Postnatal inflammation in ApoE-/- mice is associated with immune training and atherosclerosis Ellesandra C. Noye1, Siroon Bekkering2,3, Albert P. Limawan4,5, Maria U. Nguyen4,6, Lisa K. Widiasmoko4,5, Hui Lu4, Salvatore Pepe2,6, Michael M. Cheung2,6, Trevelyan R. Menheniott6,7, Megan J Wallace4, Timothy J. Moss4,8, David P. Burgner2,6,9*, Kirsty R. Short1,10* 1 School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia Queensland 4072, Australia 2Murdoch Children's Research Institute, Royal Children's Hospital, Parkville, Australia 3Department of Internal Medicine, Radboud University Medical Centre, Nijmegen, The Netherlands 4The Ritchie Centre, Hudson Institute of Medical Research, Clayton, Australia 5Fakultas Kedokteran, Universitas Indonesia, Indonesia 6Department of Paediatrics, University of Melbourne, Parkville, Australia 7Janssen Pharmaceuticals, Melbourne, Australia 8Department of Obstetrics and Gynaecology, Monash University, Clayton, Australia 9Department of Paediatrics, Monash University, Clayton, Australia 10Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, Australia INTRODUCTION: Preterm birth is associated with increased risk of cardiovascular disease (CVD). This may reflect a legacy of inflammatory exposures that characterise preterm births, including prenatal inflammation (chorioamnionitis) and recurrent early life infections. Experimental chorioamnionitis followed by postnatal inflammation has additive and deleterious effects on atherosclerosis in ApoE-/- mice. Here, we aimed to investigate whether innate immune training is a contributory inflammatory mechanism in this murine atherosclerosis model. METHODS: Bone marrow-derived macrophages and peritoneal macrophages were isolated from 13 week-old ApoE-/- mice, exposed to prenatal intra-amniotic (experimental chorioamnionitis) and/or repeated postnatal (peritoneal) lipopolysaccharide (LPS). Innate immune training was assessed by cytokine responses following 24-hour in vitro stimulation with TLR agonists (LPS, Pam3Cys), and RPMI (control) for 24 hours. Percentages of bone marrow progenitor populations, previously reported to be enhanced following immune training, were assessed by flow cytometric analysis. RESULTS: Following postnatal LPS exposure, bone marrow-derived macrophages and peritoneal macrophages produced more pro-inflammatory cytokines following TLR stimulation in vitro compared to those from unexposed mice, indicative of a trained phenotype. In addition, in peritoneal macrophages, this enhanced inflammatory response was positively associated with atherogenesis (as characterized by CD45+ cell plaque infiltration). Prenatal LPS exposure alone did not induce a trained phenotype, nor have an additive effect on in vitro cytokine responses when followed by postnatal LPS exposure. However, prenatal and postnatal LPS exposure was associated with a reduction in progenitor cells in the bone marrow. CONCLUSION: Postnatal inflammation results in a trained phenotype in atherosclerosis-prone mice that is not enhanced by prenatal inflammation. If analogous mechanisms occur in humans, then there may be novel early life opportunities to reduce CVD risk in individuals with a history of early life infections.