Antibacterial effect of human mesenchymal stem cells is mediated in part from secretion of the antimicrobial peptide LL-37

Abstract

Recent in vivo studies indicate that mesenchymal stem cells (MSCs) may have beneficial effects in the treatment of sepsis induced by bacterial infection. Administration of MSCs in these studies improved survival and enhanced bacterial clearance. The primary objective of this study was to test the hypothesis that human MSCs possessed intrinsic antimicrobial properties. We studied the effect of human MSCs derived from bone marrow on the bacterial growth of Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria. MSCs as well as their conditioned medium (CM) demonstrated marked inhibition of bacterial growth in comparison with control medium or normal human lung fibroblasts (NHLF). Analysis of expression of major antimicrobial peptides indicated that one of the factors responsible for the antimicrobial activity of MSC CM against Gram-negative bacteria was the human cathelicidin antimicrobial peptide, hCAP-18/LL-37. Both m-RNA and protein expression data showed that the expression of LL-37 in MSCs increased after bacterial challenge. Using an in vivo mouse model of E. coli pneumonia, intratracheal administration of MSCs reduced bacterial growth (in colony-forming unit) in the lung homogenates and in the bronchoalveolar lavage (BAL) fluid, and administration of MSCs simultaneously with a neutralizing antibody to LL-37 resulted in a decrease in bacterial clearance. In addition, the BAL itself from MSC-treated mice had a greater antimicrobial activity in comparison with the BAL of phosphate buffered saline (PBS)-treated mice. Human bone marrow-derived MSCs possess direct antimicrobial activity, which is mediated in part by the secretion of human cathelicidin hCAP-18/ LL-37.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest

The authors indicate no potential conflicts of interest.

Figures

Figure 1

MSC or its conditioned medium has antimicrobial activity against Gram-negative bacteria. (A): MSC directly inhibits E. coli growth after 6-hour coincubation. MSC or NHLF were incubated with live E. coli K1 for 6 hours. Bacterial growth was assessed by CFU counts. *, p < .0001 versus RPMI; √, p < .007 versus NHLF; ^, p < .02 vs. RPMI by analysis of variance (ANOVA; Bonferroni), n = 8. (B): The CM of MSC or NHLF with or without prior stimulation with live E. coli was examined for antimicrobial activity against E. coli. MSC CM after stimulation with E. coli inhibited bacterial growth by 40%, whereas CM from unstimulated cells or from NHLF did not have a significant effect. Data are mean ± SD; *, p < .0001 versus RPMI; √, p < .001 versus NHLF CM (E. coli stimulated), by ANOVA (Bonferroni), n = 8–11. (C): The CM of MSC or NHLF with or without prior stimulation with live E. coli was tested for antimicrobial activity against P. aeruginosa. MSC CM after stimulation with E. coli inhibited bacterial growth by 70%. Data are mean ± SD; *, p < .0001 versus RPMI; √, p < .003 versus NHLF CM (E. coli stimulated), ^, p < .007 versus RPMI by ANOVA (Bonferroni), n = 6. Abbreviations: CFU, colony-forming units; CM, conditioned medium; MSC, mesenchymal stem cell; NHLF, normal human lung fibroblasts; RPMI, RPMI-1640 medium.

Figure 2

MSC LL-37 expression is upregulated by E. coli stimulation. (A): Level of LL-37 mRNA in MSC is increased after E. coli stimulation as detected by semiquantitative RT-PCR. GAPDH was used as an internal control to normalize loading of the RNA samples. Lane (1) NHLF, (2) NHLF (E. coli stimulated), (3) MSC, and (4) MSC (E. coli stimulated). (B): Levels of LL-37 protein expression in MSC are increased after E. coli stimulation as indicated by immunofluorescence (green staining). (1) NHLF, (2) NHLF (E. coli stimulated), (3) MSC, and (4) MSC (E. coli stimulated). Nuclei were visualized with DAPI staining (blue). Images are representative for each condition run in triplicates. All representative examples are shown at a magnification of 1 × 1,000. (C): Using ELISA, it was found that both MSC and NHLF secreted higher levels of LL-37 after bacterial stimulation. Data are mean ± SD; *, p < .001 versus NHLF (E. coli stimulated), n = 12. Abbreviations: CM, conditioned medium; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; DAPI, 4,6-diamidino-2-phenylindole; ELISA, enzyme-linked immunosorbent assay; MSC, mesenchymal stem cell; NHLF, normal human lung fibroblasts; RT-PCR, reverse transcription polymerase chain reaction.

