Human mesenchymal stem cells reduce mortality and bacteremia in gram-negative sepsis in mice in part by enhancing the phagocytic activity of blood monocytes

Abstract

The potential therapeutic value of cell-based therapy with mesenchymal stem cells (MSC) has been reported in mouse models of polymicrobial peritoneal sepsis. However, the mechanisms responsible for the beneficial effects of MSC have not been well defined. Therefore, we tested the therapeutic effect of intravenous bone marrow-derived human MSC in peritoneal sepsis induced by gram-negative bacteria. At 48 h, survival was significantly increased in mice treated with intravenous MSC compared with control mice treated with intravenous fibroblasts (3T3) or intravenous PBS. There were no significant differences in the levels of TNF-α, macrophage inflammatory protein 2, or IL-10 in the plasma. However, there was a marked reduction in the number of bacterial colony-forming units of Pseudomonas aeruginosa in the blood of MSC-treated mice compared with the 3T3 and PBS control groups. In addition, phagocytic activity was increased in blood monocytes isolated from mice treated with MSC compared with the 3T3 and PBS groups. Furthermore, levels of C5a anaphylotoxin were elevated in the blood of mice treated with MSC, a finding that was associated with upregulation of the phagocytosis receptor CD11b on monocytes. The phagocytic activity of neutrophils was not different among the groups. There was also an increase in alternately activated monocytes/macrophages (CD163- and CD206-positive) in the spleen of the MSC-treated mice compared with the two controls. Thus intravenous MSC increased survival from gram-negative peritoneal sepsis, in part by a monocyte-dependent increase in bacterial phagocytosis.

Figures

Fig. 1.

Survival rate following Pseudomonas aeruginosa-induced peritonitis in mice treated with mesenchymal stem cells (MSC, n = 34), 3T3 fibroblasts (3T3, n = 27), or PBS (n = 42). Kaplan-Meier curves represent survival rate in the 3 groups. Survival rate at 48 h was significantly higher in the MSC group than the 3T3 and PBS groups: *P < 0.05, MSC vs. 3T3; √P < 0.01, MSC vs. PBS (by Cox model analysis).

Fig. 2.

Core body temperature following P. aeruginosa-induced peritonitis in mice treated with MSC (n = 14), 3T3 (n = 11), or PBS (n = 18). Temperature at 12 h was significantly higher in the MSC group than the 3T3 and PBS groups: *P < 0.01, MSC vs. 3T3; √P = 0.01, MSC vs. PBS (by ANOVA with Bonferroni's correction).

Fig. 3.

Plasma plasminogen activator inhibitor 1 (PAI-1) levels (A) and blood platelet counts (B, 103 cells/μl) in mice treated with MSC (n = 19), 3T3 (n = 24), or PBS (n = 33). Values are medians and 25th–75th percentiles. *P < 0.01, MSC vs. 3T3; √P < 0.01, MSC vs. PBS (by Kruskal-Wallis test followed by Wilcoxon-Mann-Whitney test).

Fig. 4.

Plasma cytokine levels. A: plasma levels of TNF-α in mice treated with MSC (n = 12), 3T3 (n = 10), or PBS (n = 11). B: plasma levels of PGE2 in mice treated with MSC (n = 11), 3T3 (n = 10), or PBS (n = 9). C: plasma levels of IL-10 in mice treated with MSC (n = 9), 3T3 (n = 11), or PBS (n = 9). Values are medians and 25th–75th percentiles. No difference was found in plasma levels of TNF-α, IL-10, or PGE2 among the 3 groups (by Kruskal-Wallis test followed by Wilcoxon-Mann-Whitney test).

Fig. 5.

TNF-α and macrophage inflammatory protein 2 (MIP-2) in peritoneal fluid. A: TNF-α in peritoneal fluid of mice treated with MSC (n = 12), 3T3 (n = 10), or PBS (n = 11). B: MIP-2 in peritoneal fluid of mice treated with MSC (n = 8), 3T3 (n = 8), or PBS (n = 7). Values are medians and 25th–75th percentiles. Level of TNF-α is lower in MSC group than the 3T3 and PBS groups; however, a statistically significant difference was reached only between the MSC and PBS groups: √P < 0.05 vs. PBS; no difference was found in MIP-2 levels among the 3 groups (by Kruskal-Wallis test followed by Wilcoxon-Mann-Whitney test).

Fig. 6.

Bacterial counts [colony-forming units (CFU)] in spleen and blood. A: bacterial counts in spleen of mice treated with MSC (n = 20), 3T3 (n = 29), or PBS (n = 29). B: bacterial counts in peripheral blood of mice treated with MSC (n = 20), 3T3 (n = 27), or PBS (n = 30). Values are medians and 25th–75th percentiles. A strong trend toward reduction of bacterial count in the spleen was observed in the MSC group compared with the 3T3 (P = 0.055 vs. 3T3) or PBS (P = 0.06 vs. PBS) group. *P < 0.05 vs. 3T3; √P < 0.05 vs. PBS.

Fig. 7.

Blood mononuclear cell phagocytic activity. A and B: percent phagocytosis and phagocytic index (PI) in mononuclear cells isolated from blood of mice treated with MSC (n = 4), 3T3 (n = 4) or PBS (n = 4). C: Escherichia coli CFU counts in the medium after 1 h of incubation with blood mononuclear cells isolated from mice treated with MSC (n = 9), 3T3 (n = 6), or PBS (n = 7). Values are means ± SD. *P < 0.05 vs. 3T3; √P < 0.05 vs. PBS (by ANOVA with Bonferroni's correction). D and E: representative flow cytometric data for percent phagocytosis within the blood monocyte (CD11bhiCD115+) population of MSC- and PBS-treated mice, respectively. F: percentage of monocyte phagocytosis. Values are means ± SD (n = 9–10 mice per group). *P < 0.05 vs. PBS.

Fig. 8.

MSC treatment is associated with enhanced expression of CD163 and CD206 (markers of alternatively activated macrophages). A–C: splenic sections stained with CD206-specific antibody (A, green), CD163-specific antibody (B, green), and hematoxylin-eosin (C). Images are representative for each condition: 2 sections per mouse and 3 mice per group. Original magnification ×200. D and E: representative data for percentage of CD11bhiCD206+ cells within CD11bhi population of splenocytes from mice treated with MSC and PBS, respectively; only CD11bhi splenocytes are shown. F: CD206+ cells in spleens from mice treated with MSC and PBS. Values are means ± SD (n = 7–8 mice per group). *P < 0.05 vs. PBS.

Fig. 9.

A and B: flow cytometry results for percent phagocytosis within CD11bhi and CD11bhiCD206+ populations of splenocytes from MSC- and PBS-treated mice. Values are means ± SD (n = 7–8 mice per group). Percent phagocytosis with both populations was similar in MSC and PBS groups. C: proportion of CD11bhiCD206+ population in blood from MSC- and PBS-treated mice. Values are means ± SD (n = 4 per group). There was no difference in the proportion of CD11bhiCD206+ population in blood between MSC and PBS groups.

Fig. 10.

A: plasma levels of C5a in mice treated with MSC or PBS. Values are means ± SD (n = 9–14 per group). *P < 0.05 vs. PBS. B: expression level of the phagocytosis receptor CD11b on blood monocytes from mice treated with MSC or PBS. MFI, mean fluorescence intensity. Values are means ± SD (n = 9–10 per group). *P < 0.05 vs. PBS.