Mesenchymal Stem Cell-Conditioned Medium Induces Neutrophil Apoptosis Associated with Inhibition of the NF-κB Pathway in Endotoxin-Induced Acute Lung Injury

Abstract

The immunomodulatory effects of mesenchymal stem cells (MSCs) are established. However, the effects of MSCs on neutrophil survival in acute lung injury (ALI) remain unclear. The goal of this study was to investigate the effect of an MSC-conditioned medium (MSC-CM) on neutrophil apoptosis in endotoxin-induced ALI. In this study, an MSC-CM was delivered via tail vein injection to wild-type male C57BL/6 mice 4 h after an intratracheal injection of lipopolysaccharide (LPS). Twenty-four hours later, bronchoalveolar lavage fluid (BALF) and lung tissue were collected to perform histology, immunohistochemistry, apoptosis assay of neutrophil, enzyme-linked immunosorbent assays, and an electrophoretic mobility shift assay. Human neutrophils were also collected from patients with sepsis-induced acute respiratory distress syndrome (ARDS). Human neutrophils were treated in vitro with LPS, with or without subsequent MSC-CM co-treatment, and were then analyzed. Administration of the MSC-CM resulted in a significant attenuation of histopathological changes, the levels of interleukin-6 and macrophage inflammatory protein 2, and neutrophil accumulation in mouse lung tissues of LPS-induced ALI. Additionally, MSC-CM therapy enhanced the apoptosis of BALF neutrophils and reduced the expression of the anti-apoptotic molecules, Bcl-xL and Mcl-1, both in vivo and in vitro experiments. Furthermore, phosphorylated and total levels of nuclear factor (NF)-κB p65 were reduced in lung tissues from LPS + MSC-CM mice. Human MSC-CM also reduced the activity levels of NF-κB and matrix metalloproteinase-9 in the human neutrophils from ARDS patients. Thus, the results of this study suggest that the MSC-CM attenuated LPS-induced ALI by inducing neutrophil apoptosis, associated with inhibition of the NF-κB pathway.

Keywords: Mesenchymal Stem Cell; Neutrophil apoptosis; acute lung injury.

Figure 1

A tail vein injection of the MSC-CM improved the histological features of LPS-induced ALI in male C57BL/6 mice. (A) H&E staining demonstrates that lung injury was significantly attenuated in the mice that received LPS + MSC-CM compared with LPS alone. The control mice and MSC alone mice exhibited minimal histopathologic abnormalities. Magnification = 100×. Scale bar = 100 μm. (B) The lung injury scores were also significantly decreased in the mice that received LPS + MSC-CM compared with LPS alone. Data are presented as the mean ± SD. * p < 0.05, compared to the controls; ** p < 0.05, compared to the LPS group; n = 6 per group.

Figure 2

Administration of the MSC-CM decreased the levels of the proinflammatory cytokines, IL-6 and MIP-2, in BALF collected from mice with ALI. In ELISA assays, significantly higher concentrations of (A) IL-6 and (B) MIP-2 were detected in BALF samples after LPS exposure (LPS) compared to the control group. In contrast, administration of the MSC-CM after LPS (LPS+MSC-CM) resulted in a significant reduction in IL-6 and MIP-2 levels compared to the LPS group. Data are presented as the mean ± SD. * p < 0.05, compared to the controls; ** p < 0.05, compared to the LPS group; n = 6 per group.

Figure 3

MSC-CM reduced neutrophil accumulation in the lungs of LPS-induced ALI mice. (A) Neutrophil accumulation was examined with immunofluorescent staining of lung tissues for Ly6G, a neutrophil marker. Neutrophil accumulation in alveoli significantly increased after LPS exposure (LPS) and was significantly reduced following administration of the MSC-CM (LPS+MSC-CM). Green: Ly6G (lymphocyte antigen 6G); Blue: 4’,6-diamidino-2-phenylindole (DAPI). Magnification = 100×. Scale bar = 100 μm. The number of neutrophils per section for lung tissue samples stained for Ly6G are presented as the mean ± SD. (B) Myeloperoxidase (MPO) activity was detected in whole lung tissue lysates and the data indicate that there was significant neutrophil accumulation. LPS mice (LPS) had a higher level of MPO in the lung compared to the control mice, whereas the mice that received LPS + MSC-CM had decreased levels of MPO compared to LPS mice. Data are presented as the mean ± SD. * p < 0.05, compared to the controls; ** p < 0.05, compared to the LPS group; n = 6 per group.

