title: Fetal microglial phenotype in vitro carries memory of prior in vivo exposure to inflammation.
abstract: OBJECTIVE
  Neuroinflammation in utero may result in life-long neurological disabilities. The molecular mechanisms whereby microglia contribute to this response remain incompletely understood.
  
  
  METHODS
  Lipopolysaccharide (LPS) or saline were administered intravenously to non-anesthetized chronically instrumented near-term fetal sheep to model fetal inflammation in vivo. Microglia were then isolated from in vivo LPS and saline (naïve) exposed animals. To mimic the second hit of neuroinflammation, these microglia were then re-exposed to LPS in vitro. Cytokine responses were measured in vivo and subsequently in vitro in the primary microglia cultures derived from these animals. We sequenced the whole transcriptome of naïve and second hit microglia and profiled their genetic expression to define molecular pathways disrupted during neuroinflammation.
  
  
  RESULTS
  In vivo LPS exposure resulted in IL-6 increase in fetal plasma 3 h post LPS exposure. Even though not histologically apparent, microglia acquired a pro-inflammatory phenotype in vivo that was sustained and amplified in vitro upon second hit LPS exposure as measured by IL-1β response in vitro and RNAseq analyses. While NFKB and Jak-Stat inflammatory pathways were up regulated in naïve microglia, heme oxygenase 1 (HMOX1) and Fructose-1,6-bisphosphatase (FBP) genes were uniquely differentially expressed in the second hit microglia. Compared to the microglia exposed to LPS in vitro only, the transcriptome of the in vivo LPS pre-exposed microglia showed a diminished differential gene expression in inflammatory and metabolic pathways prior and upon re-exposure to LPS in vitro. Notably, this desensitization response was also observed in histone deacetylases (HDAC) 1, 2, 4, and 6. Microglial calreticulin/LRP genes implicated in microglia-neuronal communication relevant for the neuronal development were up regulated in second hit microglia.
  
  
  CONCLUSIONS
  We identified a unique HMOX1 down and FBP (up) phenotype of microglia exposed to the double-hit suggesting interplay of inflammatory and metabolic pathways. Our findings suggest that epigenetic mechanisms mediate this immunological and metabolic memory of the prior inflammatory insult relevant to neuronal development and provide new therapeutic targets for early postnatal intervention to prevent brain injury.
authors: Department of Obstetrics and Gynaecology, Faculty of Medicine, CHU Ste-Justine Research Centre, Université de Montréal Montréal, QC, Canada ; Department of Neurosciences, Faculty of Medicine, CHU Ste-Justine Research Centre, Université de Montréal Montréal, QC, Canada.
authors: Mingju
authors: M
authors: Cao
authors: Faculty of Veterinary Medicine, Animal Reproduction Research Centre, Université de Montréal Montréal, QC, Canada.
authors: Marina
authors: M
authors: Cortes
authors: Neuroimmunology Unit, Montréal Neurological Institute, McGill University Montréal, QC, Canada.
authors: Craig S
authors: CS
authors: Moore
authors: Neuroimmunology Unit, Montréal Neurological Institute, McGill University Montréal, QC, Canada.
authors: Soo Yuen
authors: SY
authors: Leong
authors: Department of Obstetrics and Gynaecology, Faculty of Medicine, CHU Ste-Justine Research Centre, Université de Montréal Montréal, QC, Canada ; Department of Neurosciences, Faculty of Medicine, CHU Ste-Justine Research Centre, Université de Montréal Montréal, QC, Canada.
authors: Lucien D
authors: LD
authors: Durosier
authors: Department of Clinical Sciences, Faculty of Veterinary Medicine, Université de Montréal QC, Canada.
authors: Patrick
authors: P
authors: Burns
authors: Department of Clinical Sciences, Faculty of Veterinary Medicine, Université de Montréal QC, Canada.
authors: Gilles
authors: G
authors: Fecteau
authors: Department of Clinical Sciences, Faculty of Veterinary Medicine, Université de Montréal QC, Canada.
authors: Andre
authors: A
authors: Desrochers
authors: Département de Pathologie, University Hospital Ste-Justine, Université de Montréal QC, Canada.
authors: Roland N
authors: RN
authors: Auer
authors: Department of Pediatrics, Faculty of Medicine, CHU Ste-Justine Research Centre, Université de Montréal Montréal, QC, Canada.
authors: Luis B
authors: LB
authors: Barreiro
authors: Neuroimmunology Unit, Montréal Neurological Institute, McGill University Montréal, QC, Canada.
authors: Jack P
authors: JP
authors: Antel
authors: Department of Obstetrics and Gynaecology, Faculty of Medicine, CHU Ste-Justine Research Centre, Université de Montréal Montréal, QC, Canada ; Department of Neurosciences, Faculty of Medicine, CHU Ste-Justine Research Centre, Université de Montréal Montréal, QC, Canada ; Faculty of Veterinary Medicine, Animal Reproduction Research Centre, Université de Montréal Montréal, QC, Canada.
authors: Martin G
authors: MG
authors: Frasch
keywords: RNAseq; bioinformatics; brain; cytokines; epigenetics; metabolism; neuroinflammation; sheep