tissue samples previously collected from non-GFP reporter mice at the same timepoint. In addition to altering monocyte migration, phenotype of tissue macrophages was also altered in the three groups of mice (control, MSC, aMSC). In tissues of untreated mice (Ctrl) the macrophages were mostly of the M1 phenotype (E) and fluoresced in the red channel.  Mice treated with resting MSC (MSC) had a combination of M1 and M2 (green) macrophages and activated MSC (aMSC) contained mainly M2 macrophages. Monocytes were stained with F4/80, anti-iNOS antibody, and anti arginase antibody. The cells were quantified using Cell Sens software, Olympus) Statistical analysis performed using ANOVA with Tukey multiple means comparison, *denotes p < 0.05.

Figure 7

Illustration of Study Design. Dogs with naturally occuring infections were selected for treatment with activated canine allogeneic MSC according to inclusion and exclusion criteria explained in methods. The wounds were quantitatively cultured prior to the start of treatment and every 2 weeks prior to MSC injection. Dogs were evaluated 2 weeks after the last treatment and a culture performed at that time. Dogs were kept on the antibiotic they were taking prior to enrollment in the trial for the length of the trial.

Figure 8

Effects of antimicrobial treatment with activated allogeneic canine MSC in pet dogs with spontaneous wound infections with MDR bacteria. Pet dogs with chronic wound infections unresponsive to prolonged antibiotic therapy were enrolled in a clinical trial designed to evaluate the effects of systemic administration of activated canine MSC, as described in Fig. 7. In (A) photographs of the paw wound in a dog before and 4 weeks after MSC treatment. In (B) serial cytological evaluation of joint fluid samples obtained from a dog with septic arthritis prior to treatment and at week 4 of treatment with activated MSC. The dog developed post-operative septic arthritis with MDR S. pseudointermedius and had been unresponsive to several month long courses of antibiotic treatment over a period of 12 months. The animal became clinically normal at the completion of the 6 week study and the joint fluid remained cytologically normal.  In (C) serial quantitative cultures of a soft tissue infection from traumatic paw injury displayed in A shows resolution of two multidrug resistant infections throughout the treatment period.