L. K. Winer1, A. M. Pugh1, V. Nomellini1,2, C. Caldwell1 1University Of Cincinnati,Division Of Research, Department Of Surgery,Cincinnati, OH, USA 2University Of Cincinnati,Division of Trauma, Critical Care, and Acute Care Surgery, Department Of Surgery,Cincinnati, OH, USA
Introduction: Hypothermia can predict persistent lymphopenia in critically ill patients. In a murine model of intraabdominal sepsis that includes both surgical and antibiotic source control, these interventions were shown to normalize core body temperature, reverse lymphopenia, and improve survival of septic mice. However, the mechanisms linking hypothermia, lymphopenia, and mortality are still unknown. The objective of this study was to investigate the relationship between hypothermia and immunosuppression. We hypothesized that a hypothermic environment would induce lymphocyte apoptosis and dysfunction.
Methods: Single cell suspensions were prepared from the spleens of healthy CD-1 male mice. Lymphocytes were stimulated with an anti-CD3 antibody, and in vitro studies were performed at both 32 and 37° C. Lymphocytes were enumerated by flow cytometry. A fluorescent glucose analog was used to measure intracellular glucose uptake. Carboxyfluorescein succinimidyl ester (CFSE) was used to capture lymphocyte proliferation. Cytokines were quantified by a cytometric bead array assay.
Results: To determine whether hypothermia directly leads to lymphopenia, Annexin V staining was performed. This showed an approximately 1.5 fold increase in splenic CD4 apoptosis in hypothermic (32° C) compared to normothermic (37° C) conditions (p<0.05). Because septic mice are susceptible to secondary infection, suggestive of immunosuppression, we next characterized whether the remaining lymphocyte population has altered function. We found that hypothermia is associated with a significant decrease in CD4 activation and cellular division based on CFSE quantification. To further delineate the mechanism underlying diminished CD4 division, we examined cell signaling and metabolic pathways. This revealed that hypothermic CD4 cells secrete significantly less IL-2, a cytokine imperative for lymphocyte proliferation. Correspondingly, there was a significant reduction in IL-2 receptor alpha chain CD25 expression on CD4 lymphocytes. Finally, splenic CD4 lymphocytes had a 25% decrease in glucose uptake under hypothermic conditions (p<0.05).
Conclusion: Our study demonstrates that hypothermia causes lymphopenia through a combination of increased splenic CD4 apoptosis and decreased cellular proliferation. The remaining pool of available splenic CD4 lymphocytes is dysfunctional, with significantly reduced activation and glucose utilization. These data suggest an integral relationship between hypothermia and immune compromise, stressing the importance of early source control and thermoregulation to reduce the morbidity and mortality from sepsis.