S. D. Larson1, A. L. Cuenca1, B. E. Szpila2, B. Mathias1, A. G. Cuenca2, L. F. Gentile2, P. A. Efron2, L. L. Moldawer2 1University Of Florida,Pediatric Surgery, Department Of Surgery, UF College Of Medicine,Gainesville, FL, USA 2University Of Florida,Department Of Surgery, UF College Of Medicine,Gainesville, FL, USA
Introduction: Severe infection and sepsis during the neonatal period is a global health care issue with over 1 million deaths annually. Contributing to this high mortality is the neonate’s functionally distinct innate immune response. In previous studies, we have demonstrated that adjuvant activation via the TLR4 receptor improves outcomes in neonatal sepsis. Unfortunately, TLR agonists (e.g. LPS, resiquimod) are contraindicated in clinical use due to adverse side-effects. Aluminum (alum) salts are currently used as adjuvants in pediatric vaccines to improve immune responses; however, the mechanisms by which alum mediate these responses are incompletely understood. Therefore, the purpose of our study was two-fold: 1) As the neonate is more dependent on innate immunity, we examined whether pretreating neonates with alum wound improve survival and 2) to determine if alum enhances the myelopoietic response to polymicrobial sepsis.
Methods: 5-7 day old (neonate) C57BL/6J (B6) mice received intraperitoneal (IP) administration of cecal slurry (CS; LD25-45) to induce intra-abdominal polymicrobial sepsis. Neonates received either no pretreatment (control) or 20 µg aluminum hydroxide (alum) via IP injection 24 h prior to CS administration. Following injection of CS, mice were observed for 7 days to determine survival. Bone marrow and splenocytes were harvested at baseline (0 h) and 36 h following CS. Peritoneal washes and blood samples were collected at 0, 2, 6 and 24 h following sepsis. Harvested cells were analyzed by flow cytometry for phenotype.
Results: Neonates pretreated with alum had significantly improved survival compared to groups receiving CS alone (p<0.001). Alum pretreated mice had significant expansion of hematopoietic stem cells (Lin–ska+c-kit+ or LSKs) in the bone marrow and spleen 36 h following sepsis compared to controls (1.98±0.25 vs. 0.61±0.05 (p<0.0001), 1.59±0.51 vs. 0.42±0.04 (p<0.001), respectively). At 2 h following sepsis, pretreated neonates had increased populations of macrophages and natural killer (NK) cells in the peritoneum compared to controls (7.57±0.81% vs. 1.86±0.92% (p=0.001), 11.6±2.97% vs. 0.94±0.46% (p<0.003), respectively). Total percentage of peritoneal NK cells remained increased at 6 h following sepsis in pretreated neonates (4.97±2.37% vs. 0.66±0.27%; p=0.03).
Conclusion: We demonstrate here for the first time that neonates pretreated with alum have improved survival following polymicrobial sepsis. Alum pretreatment leads to a dramatic increase in bone marrow and splenic LSKs, peritoneal macrophages and NK cells, likely influencing this survival advantage. Finally, alum pretreatment appears to play a previously undescribed role in the neonatal myelopoietic response to sepsis.