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. 2016 Mar;18(3):424-36.
doi: 10.1111/cmi.12522. Epub 2015 Oct 27.

Antibacterial effect of mesenchymal stem cells against Escherichia coli is mediated by secretion of beta- defensin- 2 via toll- like receptor 4 signalling

Dong Kyung Sung  1 , Yun Sil Chang  2   1 , Se In Sung  2   1 , Hye Soo Yoo  2   1 , So Yoon Ahn  2   1 , Won Soon Park  2   1
Affiliations

Antibacterial effect of mesenchymal stem cells against Escherichia coli is mediated by secretion of beta- defensin- 2 via toll- like receptor 4 signalling

Dong Kyung Sung et al. Cell Microbiol. 2016 Mar.

Abstract

Recently, we demonstrated that intratracheal transplantation of human umbilical cord blood- derived mesenchymal stem cells (MSCs) attenuates Escherichia (E) coli- induced acute lung injury primarily by down- modulating inflammation and enhancing bacterial clearance iQn mice. This study was performed to elucidate the mechanism underlying the antibacterial effects of MSCs. The growth of E. coli in vitro was significantly inhibited only by MSCs or their conditioned medium with bacterial preconditioning, but not by fibroblasts or their conditioned medium. Microarray analysis identified significant up- regulation of toll- like receptors (TLR)- 2 and TLR- 4, and β- defensin 2 (BD2) in MSCs compared with fibroblasts after E. coli exposure. The increased BD2 level and the in vitro antibacterial effects of MSCs were abolished by specific antagonist or by siRNA- mediated knockdown of TLR- 4, but not TLR- 2, and restored by BD2 supplementation. The in vivo down- modulation of the inflammatory response and enhanced bacterial clearance, increased BD2 secretion and the resultant protection against E. coli- induced pneumonia observed only with MSCs, but not fibroblasts, transplantation in mice, were abolished by knockdown of TLR- 4 with siRNA transfection. Our data indicate that BD2 secreted by the MSCs via the TLR- 4 signalling pathway is one of the critical paracrine factors mediating their microbicidal effects against E. coli, both in vitro and in vivo. Furthermore, TLR- 4 from the transplanted MSCs plays a seminal role in attenuating in vivo E. coli- induced pneumonia and the ensuing acute lung injury through both its anti- inflammatory and antibacterial effects.

Keywords: Escherichia coli; acute lung injury; beta- defensin 2; mesenchymal stem cells; toll- like receptor 4.