Figure 4

Apoptosis of pulmonary neutrophils were significantly induced by MSC-CM treatment in vivo and vitro models. (A) In vivo model, the ratio of apoptotic neutrophils present in BALF samples collected from the mice with LPS-induced ALI (LPS) (4.6 ± 2%) contained a lower ratio of apoptotic neutrophils compared with control mice (Control) (10.3 ± 0.15%). In contrast, the ratio of apoptotic neutrophils was higher in the mice that received LPS + MSC-CM (LPS+MSC-CM) (33.4 ± 2%) than LPS alone (LPS). The bar graph below presents the percentage apoptosis for each experimental group. (B) In vitro model, the neutrophils isolated from BALF samples collected from the mice with LPS-induced ALI and cultured 2 h with or without LPS and MSC-CM treatment in vitro. Mouse neutrophils with LPS in vitro stimulation (LPS) (53.7 ± 14.3%) contained a lower ratio of apoptosis compared with control neutrophils (Control) (92.7 ± 5.5%). In contrast, the ratio of apoptotic neutrophils was higher in neutrophils that received LPS + MSC-CM (90.4 ± 8.6%) compared with neutrophils receiving LPS only (LPS). The bar graph below presents the percentage apoptosis for each experimental group. (C) Expression of Bcl-xL and Mcl-1 in pulmonary neutrophils of BALF samples increased in the mice with LPS-induced ALI (LPS) compared to the control mice (Control), while treatment with MSC-CM(LPS+MSC-CM)markedly reduced both the expression of Bcl-xL and Mcl-1. Data are presented as the mean ± SD. PI: propidium iodine; Blue: DAPI (4’,6-diamidino- 2-phenylindole); Green (FITC): Ly6G (lymphocyte antigen 6G); Red (Cy5): Bcl-xL or Mcl-1. Magnification = 400×. Scale bar = 100 μm. Arrows indicate positive cells. Immunofluoresence intensity was quantitated for the control, LPS, and LPS-MSC tissue sections for the staining of Bcl-xL and Mcl-1 and the data are presented as the mean ± SD. * p < 0.05, compared to the controls; ** p < 0.05, compared to the LPS group; n = 6 per group.

Figure 5

Administration of the MSC-CM reduced the expression levels of total and phosphorylated NF-kB p65 in mice with LPS-induced ALI. (A) Lung tissues from the mice challenged with LPS (LPS) exhibited more positive staining for total NF-kB p65 than control mice (Control). In contrast, lung tissues from mice that received LPS + MSC-CM exhibited a significant decrease in total NF-kB p65 staining compared to the LPS mice. Staining of phospho-NF-kB p65 was enhanced in lung tissues from the LPS mice compared with control mice, and staining was significantly reduced in lung tissues from the mice that received LPS + MSC-CM compared to the LPS mice. (B) The quantification of IHC staining showed the similar results. Magnification = 100× and 200× (red box). Scale bar = 100 μm. For both sets of staining, quantitation of staining intensity is presented as the mean ± SD in bar graphs. * p < 0.05, compared to the controls; ** p < 0.05, compared to the LPS group; n = 6 per group.

Figure 6

The human MSC-CM (hMSC-CM) reduced NF-kB activation and the expression of MMP-9 in human neutrophils isolated from patients with ARDS. (A) Human neutrophils that were treated with LPS for 30 min or 60 min in vitro exhibited a significant increase in NF-kB activity in EMSA analyses compared to the control neutrophils. In contrast, human neutrophils that underwent LPS treatment (30 min or 60 min) and hMSC-CM (LPS+hMSC-CM) showed a marked reduction in NF-kB activation compared to LPS-treated neutrophils. (B) In ELISA assays of MMP-9 activity from culture mediums of human neutrophils in vitro, greater activity was observed after LPS treatment, while a marked reduction in MMP-9 activity was observed after LPS + hMSC-CM treatment. Data are presented as the mean ± SD. * p < 0.05, compared to the controls; ** p < 0.05, compared to the LPS group; n = 6 per group.