REGULATORY T-CELLS SUPPRESS THE DEVELOPMENT OF THE 2-HIT RESPONSE AFTER INJURY

Abstract

Introduction: We recently reported that injury induces increased CD4 regulatory T cell (Treg) activity and that Tregs play a role in controlling the host inflammatory response to burn injury. In this study, we address the potential for Tregs to actively influence the development of enhanced innate immune system activation and the associated predisposition to the 2-hit response after burn injury. Methods: This was studied by testing the effects of in vivo Treg cell depletion on the responsiveness of sham versus burn mice to re-purified bacterial lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) specific agonist. Groups of male, C57BL/6J mice were depleted of Tregs by anti-CD25 antibody treatment or were given control antibody at 3 days before sham or burn injury. To examine mortality, mice were challenged with 5 mg/kg or 10 mg/kg of LPS by intraperitoneal injection at 7 days after sham or burn injury. The influence of Treg deficiency on TLR4 reactivity in vivo was determined by measuring plasma and organ cytokine levels after LPS challenge. Parallel studies tested the influence of Treg depletion on TLR4-induced cytokine production by spleen cells in vitro. Results: We found that burn mice that were Tregdeficient at the time of injury showed significantly higher mortality to LPS challenge at 7 days after injury (p<0.05, Log rank test). Burn-injured Treg-deficient mice had higher plasma and lung levels of TNFα, IL-6, and MCP-1after LPS challenge than control burn mice, sham mice, and Treg-deficient sham mice (P < 0.001, ANOVA). LPS-stimulated spleen cells prepared from Treg-deficient burn-injured mice also produced higher levels of TNFα and MCP-1 than control burn-injured mice, sham mice, and Treg-deficient sham mice. Conclusion: Taken together, these findings indicate that Tregs play an active role in suppressing the innate inflammatory response to injury. We also demonstrate that in their absence the injured-host becomes more predisposed to a heightened 2-hit response.