Antimicrobial Properties of Mesenchymal Stem Cells: Therapeutic Potential for Cystic Fibrosis Infection, and Treatment

Abstract

Cystic fibrosis (CF) is a genetic disease in which the battle between pulmonary infection and inflammation becomes the major cause of morbidity and mortality. We have previously shown that human MSCs (hMSCs) decrease inflammation and infection in the in vivo murine model of CF. The studies in this paper focus on the specificity of the hMSC antimicrobial effectiveness using Pseudomonas aeruginosa (gram negative bacteria) and Staphylococcus aureus (gram positive bacteria). Our studies show that hMSCs secrete bioactive molecules which are antimicrobial in vitro against Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumonia, impacting the rate of bacterial growth and transition into colony forming units regardless of the pathogen. Further, we show that the hMSCs have the capacity to enhance antibiotic sensitivity, improving the capacity to kill bacteria. We present data which suggests that the antimicrobial effectiveness is associated with the capacity to slow bacterial growth and the ability of the hMSCs to secrete the antimicrobial peptide LL-37. Lastly, our studies demonstrate that the tissue origin of the hMSCs (bone marrow or adipose tissue derived), the presence of functional cystic fibrosis transmembrane conductance regulator (CFTR: human, Cftr: mouse) activity, and response to effector cytokines can impact both hMSC phenotype and antimicrobial potency and efficacy. These studies demonstrate, the unique capacity of the hMSCs to manage different pathogens and the significance of their phenotype in both the antimicrobial and antibiotic enhancing activities.

Figures

Figure 1

hMSCs in the PA (a) and SA (b) infection model: Cftrtm1Kth (CF) and wild type (WT) controls were infected with 105 CFUs of either Pseudomonas aeruginosa or Staphylococcus aureus impregnated into agarose beads to generate chronic gram negative or gram positive chronic infection models in CF. hMSCs were administered on day 1, 24 hours after infection. Mice were followed up to 10 days and were then euthanized for bacteria burden (BAL CFUs+ whole lung homogenate CFUs, n = 4 experiments with 10 animals in each group). hMSCs decreased bacteria burden (P ≤ 0.05) in response to both pathogens.

Figure 2

MSCs products decrease Pseudomonas aeruginosa growth. Bone marrow derived hMSCs supernatants were cultured with different dosages of Pseudomonas aeruginosa with and without the addition of geneticin (100 μg/mL). Aliquots of the bacteria were streaked onto TSA plates for CFUs (a) or evaluated for ATP production (b). hMSC supernatants (n = 8 different donors) significantly decreased both Pseudomonas aeruginosa growth kinetics (P ≤ 0.05) and CFUs (P ≤ 0.05). Geneticin was used as a positive control which also significantly decreased both CFUs (P ≤ 0.05) and growth rate (P ≤ 0.05) which was enhanced by the addition of hMSCs (P ≤ 0.05 versus antibiotic alone for both CFUs and growth kinetics). PA = Pseudomonas aeruginosa growth without treatment. Geneticin = treatment with the antibiotic geneticin, +hMSCs = treatment with hMSC derived supernatant, and Both = treatment with both hMSC supernatants and geneticin.

Figure 3

MSCs and their products alter Staphylococcus aureus growth. Bone marrow derived hMSCs supernatants were cultured with different dosages of Staphylococcus aureus with and without the addition of geneticin (100 μg/mL). Aliquots of the bacteria were streaked onto TSA plates for CFUs (a) or evaluated for ATP production (b). hMSC supernatants (n = 8 different donors) significantly decreased both Staphylococcus aureus CFUs (P ≤ 0.05) and growth kinetics (P ≤ 0.05). Geneticin was used as a positive control which also significantly decreases both CFUs (P ≤ 0.05) and growth rate (P ≤ 0.05) which was enhanced by the addition of hMSCs (P ≤ 0.05 versus antibiotic alone for both CFUs and growth rate). SA = Staphylococcus aureus growth without treatment. Geneticin = treatment with the antibiotic geneticin, +hMSCs = treatment with hMSC derived supernatant, and Both = treatment with both hMSC supernatants and geneticin.