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Figures

Figure 1
Figure 1
Bacterial growth inhibition assay in vitro. A. Bacterial colony forming units (CFUs) in the culture media (DMEM) only, human fibroblasts (MRC‐ 5), or human umbilical cord blood‐ derived mesenchymal stem cells (MSC) after infection with 103 colony‐ forming unit (CFU) E. coli and incubation for 6 h. B. Bacterial CFUs in the conditioned media (CM) obtained from MRC‐ 5 or MSC with or without bacterial preconditioning. Note the antibacterial effects are specific to MSC and MSC CM only with bacterial preconditioning. Data are presented as mean ± SEM. The asterisk indicates the P < 0.05 compared with E. coli control group, the dagger sign the P < 0.05 compared with MRC‐ 5 group, the number sign the P < 0.05 compared with MSC CM without bacterial preconditioning group, the double dagger sign the P < 0.05 compared to MRC‐ 5 CM without bacterial preconditioning group, and the dollar sign the P < 0.05 compared with MRC‐ 5 CM with bacterial preconditioning. group (n = 6 per each analysis).
Figure 2
Figure 2
Venn diagram, functional annotation, and KEGG molecular pathway analysis of microarray. A. Venn diagram analyses showing significantly up‐ regulated genes in the human umbilical cord blood‐ derived mesenchymal stem cells (MSC) and/or human fibroblast (MRC‐ 5) in the microarray analyses performed after infection with 103 CFU E. coli and incubation for 6 h. B. Enriched functional categories and the number of genes in each category among the 400 significantly up‐ regulated genes in the MSC compared with MRC‐ 5 after infection with 103 CFU E. coli and incubation for 6 h. C. KEGG molecular pathway analysis and the number of genes in each pathway among the 400 significantly up‐ regulated genes in the MSC compared with MRC‐ 5 after infection with 103 CFU E. coli and incubation for 6 h. D. Gene ontogeny analysis up‐ regulated in MSCs using Venn diagram. Note that both the toll‐ like receptor (TLR)‐ 2 and TLR‐ 4 genes are commonly involved in the intracellular signaling cascade, defence response, and immune response, and β‐ defensin (BD)2 is the only significantly up‐ regulated antibacterial protein observed in the MSC, but not in the MRC‐ 5, only after infection with 103 CFU E. coli and incubation for 6 h (n = 3 per MSC or MRC‐ 5 respectively).
Figure 3
Figure 3
Validation of the TLR‐ 2 and TLR‐ 4 up‐ regulation by reverse transcription‐ polymerase chain reaction (RT‐ PCR) (A–D), Western blotting (E–H) and immunofluorescence staining (I and J) in the human umbilical cord blood‐ derived mesenchymal stem cells (MSC) or fibroblasts (MRC‐ 5) with or without infection with 103 CFU E. coli and incubation for 6 h. Note increased TLR‐ 2 and TLR‐ 4 expression by RT‐ PCR, Western blot, and immunofluorescence staining (green colour denotes TLR‐ 2 or TLR‐ 4; blue colour denotes nuclei stained by DAPI) in the MSC, but not in MRC‐ 5 only after infection with 103 CFU E. coli and incubation for 6 h. Data are presented as mean ± SEM. The asterisk indicates the P < 0.05 compared with MSC without E. coli group, the number sign the P < 0.05 compared with MSC with E. coli group, the dollar sign the P < 0.05 compared with MRC‐ 5 without E. coli group, and the ampersand the P < 0.05 compared with MRC‐ 5 with E. coli group (n = 7 per each analysis).
Figure 4
Figure 4
Validation of the β‐ defensin (BD)2 up‐ regulation by reverse transcription‐ polymerase chain reaction (RT‐ PCR) (A), ELISA (B), and immunofluorescence staining (C and D) in the human umbilical cord blood‐ derived mesenchymal stem cells (MSC) or fibroblasts (MRC‐ 5) with or without infection with 103 CFU E. coli and incubation for 6 h. Note increased BD2 expression by RT‐ PCR, ELISA, and immunofluorescence staining (green colour denotes BD2; blue colour denotes nuclei stained by DAPI) in the MSC, but not in MRC‐ 5 only after infection with 103 CFU E. coli and incubation for 6 h. Data are presented as mean ± SEM. The asterisk indicates the P < 0.05 compared with MSC without E. coli group, the number sign the P < 0.05 compared with MSC with E. coli group, the dollar sign the P < 0.05 compared with MRC‐ 5 without E. coli group, and the ampersand the P < 0.05 compared with MRC‐ 5 with E. coli group (n = 7 per each analysis).
Figure 5
Figure 5
Bacterial quantification and β‐ defensin (BD)2 levels in vitro in the MSC with toll‐ like receptor (TLR)‐ 2/TLR‐ 4 antagonist (A and B), or with TLR‐ 2/TLR‐ 4 siRNA transfection (C and D), or with dose titrated supplementation of BD2 (E and F), or with bacterial preconditioned media (CM) (G and H) after infection with 103 CFU E. coli and incubation for 6 h. Note the abolishment of antibacterial effects and the simultaneously increased BD2 levels of MSC or its bacterial preconditioned media only with TLR‐ 4 antagonist or siRNA transfection, but not with TLR‐ 2 antagonist or siRNA transfection, and the restoration of the antibacterial effects with dose titrated supplementation of BD2. Data are mean ± SD. The asterisk indicates the P < 0.05 versus E. coli treated group, the number sign the P < 0.05 versus E. coli + MSC treated group, the dagger sign the P < 0.05 versus E. coli + MSC + TLR‐ 4 antagonist treated group, the double dagger sign the P < 0.05 versus E. coli + MSC + TLR‐ 4 siRNA treated group, the dollar sign the P < 0.05 versus E. coli + MSC + TLR‐ 4 siRNA +0.1 ng BD treated group, the ampersand the P < 0.05 versus E. coli + MSC + TLR‐ 4 siRNA +0.5 ng BD treated group, the commercial at sign the P < 0.05 versus E. coli + MSC‐ CM, and the percent sign the P < 0.05 versus E. coli + MSC + TLR4 siRNA‐ CM. By ANOVA (Tukey) (n = 5 per each analysis).
Figure 6
Figure 6
Representative optical microscopy photomicrographs of the mice lungs stained with hematoxylin and eosin (A–F), and lung injury scores such as alveolar congestion, alveolar wall thickening, alveolar haemorrhage, and neutrophil infiltration (G–J) in each experimental group at 24 h after intratracheal inoculation of 107 E. coli. Note that the significant attenuation of the increase in E. coli‐ induced acute lung injury scores observed with human umbilical cord blood‐ derived mesenchymal stem cells (MSC) transplantation was abolished with TLR‐ 4 siRNA transfection, but not with scrambled siRNA transfection, of MSC. Data are presented as mean ± SEM. Data are mean ± SD. The asterisk indicates the P < 0.05 versus E. coli treated group, the number sign the P < 0.05 versus E. coli + MSC treated group, and the double dagger sign the P < 0.05 versus E. coli + MSC + TLR4 siRNA treated group. By ANOVA (Tukey) (n = 5 per each analysis).
Figure 7
Figure 7
Bacterial counts (A), protein (B), cytokine (C–F) and β‐ defensin (BD)2 (G) concentrations in the bronchoalveolar lavage (BAL) fluid of mice at 24 h after intratracheal inoculation of 107 CFU E. coli. Note that the significant attenuation of the E. coli‐ induced increase in protein and cytokine concentrations along with the simultaneous increase in BD2 levels in the BAL fluid observed with human umbilical cord blood‐ derived mesenchymal stem cells (MSC) transplantation was abolished with TLR‐ 4 siRNA transfection, but not with scrambled siRNA transfection, of MSC. Data are presented as mean ± SEM. Data are mean ± SD. The asterisk indicates the P < 0.05 versus E. coli treated group, the number sign the P < 0.05 versus E. coli + MSC treated group, and the double dagger sign the P < 0.05 versus E. coli + MSC + TLR4 siRNA treated group. By ANOVA (Tukey) (n = 5 per each analysis).
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