Mesenchymal stem cells enhance survival and bacterial clearance in murine Escherichia coli pneumonia

Abstract

Rationale: Bacterial pneumonia is the most common infectious cause of death worldwide and treatment is increasingly hampered by antibiotic resistance. Mesenchymal stem cells (MSCs) have been demonstrated to provide protection against acute inflammatory lung injury; however, their potential therapeutic role in the setting of bacterial pneumonia has not been well studied.

Objective: This study focused on testing the therapeutic and mechanistic effects of MSCs in a mouse model of Gram-negative pneumonia.

Methods and results: Syngeneic MSCs from wild-type mice were isolated and administered via the intratracheal route to mice 4 h after the mice were infected with Escherichia coli. 3T3 fibroblasts and phosphate-buffered saline (PBS) were used as controls for all in vivo experiments. Survival, lung injury, bacterial counts and indices of inflammation were measured in each treatment group. Treatment with wild-type MSCs improved 48 h survival (MSC, 55%; 3T3, 8%; PBS, 0%; p<0.05 for MSC vs 3T3 and PBS groups) and lung injury compared with control mice. In addition, wild-type MSCs enhanced bacterial clearance from the alveolar space as early as 4 h after administration, an effect that was not observed with the other treatment groups. The antibacterial effect with MSCs was due, in part, to their upregulation of the antibacterial protein lipocalin 2.

Conclusions: Treatment with MSCs enhanced survival and bacterial clearance in a mouse model of Gram-negative pneumonia. The bacterial clearance effect was due, in part, to the upregulation of lipocalin 2 production by MSCs.

Figures

Figure 1

Intratracheal mesenchymal stem cell (MSC) treatment improved survival in the Escherichia coli pneumonia model of acute lung injury. Mice were injured with 106 cfu of E coli instilled intratracheally and then were given either MSCs, 3T3 fibroblasts (750 000 MSCs or 3T3 fibroblasts/30 µl phosphate-buffered saline (PBS)) or PBS (30 µl) as treatment 4 h later. Survival over 48 h was determined in each group, which was significantly higher in the MSC-treated mice (n = 11–12 per group, *p<0.01 for MSC vs PBS and #p = 0.03 for MSC vs 3T3 group, using a log-rank test).

Figure 2

Mesenchymal stem cell (MSC) treatment reduced the severity of lung injury in Escherichia coli pneumonia. (A) Mice treated with MSCs had a lower quantity of excess lung water compared with mice treated with 3T3 fibroblasts and phosphate-buffered saline (PBS) (n = 14–15 per group, *, #p<0.0001 for MSC vs PBS and MSC vs 3T3 group, respectively). Data are mean±SD. (B) Haematoxylin and eosin staining of lung tissue demonstrated that MSC-treated mice had qualitatively less lung injury than mice treated with 3T3 fibroblasts or PBS.

Figure 3

Mesenchymal stem cell (MSC) treatment led to enhanced bacterial clearance after infection with intratracheal Escherichia coli. (A) Mice treated with MSCs had fewer E coli cfu in their bronchoalveolar lavage (BAL) at 8 h post infection compared with control-treated mice (n = 12 per group, *p = 0.002 for MSC vs phosphate-buffered saline (PBS) and #p = 0.04 for MSC vs 3T3 group). Data are mean±SD. (B) MSC treatment also led to a reduction in the number of E coli cfu in the whole lung homogenate 24 h after infection (n = 8–9 per group, *p = 0.01). Data are mean±SD.

Figure 4

Mesenchymal stem cell (MSC) treatment reduced the early pro-inflammatory response to Escherichia coli pneumonia. (A) Macrophage inflammatory protein 2 (MIP-2) was significantly lower in MSC-treated mice versus phosphate-buffered saline (PBS)-treated mice (n = 7–8 per group, *p = 0.03) and (B) tumour necrosis factor α (TNFα) was reduced in MSC-treated mice compared with 3T3-treated and PBS-treated mice 8 h after infection (n = 8–10 per group, *, #p = 0.04 for MSC vs PBS and 3T3 groups). Data are mean±SD. (C) Neutrophil influx was reduced in MSC-treated mice compared with controls as measured by bronchoalveolar lavage (BAL) levels of myeloperoxidase (MPO) 8 h after infection with E coli (n = 4 per group, *p = 0.04 for MSC vs PBS and #p = 0.03 for MSC vs 3T3). Data are mean±SD.

Figure 5

Lipocalin 2 production is upregulated with mesenchymal stem cell (MSC) treatment and accounts for part of the antimicrobial effect. (A) MSC-treated mice had higher levels of lipocalin 2 in their bronchoalveolar lavage (BAL) fluid at 8 h post infection (n = 4–6 per group, *p = 0.005 for MSC vs phosphate-buffered solution (PBS) and #p = 0.01 for MSC vs 3T3). Data are mean±SD. (B) Co-administration of a lipocalin 2 blocking antibody (anti-lcn-2 Ab) with MSCs significantly reduced the antibacterial effect of MSC treatment in vivo. MSCs retained significant antibacterial activity when co-administered with an isotype control antibody (n = 3–7 per group, √p

Figure 6

Mesenchymal stem cells (MSCs) upregulate their gene expression and secretion of lipocalin 2 with lipopolysaccharide (LPS) stimulation. (A) Quantitative reverse transcriptase PCR on RNA isolated from unstimulated and LPS-stimulated MSCs demonstrated a significant upregulation of mRNA for the lipocalin 2 gene with LPS stimulation (n = 5 per group, *p

Figure 7

Mesenchymal stem cells (MSCs) enhance their secretion of lipocalin 2 in response to a combination of inflammatory signals including tumour necrosis factor α (TNFα), produced by activated macrophages, and lipopolysaccharide (LPS). (A) Co-culture of MSCs and alveolar macrophages (AMs) led to a significant increase in the quantity of lipocalin 2 produced (n = 8–12 per group, *p−11 vs MSC + LPS group, √p = 0.0003 vs MSC + LPS + 200 pg/ml TNF-α group, #p<0.05 vs MSC + 1000 pg/ml TNFα). Data are mean±SD.