Figure 3

Antimicrobial activity of MSC CM is mediated by LL-37 secretion. (A): Synthetic LL-37 showed significant antimicrobial activity against E. coli in a dose-dependent manner. Data are mean ± SD; *, p < .001 versus RPMI by analysis of variance (ANOVA; Bonferroni) n = 3. (B): Synthetic LL-37 displayed dose-dependent antimicrobial effect against P. aeruginosa. Data are mean ± SD; *, p < .001 versus RPMI by ANOVA (Bonferroni) n = 3. Preincubation of MSC CM with anti-LL-37 antibody (1 μ;g/ml), but not with mouse IgG (1 μg/ml), significantly reduced the antimicrobial effect of MSC CM against E. coli(C) and P. aeruginosa(D). Data are mean ± SD; *, p < .0002 versus MSC CM + anti-LL-37 by ANOVA (Bonferroni), n = 5–7. Abbreviations: CFU, colony-forming unit; MSC CM, mesenchymal stem cell conditioned medium; RPMI, RPMI-1640 medium.

Figure 4

MSCs reduce bacterial growth in an E. coli pneumonia mice model. Intratracheal administration of MSCs 4 hours following E. coli instillation significantly reduced E. coli CFU growth in mouse lung homogenates (A) and BAL fluid (B) 18 hours after infection. Data are mean ± SD; *, p < .03 versus PBS-treated control mice for lung homogenates; √, p < .04 versus PBS-treated control mice for BAL; n = 12–14. Abbreviations: BAL, bronchoalveolar lavage; CFU, colony-forming unit; LH, lung homogenates; MSC, mesenchymal stem cell; PBS, phosphate buffered saline.

Figure 5

Effect of MSCs on cellular responses and protein influx in the airspaces of E. coli pneumonia in mice. (A): MSC-treated mice had reduced levels of cells, blood, and edema in the BAL as compared with PBS treated following E. coli pneumonia in mice. Images are representative for each condition performed at least in triplicates. (B): Mice treated with MSCs had reduced total BAL cell and neutrophil counts. *, p < .04 versus PBS-treated mice for total cell counts; √, p < .05 versus PBS-treated mice for absolute neutrophil counts; n = 12–14. (C): Levels of the neutrophil chemokine, MIP-2, were significantly lower in the BAL of MSC-treated group as compared with control. *, p < .05 versus PBS-treated mice; n = 15–16. (D): MSC significantly improved lung endothelial/epithelial permeability to protein as represented by total BAL protein at 18 hours. Data as mean ± SD; *, p < .05 versus PBS-treated mice; n = 12–14. Abbreviations: BAL, bronchoalveolar lavage; MIP-2, macrophage inflammatory protein; MSC, mesenchymal stem cells; PBS, phosphate buffered saline.

Figure 6

Intratracheal administration of anti-LL 37 antibody reduced the therapeutic effect of MSC in E. coli pneumonia. Coadministration of MSC together with an anti-LL 37 neutralizing antibody (10 μg), but not with mouse IgG isotype antibody, inhibited the therapeutic effect of MSC in bacterial clearance in lung homogenates (A) and BAL fluid (B). Values are mean CFU ± SD; *, p < .002 versus MSC + anti-Ll-37 antibody treated mice; √, p < .005 versus MSC + anti-LL-37 antibody-treated mice by analysis of variance (ANOVA; Bonferroni), n = 5. (C): MSC administration enhanced antimicrobial activity of mouse BAL. BAL samples were incubated with E. coli (105 CFU/ml) for 2 hours, and CFU growth was counted. Data are mean ± SD; *, p < .002 versus BAL of PBS-treated mice by ANOVA (Bonferroni), n = 8–9. Abbreviations: BAL, bronchoalveolar lavage; CFU, colony-forming unit; LH, lung homogenates; MSC, mesenchymal stem cell; PBS, phosphate buffered saline.

Figure 7

MSCs are the primary source of LL-37 secretion in cocultures of MSCs and human ATII cells. (A): Human MSCs and ATII cells were cocultured, with or without contact, in the presence of LPS. (B): LL-37 was measured following each condition. *, p < .02 versus ATII alone by analysis of variance (ANOVA; Bonferroni), n = 8–9. (C): Conditioned medium from cocultures of ATII cells and MSC in the presence of LPS (100 ng/ml) significantly inhibited bacterial growth. Data are mean ± SD; *, p < .002 by ANOVA (Bonferroni) versus ATII + LPS, n = 8–9. Abbreviations: ATII, alveolar epithelial type II cells; CFU, colony-forming unit; CM, conditioned medium; LPS, lipopolysaccharide; MSC, mesenchymal stem cell.