Regulatory T cells are protective in systemic inflammation response syndrome induced by zymosan in mice

Abstract

Systemic inflammation response syndrome (SIRS) is a key and mainly detrimental process in the pathophysiology of multiple organ dysfunction syndrome. The balance of pro-inflammation and anti-inflammation controls the initiation and development of SIRS. However, the endogenous counterregulatory immune mechanisms that are involved in the development of SIRS are not well understood. CD4(+)CD25(+)Foxp3 (forkhead box P3)(+) regulatory T lymphocytes (Treg cells) play a key role in the immunological balance of the body. Thus, our aim was to investigate the contribution of these key immunomodulators (Treg cells) to the immune dysfunction that is observed in zymosan-induced SIRS in mice. We first evaluated the level of Treg cells in the lung of mice 6 h, 1 d, 2 d, 3 d, 5 d, and 7 d after the injection of zymosan or normal saline by western blot, real-time PCR and flow cytometry. We found that the number of Treg cells and the levels of the Treg cell-related transcription factor (Foxp3) and cytokines (IL-10) in the zymosan-treated group significantly decreased on day 1 and day 2 and significantly increased on day 5 compared with the NS-treated group. In the next experiment, the mice were injected with 200 μg of anti-CD25 mAb (clone PC61) to deplete the Treg cells and then injected with zymosan 2 days later. The number of Treg cells decreased by more than 50% after the injection of the PC61 mAb. In addition, the expression of the anti-inflammatory cytokine IL-10 also decreased. Moreover, the depletion of the Treg cells profoundly increased the mice'mortality and the degree of lung tissue injury. In conclusion, Treg cells tend to play a protective role in pathogenesis of the zymosan-induced generalized inflammation, and IL-10 signaling is associated with their immunomodulatory effect.

Figure 1. Lung W/D ratio (A) and histology (B).

The lungs were stained with hematoxylin-eosin (original magnification: 40×). NS (a), 1d after the injection of zymosan (b), 2d after the injection of zymosan (c), 3d after the injection of zymosan (d), 5d after the injection of zymosan (e), and 7d after the injection of Zymosan (f). The values show the means ± SEM (n = 6 for each group). *P<0.05 compared with the NS-treated group; **P<0.01 compared with the NS-treated group.

Figure 2. The IL-6, TNF-α and IL-10 mRNA expression levels in the lung tissues.

Expression levels of IL-6 mRNA (A), TNF-α mRNA (B) and IL-10 mRNA in the lung tissues (C). The values show the means ± SEM (n = 6 for each group). *P<0.05 compared with the NS-treated group; **P<0.01 compared with the NS-treated group.

Figure 3. The Foxp3 mRNA expression level and the Foxp3 protein level in the lung tissues and flow cytometry analysis of the Treg cells in the spleen.

Expression level of Foxp3 mRNA (A), Western blot of Foxp3 protein level (B) and flow cytometry of Treg cells (C). The values show the means ± SEM (n = 6 for each group). *P<0.05 compared with the NS-treated group; **P<0.01 compared with the NS-treated group.

Figure 4. Effect of the injection of PC61 mAb on the Treg cells of normal mice.

Mice (6 animals per group) were intraperitoneally injected with PBS (a), 200 μg of IgG1 mAb (b), or 200 μg of purified anti-CD25 mAb (PC61) (c). Two days after the injection, the spleen cells were collected. The numbers indicate the percentage of CD4⁺CD25⁺cells within the CD4⁺population and the percentage of Foxp3⁺cells within the CD4⁺population. The values show the means ± SEM. **P<0.01 compared with the PBS- and the IgG1-treated groups.

Figure 5. Effect of the injection of PC61 mAb on the Treg cells of ZIGI mice.

Mice (6 animals per group) were injected intraperitoneally with 200 μg of IgG1 mAb (a, c) or 200 μg of purified anti-CD25 mAb (PC61) (b, d) on day −2. Zymosan was administered intraperitoneally on day 0. The spleen cells were collected on day 2 and day 5. The numbers indicate the percentage of CD4⁺CD25⁺cells within the CD4⁺population and the percentage of Foxp3⁺cells within the CD4⁺population. The values show the means ± SEM. **P<0.01 compared withthe ZYM and the IgG1+ZYM groups.

Figure 6. Lung W/D ratio and histology of ZIGI mice with depleted levels of Treg cells.

Lung W/D ratio (A, n = 6 for each group). The lungs of ZIGI mice, which had depleted levels of Treg cells, were collected and stained with hematoxylin-eosin (B, original magnification: 10×). The values show the means ± SEM. *P<0.05compared with the ZYM and the IgG1+ZYM groups.

Figure 7. The IL-10, IL-6, and TNF-α mRNA expression levels in the lung tissues of ZIGI mice with depleted levels of Treg cells.

Expression levels of IL-10 mRNA, IL-6 mRNA and TNF-α mRNA on day 2 (A) and day 5 (B) in the lung tissues of ZIGI mice with depleted levels of Treg cells. The values show the means ± SEM (n = 6 for each group). *P<0.05 compared with ZYM and IgG1+ZYM; **P<0.01 compared with ZYM and IgG1+ZYM.

Figure 8. Survival of ZIGI mice with depleted levels of Treg cells.

Survival of ZIGI mice that were administered the PC61 mAb. The following groups were analyzed: ZYM (n = 80), IgG1+ZYM (n = 80), and PC61+ZYM (n = 80). The survival curves were compared using the logrank test (P<0.05).