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. 2014 Sep 19;9(9):e106903.
doi: 10.1371/journal.pone.0106903. eCollection 2014.

Mesenchymal stem cells augment the anti-bacterial activity of neutrophil granulocytes

Sven Brandau  1 , Mark Jakob  2 , Kirsten Bruderek  1 , Friedrich Bootz  2 , Bernd Giebel  3 , Stefan Radtke  3 , Katharina Mauel  4 , Marcus Jäger  4 , Stefanie B Flohé  4 , Stephan Lang  1
Affiliations

Mesenchymal stem cells augment the anti-bacterial activity of neutrophil granulocytes

Sven Brandau et al. PLoS One. .

Erratum in

  • PLoS One. 2014;9(11):e114201

Abstract

Background: Mesenchymal stem cells (MSCs) participate in the regulation of inflammation and innate immunity, for example by responding to pathogen-derived signals and by regulating the function of innate immune cells. MSCs from the bone-marrow and peripheral tissues share common basic cell-biological functions. However, it is unknown whether these MSCs exhibit different responses to microbial challenge and whether this response subsequently modulates the regulation of inflammatory cells by MSCs.

Methodology/principal findings: We isolated MSCs from human bone-marrow (bmMSCs) and human salivary gland (pgMSCs). Expression levels of TLR4 and LPS-responsive molecules were determined by flow cytometry and quantitative PCR. Cytokine release was determined by ELISA. The effect of supernatants from unstimulated and LPS-stimulated MSCs on recruitment, cytokine secretion, bacterial clearance and oxidative burst of polymorphonuclear neutrophil granulocytes (PMN) was tested in vitro. Despite minor quantitative differences, bmMSCs and pgMSCs showed a similar cell biological response to bacterial endotoxin. Both types of MSCs augmented anti-microbial functions of PMNs. LPS stimulation, particularly of bmMSCs, further augmented MSC-mediated activation of PMN [corrected].

Conclusions/significance: This study suggests that MSCs may contribute to the resolution of infection and inflammation by promoting the anti-microbial activity of PMNs. This property is exerted by MSCs derived from both the bone-marrow and peripheral glandular tissue.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Immunophenotyping of bmMSCs and pgMSCs.
(A/B) Flow cytometric analysis of MSCs; data are presented as Δ Median defined as the mean fluorescence intensity (MFI) of the specific antibody minus MFI of the isotype control antibody. Bar graphs show the median from 4–5 independent experiments (bmMSCs, n = 4; pgMSCs, n = 5). (B/C) TLR4 expression of bmMSCs and pgMSCs was determined by flow cytometry (five independent experiments). The Mann-Whitney U test was used for statistical analysis; significance was set at the level of P≤0.05. (C) Data are shown as an overlay histogram: isotype control (gray) and specific cell surface marker for TLR4 (white). Representative result from 1 out of 5 independent experiments (bmMSCs, n = 5; pgMSCs, n = 5).
Figure 2
Figure 2. Expression of adhesion molecules after LPS stimulation.
MSCs were left untreated or were stimulated with LPS [10 ng/ml]. Flow cytometry data are presented as ΔMedian of 5–7 independent experiments (bmMSCs, n = 5; pgMSCs, n = 7). (A) Absolute fluorescence intensity is shown. (B) Relative induction of fluorescence intensity with untreated MSCs set as 1 (reference value) is shown for visualization of LPS-induced up-regulation; Mann-Whitney U test was used for statistical analysis; significance was set at the level of P≤0.05.
Figure 3
Figure 3. Cytokine secretion profile of bmMSCs and pgMSCs.
Cytokines were quantified by ELISA. MSCs were (A) left untreated or (B/C) were stimulated with LPS [10 ng/ml] (bmMSCs, n = 7; pgMSCs, n = 5; passage 3). (C) Cytokine secretion of untreated MSCs was set as 1 (reference value). Mann-Whitney test was used for statistical analysis; significance was set at the level of P≤0.05.
Figure 4
Figure 4. Activation of the NF-κB, JAK-STAT and TRAF pathways.
(A/B) mRNA expression of bmMSCs and pgMSCs was determined in unstimulated and stimulated (LPS, 10 ng/mL for 4 h) MSCs by qPCR. mRNA was pooled from 4 different donors respectively for bmMSCs and pgMSCs. Gene expression of unstimulated MSCs was set as 1 (reference value). Values ≥ 2.0 are set as a significant increase of gene expression and values≤0.5 are set as a significant decrease of gene expression after LPS stimulation. 2−ddCT = 2−(dCT Target – dCT Reference) LPS stimulated – (dCT Target – dCT Reference) unstimulated. (C/D) Classical (canonical) NF-κB pathway: immunofluorescence analysis of translocation of p65 (C) and c-REL (D) was determined in unstimulated and stimulated (LPS, 10 ng/mL for 0, 30, 60, 90, 120 min) bmMSCs and pgMSCs. One representative experiment of two independent experiments is shown. (E) Analysis of the phosphorylation status of Acetyl-NF-κB p65 and phospho-NF-κB (p-NF-κB) detected by western blot (unstimulated and stimulated with LPS for 0, 30, 60, 90, and 120 min).
Figure 5
Figure 5. Activated bmMSCs and pgMSCs induced the recruitment of PMNs.
A migration assay using a Transwell insert culture system was used to evaluate the migration of PMNs toward medium, MSCs (bmMSCs and pgMSCs, each n = 5) supernatant (SN), and supernatant of LPS-stimulated MSCs. Data are presented as median of 5 independent experiments. Spontaneous migration toward control medium was set as 1 (reference value); statistical analysis by ANOVA test; significance was set at the level of P≤0.05.
Figure 6
Figure 6. CCL4 secretion by PMNs.
PMNs were cultured in control medium or in conditioned medium of unstimulated or LPS-stimulated MSCs for 24 hours. CCL4 secreted by PMNs was measured by ELISA. Data are presented as median of 3 independent experiments. ANOVA test was used for statistical analysis; significance was set at the level of P≤0.05.
Figure 7
Figure 7. Phagocytic activity of PMNs.
(A) PMNs were pre-incubated in MSCs (bmMSCs or pgMSCs) supernatant or standard culture medium (1 h). Then PMNs were infected with Escherichia coli (MOI 50) for 30 minutes. Phagocytosis was assessed and counted after Pappenheim's staining. Examples of Pappenheim's-stained PMNs (arrow: phagocytized bacteria). One representative photomicrograph from 3 different donor cultures is shown (63× magnification). MOI, multiplicities of infection. (B) Influence of bmMSC or pgMSC supernatants (SN) on the phagocytic activity of PMNs. Data are depicted as percentage of infected PMNs (n = 3).
Figure 8
Figure 8. Respiratory Burst.
PMNs were pre-incubated in bmMSCs or pgMSCs supernatant or standard culture medium (1 h). PMNs were incubated in the presence or absence of Escherischia coli with MOI 25, 50, 100, and 200, followed by addition of dihydrorhodamine-123. Superoxide anion (O2 −) release was measured by flow cytometry. Each panel of the figure shows the mean of 3 independent experiments.
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