Figure 4

hMSCs and their products on Streptococcus pneumoniae growth. Bone marrow derived hMSCs supernatants were cultured with different dosages of Streptococcus pneumoniae with and without the addition of geneticin (100 μg/mL). Aliquots of the bacteria were streaked onto MacConkey plates for CFUs (a) or evaluated for ATP production (b). hMSC supernatants (n = 8 different donors) significantly decreased both Streptococcus pneumoniae CFUs ((a), P ≤ 0.05) and growth rates ((b), P ≤ 0.05). Geneticin was used as a positive control, decreasing both CFUs (P ≤ 0.05) and growth rates (P ≤ 0.05). hMSC supernatants decreased CFUs and enhanced antibiotic sensitivity when measuring CFUs. However, hMSCs supernatants had minimal antibiotic enhancing effect on Streptococcus pneumonia growth rate (b). ST = Streptococcus pneumoniae growth without treatment. Geneticin = treatment with the antibiotic geneticin, +hMSCs = treatment with hMSC derived supernatant, and Both = treatment with both MSC supernatants and geneticin.

Figure 5

Antibiotic enhancing activity. To determine whether the antibiotic enhancing effects of the bone marrow derived hMSCs were antibiotic specific, we tested the ability of the hMSCs to enhance the impact of other antibiotics which are often used to treat CF: ceftazadine (100 μg/mL) and tobramycin (100 μg/mL). (a) Ceftazadine and tobramycin decreased Pseudomonas aeruginosa CFUs (P ≤ 0.05, n = 3) which were enhanced by the addition of hMSCs derived supernatants (P = 0.06 for ceftazadine and P = 0.07 for tobramycin). (b) When each experiment is used as its own control for Pseudomonas aeruginosa growth (100%), the MSCs and the antibiotics had statistically significant effect on Pseudomonas growth (100 μg/mL, P ≤ 0.05).

Figure 6

hMSC origin and impact on Pseudomonas aeruginosa growth. The availability of different hMSC sources provides the opportunity to explore whether the antimicrobial effectiveness of the hMSC supernatant was dependent on the tissue origin of the hMSC. Like the bone marrow derived hMSCs, hMSCs derived from adipose tissue significantly decreased Pseudomonas aeruginosa CFUs (P ≤ 0.05) and enhanced geneticin (100 μg/mL) effectiveness (P = 0.08, n = 3). PA = Pseudomonas aeruginosa growth without treatment. Geneticin = treatment with the antibiotic geneticin, +hMSCs = treatment with hMSC derived supernatant, and Both = treatment with both hMSC supernatants and geneticin. hMSC supernatants enhance antibiotic effectiveness against Pseudomonas aeruginosa.

Figure 7

Impact of blocking CFTR function on antimicrobial activity of MSCs. To mimic CF cells, healthy bone marrow derived hMSCs were cultured in the presence and absence of CFTR blocker I-172 (10 μg/mL) without antibiotics for 24 hours. The hMSC supernatants were evaluated for the ability to impact Pseudomonas aeruginosa PA CFUs (a) and growth rate (b). Supernatants generated from CFTR deficient hMSCs were more inefficient at decreasing Pseudomonas aeruginosa CFUs ((b), P ≤ 0.05) and growth rate ((c), P ≤ 0.05) than hMSCs without CFTR activity blocked. Further, hMSCs with deficient CFTR activity had less ability to secrete LL-37 ((c), P ≤ 0.05) relative to controls. LL-37 production by bone marrow derived hMSCs is decreased when CFTR is blocked but can be increased by treating the cells with a variety of cytokine stimulators. hMSCs stimulated with cytokines IFNγ (100 ng/mL), IL-1B (50 ng/mL), and IL-12 (100 ng/mL) secreted significantly more LL-37 than unstimulated controls ((d), P ≤ 0.05, n = 4 different hMSC preparations).