Effects of hypertonic saline on CD4+CD25+Foxp3+ regulatory T cells after hemorrhagic shock in relation to iNOS and cytokines.

Background: Hemorrhagic shock and resuscitation induce immunosuppression. CD4(+)CD25(+)Foxp3(+) regulatory T Cells (Foxp3(+) Tregs), iNOS and cytokines may affect these severe conditions such as acute respiratory distress syndrome and multiple organ failure after hemorrhagic shock and resuscitation. Foxp3(+) Tregs have been described to be specific and play a key role in the control of the immune system. Immune condition may be restored by hypertonic saline resuscitation that inhibits pro-inflammatory effects of cytokine. Our aim was to investigate how hypertonic saline resuscitation affected Foxp3(+) Tregs after hemorrhagic shock and resuscitation in relation to iNOS and cytokines.

Methods: Male C57BL6/J and B6.129P2- NOS2(tm1Lau)/J (iNOS gene knockout) mice were used in creating hemorrhagic shock model. Mice were divided into two groups, each according to the type of resuscitation. (1) Wild HS: resuscitation with hypertonic saline (4 mL/Kg of 7.5% NaCl) and the shed blood (SB); (2) wild 2LR: resuscitation with lactated Ringer's solution and the SB; (3) iNOS knockout HS: similarly resuscitated as wild HS; (4) iNOS knockout 2LR: similarly resuscitated as wild 2LR. Samples of thymus and spleen were harvested at 2, 6, 24, 48, and 72 h after resuscitation. CD4(+) T cells and Foxp3(+) Tregs were analyzed at 24, 48, and 72 h. At 2, 6, 24, and 48 h, plasma cytokines were assayed and expression of iNOS (NOS2) was also measured by immunofluorescence.

Results: NOS2 of HS and 2LR wild groups at 2 and 6 h in spleen increased compared with the control group. At 6h, NOS2 in HS wild group was significantly lower than in 2LR wild group. Plasma levels of interleukin (IL)-6, TNF- α, MCP-1, and IL-10 increased at 2 h. Both in wild type and iNOS knockout mice, hypertonic saline resuscitation decreased plasma IL-6, TNF-α, and MCP-1 levels at 2 h; CD4(+) T cells in spleen and thymus decreased at 24, 48, and 72 h, and Foxp3(+) Tregs in spleen at 48 h increased, however, hypertonic saline resuscitation did not affect the Foxp3(+).

Conclusions: These results show that in early phase, the inflammatory cytokines in plasma might affect iNOS expression and cytokines. Further, this study showed that hypertonic saline resuscitation and suppression of iNOS might improve immunosuppressive reaction after hemorrhagic shock.