TSG-6 Secreted by Human Adipose Tissue-derived Mesenchymal Stem Cells Ameliorates DSS-induced colitis by Inducing M2 Macrophage Polarization in Mice

Abstract

Previous studies have revealed that mesenchymal stem cells (MSCs) alleviate inflammatory bowel disease (IBD) by modulating inflammatory cytokines in the inflamed intestine. However, the mechanisms underlying these effects are not completely understood. We sought to investigate the therapeutic effects of human adipose tissue-derived (hAT)-MSCs in an IBD mouse model and to explore the mechanisms of the regulation of inflammation. Dextran sulfate sodium-induced colitis mice were infused with hAT-MSCs intraperitoneally and colon tissues were collected on day 10. hAT-MSCs were shown to induce the expression of M2 macrophage markers and to regulate the expression of pro- and anti-inflammatory cytokines in the colon. Quantitative real time-PCR analyses demonstrated that less than 20 hAT-MSCs, 0.001% of all intraperitoneally injected hAT-MSCs, were detected in the inflamed colon. To investigate the effects of hAT-MSC-secreted factors in vitro, transwell co-culture system was used, demonstrating that tumour necrosis factor-α-induced gene/protein 6 (TSG-6) released by hAT-MSCs induces M2 macrophages. In vivo, hAT-MSCs transfected with TSG-6 small interfering RNA, administered intraperitoneally, were not able to induce M2 macrophage phenotype switch in the inflamed colon and had no significant effects on IBD severity. In conclusion, hAT-MSC-produced TSG-6 can ameliorate IBD by inducing M2 macrophage switch in mice.

Figure 1

Intraperitoneally injected hAT-MSCs ameliorate IBD. DSS-induced colitis mice were infused hAT-MSCs or PBS (vehicle control) on day 1. (a) Body weight, measured every day and expressed as the relative change from day 0. (b–d) Mice were sacrificed on day 10 and (b) DAI, and (c) colon length, were assessed. (d) Representative H&E staining of the colon sections, and their histological scores are shown. Bars = 100 µm. Six to eight mice per group were used. Results are shown as mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

hAT-MSCs inhibit inflammatory response in the colon. (a) mRNA expression levels of pro- and anti-inflammatory cytokines in colons were determined by qRT-PCR. (b) Levels of TNF-α and IL-10 in colons were assessed using ELISA. Six to eight mice per group were used. Results are presented as mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

hAT-MSC administration leads to an increase in the percentage of M2 macrophages in the colon. (a) Expression levels of CD206, Arg1, Fizz1, and Ym1 were determined using qRT-PCR. (b) Representative immunofluorescence staining using anti-CD11b or anti-CD206 antibodies. Bars = 30 µm. (c) The number of CD11b- or CD206-positive cells within the inflammatory infiltrates, and the calculated percentage of CD206-positive cells among the CD11b-positive cells. Six to eight mice per group were used. Results are presented as mean ± standard deviation. *P < 0.05, ***P < 0.001.

Figure 4

Intraperitoneally administered hAT-MSCs do not migrate into the colon. Serial dilutions of hAT-MSCs were administered to the investigated mice and the expression of human-specific GAPDH was evaluated. The results are representative of three independent experiments.

Figure 5

TNF-α-stimulated hAT-MSCs induce the expression of the M2 macrophage markers. (a,b) TSG-6 (a) gene and (b) protein expression levels in TNF-α-stimulated hAT-MSCs and naïve hAT-MSCs. (c,d) LPS-stimulated Raw 264.7 macrophages were co-cultured with naïve or TNF-α-stimulated hAT-MSCs in a transwell system for 48 h. (c) CD206, Arg1, Fizz1, and Ym1 mRNA expression levels. (d) CD206 and Arg1 protein expression levels in these cells. Results are presented as mean ± standard deviation of the data obtained in three independent experiments. *P < 0.05, ***P < 0.001.

Figure 6

Expression of M2 macrophage markers decreases in hAT-MSCs transfected with TSG-6 siRNA. (a,b) TSG-6 (a) mRNA and (b) protein expression levels in hAT-MSCs transfected with TSG-6 siRNA (siTSG6-hAT-MSCs), hAT-MSCs transfected with control siRNA (siCTL-hAT-MSCs), hAT-MSCs with transfection reagent, and naïve hAT-MSCs. (c,d) LPS-stimulated Raw 264.7 macrophages were co-cultured with naïve or siCTL- or siTSG6-hAT-MSCs for 48 h. (c) CD206, Arg1, Fizz1, and Ym1 mRNA expression levels in these cells. (d) CD206 and Arg1 protein expression levels. Results are presented as mean ± standard deviation obtained in three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7

TSG-6 knockdown in hAT-MSCs inhibits their effect on IBD. (a) Body weight of animals injected with siTSG6-hAT-MSCs, siCTL-hAT-MSCs, or naïve hAT-MSCs was measured every day during the experiment, and expressed in terms of the relative change from the weight measured on day 0. (b–d) Mice were sacrificed on day 10 to evaluate clinical severity. (b) DAI, (c) colon length, and (d) representative colon tissue sections stained with H&E and histological scores are shown. Bars = 100 µm. Six to eight mice per group were used. Results are presented as mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 8

TSG-6 knockdown in hAT-MSCs inhibits their effect on M2 macrophage phenotypic switch. (a) The gene expression levels of CD206, Arg1, Fizz1, and Ym1 in the colon samples of mice injected with siTSG6-hAT-MSCs, siCTL-hAT-MSCs, or naïve hAT-MSCs. (b) Representative immunofluorescence staining using CD11b- or CD206-specific antibodies. Bars = 30 µm. (c) The number of CD11b- or CD206-positive cells within the inflammatory infiltrates, and the calculated percentage of CD206-positive cells among the CD11b-positive cells. Six to eight mice per group were used. Results are presented as mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001.