An increase in CD3+CD4+CD25+ regulatory T cells after administration of umbilical cord-derived mesenchymal stem cells during sepsis

Abstract

Sepsis remains an important cause of death worldwide, and vigorous immune responses during sepsis could be beneficial for bacterial clearance but at the price of collateral damage to self tissues. Mesenchymal stem cells (MSCs) have been found to modulate the immune system and attenuate sepsis. In the present study, MSCs derived from bone marrow and umbilical cord were used and compared. With a cecal ligation and puncture (CLP) model, the mechanisms of MSC-mediated immunoregulation during sepsis were studied by determining the changes of circulating inflammation-associated cytokine profiles and peripheral blood mononuclear cells 18 hours after CLP-induced sepsis. In vitro, bone marrow-derived MSCs (BMMSCs) and umbilical cord-derived MSCs (UCMSCs) showed a similar morphology and surface marker expression. UCMSCs had stronger potential for osteogenesis but lower for adipogenesis than BMMSCs. Compared with rats receiving PBS only after CLP, the percentage of circulating CD3+CD4+CD25+ regulatory T (Treg) cells and the ratio of Treg cells/T cells were elevated significantly in rats receiving MSCs. Further experiment regarding Treg cell function demonstrated that the immunosuppressive capacity of Treg cells from rats with CLP-induced sepsis was decreased, but could be restored by administration of MSCs. Compared with rats receiving PBS only after CLP, serum levels of interleukin-6 and tumor necrosis factor-α were significantly lower in rats receiving MSCs after CLP. There were no differences between BMMSCs and UCMSCs. In summary, this work provides the first in vivo evidence that administering BMMSCs or UCMSCs to rats with CLP-induced sepsis could increase circulating CD3+CD4+CD25+ Treg cells and Treg cells/T cells ratio, enhance Treg cell suppressive function, and decrease serum levels of interleukin-6 and tumor necrosis factor-α, suggesting the immunomodulatory association of Treg cells and MSCs during sepsis.

Figure 1. Comparison of BMMSCs and UCMSCs.

(A) In vitro culture, BMMSCs and UCMSCs showed a similar spindle-shaped morphology (100×). (B) UCMSCs had stronger potential for osteogenesis than BMMSCs after 2-week induction (von Kossa staining, 100×). (C) UCMSCs had lower potential for adipogenesis than BMMSCs after 2-week adipogenic induction (Oil red O staining, 200×).

Figure 2. The beneficial effects from MSC administration on survival in rats with CLP-induced sepsis.

Survival was evaluated for 14 days. Although no statistical significance, it appeared that rats receiving UCMSCs or BMMSCs after CLP had lower mortality rates than rats receiving PBS only after CLP. n = 10 rats/group.

Figure 3. Changes of circulating inflammation-associated cytokine profiles in rats 18 hours after CLP.

Compared with sham-operated rats, serum levels of IL-6 and TNF-α were elevated in rats undergoing CLP (i.e. BMMSC, UCMSC and PBS groups). Compared with rats receiving PBS only after CLP, the levels of IL-6 and TNF-α were significantly lower in rats receiving MSCs, either BMMSCs or UCMSCs. There were no significant differences in these two parameters between rats receiving BMMSCs and UCMSCs. There were no significant differences in serum levels of GM-CSF, MCP-1, IL-1 and IL-10 among sham control, BMMSC, UCMSC and PBS groups. Data are presented as mean ± SEM. n = 6–9 rats/group. *p<0.05 versus sham control group. # p<0.05 versus PBS group.

Figure 4. Analysis of peripheral blood mononulcear cells in rats 18 hours after CLP.

Compared with sham-operated rats, the percentages of circulating CD11b+CD3- monocytes and CD11b/c+CD3- dendritic cells were elevated in rats undergoing CLP (i.e. BMMSC, UCMSC and PBS groups). Compared with rats receiving PBS only after CLP, the percentage of circulating CD3+CD4+CD25+ Treg cells was significantly higher in rats receiving BMMSCs or UCMSCs after CLP, and so was the ratio of Treg cells/T cells. There were no significant differences in these two parameters between rats of BMMSC and UCMSC groups. Data are presented as mean ± SEM. n = 6–9 rats/group. *p<0.05 versus sham control group. #p<0.05 versus PBS group.

Figure 5. Treg cell suppressive function in rats 18 hours after CLP.

(A) Analysis of CFSE-labeled CD4+CD25- cell proliferation by flow cytometry after pre-treatment with a IL-2/CD3/CD28 mixture was shown. (B) Proliferation of CFSE-labeled CD4+CD25- cells was decreased when cocultured with CD4+CD25+ Treg cells from rats of Sham, BMMSC, and UCMSC groups. The suppressive capacity of CD4+CD25+ cells from rats receiving PBS only after CLP nearly disappeared. Data are presented as mean ± SEM. n = 6–9 rats/group. #p<0.05 versus PBS group.

Figure 6. Evaluation of lung immunohistochemistry in rats 18 hours after CLP.

Positive cells for CD44 or CD105 which represented the transferred human MSCs can be found in the perivascular interstitial area 14 hours after administration of BMMSCs or UCMSCs (200×).