The Regulatory Roles Of Regulatory T Cells In The Development Of Immunosuppression At The Late Phase Of Sepsis

Sepsis, a disorder of systemic inflammation response to severe infection or injury, is the leading cause of death of the severe infection patients and animals. In recent years, late death occurred in immunosuppressive phase of sepsis patients often exceeds the death occurred in acute phase of sepsis. Immunosuppression-associated immunopathologic alterations have been considered as a predominant factor in induction of late death in the majority of patients who survive the initial hypeinflammatory state. In this study, we established an immunosuppressed sepsis model of mice by LPS injection and tested the immune status of mice at different times after LPS injection to identify the time caused severe immunosuppression. Then we explored the roles of Tregs in the formation of immunesuppression and its influence to the death ratio of sepsis mice undergoing chronic or acute peritonitis CLP.The following results were obtained in the present study:1. LPS-induced septic mice have the typical immunological features of sepsis.We induced the sepsis model of mice by injecting different doses of LPS. The results showed that injection of mice with 1.25 mg/kg LPS leads to classical clinical signs and immunosuppression-associated alterations in the late phase. Mice injected with 5 mg/kg LPS showed more serious clinical signs with extended time to recover compared with the mice injected with 1.25 mg/kg LPS. We examined the inflammatory cytokines in serum. A detailed time-course showed that, after 5 mg/kg LPS challenge, IL-12, TNF-α, IL-6, IFN-γ, MCP-1, IL-6, IL-10 increase at 1~12 h, and followed by a rapid decline at 24 h post-LPS treatment. We then detected the changes of spleen lymphocytes and blood lymphocytes. The results showed that lymphocytes decreased at 24 h post-LPS treatment. Mice given 5 mg/kg of LPS followed by challenging with bacterial infections at 24 h post-LPS exhibited a decreased inflammatory cytokines and weakened bacteria clearance ability compared with the control mice. These data indicated that injection of mice with 5 mg/kg LPS leads to classical clinical signs and immunosuppression-associated alterations at 24 h post-LPS treatment.2. Regulatory T cells(Tregs) play an important role in promoting the development of immunosuppression in the late sepsis.We used anti-CD25 mAb to deplete Tregs in sepsis mice and detected the effects of the depletion of Tregs on the formation of immunosuppression in the late sepsis. The results showed that the percentages of CD4+ T cells, CD8+ T cells and NK cells in the splenocytes of mice injected anti-CD25 mAb were significantly increased compared to those of isotype control-treated mice. Splenocytes from Treg-depleted sepsis mice exhibited significantly higher percentages of CD4+ CD69+, CD8+ CD69+ activated T cells and CD69+ activated NK cells compared with isotype-matched controls. Upon IL-12 stimulation in vitro, the percentages of IFN-γ-producing T cells and NK cells from spleen of anti-CD25 mAb-treated sepsis mice significantly increased at 24 h post-LPS compared to that of isotype control mAb-treated sepsis mice. These results suggested that the depletion of Tregs could significantly reverse the decrease of CD4+ T cells, CD8+ T cells and NK cells induced by LPS and increase the activity and reactivity of the T cells and NK cells in late sepsis.3. The depletion of Tregs during sepsis can enhance resistance to subsequent chronic infection.We assessed the bacterial clearance rate of Treg-depleted LPS mice undergoing a chronic peritonitis induced using CLP at 24 hours post-LPS. We found that bacterial counts in the peritoneal cavity, blood and lungs increased about two orders of magnitude in the LPS-induced sepsis mice compared to that in control mice at 72 h after chronic CLP. Depletion of Tregs during sepsis decreased bacterial counts in the peritoneal cavity, blood and lungs after chronic CLP. The survival differences in animals undergoing CLP were also significant between Treg-depleted mice and isotype matched control mice. The survival rate of LPS mice given isotype control mAb was 40% at 14 days after undergoing a chronic peritonitis induced by CLP, whereas the survival rate of LPS mice given anti-CD25 mAb was over 70%, suggesting that Treg-depletion using anti-CD25 mAb increased the survival of LPS sepsis mice over 30%. These results suggested that the depletion of Tregs enhances resistance to subsequent chronic infections and improves the survival rate of LPS sepsis mice upon second chronic infections.4. The depletion of Tregs during sepsis could enhance susceptibility to secondary infection and increase early mortality upon sever subsequent infection.We evaluated the survival of LPS mice given anti-CD25 mAb or isotype control mAb after undergoing a medium-grade CLP severity. Treg-depleted mice exhibited a significantly decreased survival rate compared to the isotype control mAb-injected mice. These results suggested that depletion of Tregs leads to enhanced susceptibility to secondary infection and increases mortality upon sever subsequent infection.This study established mouse model of LPS-induced sepsis, and bacterial infections and CLP were used as a way of secondary infection to study the role of Tregs in the formation of immunesuppression in late sepsis. We found that although the depletion of Tregs during sepsis could enhances resistance to subsequent chronic infections and improves the survival rate of LPS sepsis mice upon second chronic infections, it leads to enhanced susceptibility to secondary infection and increases mortality upon sever subsequent infection. This work provides a theoretical support for recognizing the roles and mechanism of the regulation of Tregs in in severe infections of sepsis, and further and better understanding of the regulation mechanisms of Tregs in sepsis should help to design more effective immune-based therapies and improve the outcome of post septic patients.