AUTHOR=Cao Mingju , Cortes Marina , Moore Craig S., Leong Sooyuen , Durosier Lucien D., Burns Patrick , Fecteau Gilles , Desrochers Andre , Auer Roland N., Barreiro Luis , Antel Jack , Frasch Martin G.

TITLE=Fetal microglial phenotype in vitro carries memory of prior in vivo exposure to inflammation

JOURNAL=Frontiers in Cellular Neuroscience

VOLUME=9

YEAR=2015

URL=https://www.frontiersin.org/journals/cellular-neuroscience/articles/10.3389/fncel.2015.00294

DOI=10.3389/fncel.2015.00294

ISSN=1662-5102

ABSTRACT=<p><bold>Objective:</bold> Neuroinflammation <italic>in utero</italic> may result in life-long neurological disabilities. The molecular mechanisms whereby microglia contribute to this response remain incompletely understood.</p><p><bold>Methods:</bold> Lipopolysaccharide (LPS) or saline were administered intravenously to non-anesthetized chronically instrumented near-term fetal sheep to model fetal inflammation <italic>in vivo</italic>. Microglia were then isolated from <italic>in vivo</italic> LPS and saline (naïve) exposed animals. To mimic the second hit of neuroinflammation, these microglia were then re-exposed to LPS <italic>in vitro</italic>. Cytokine responses were measured <italic>in vivo</italic> and subsequently <italic>in vitro</italic> 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.</p><p><bold>Results:</bold><italic>In vivo</italic> 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 <italic>in vivo</italic> that was sustained and amplified <italic>in vitro</italic> upon second hit LPS exposure as measured by IL-1β response <italic>in vitro</italic> and RNAseq analyses. While NFKB and Jak-Stat inflammatory pathways were up regulated in naïve microglia, heme oxygenase 1 (<italic>HMOX1</italic>) and Fructose-1,6-bisphosphatase (<italic>FBP</italic>) genes were uniquely differentially expressed in the second hit microglia. Compared to the microglia exposed to LPS <italic>in vitro</italic> only, the transcriptome of the <italic>in vivo</italic> LPS pre-exposed microglia showed a diminished differential gene expression in inflammatory and metabolic pathways prior and upon re-exposure to LPS <italic>in vitro</italic>. Notably, this desensitization response was also observed in histone deacetylases (<italic>HDAC</italic>) <italic>1, 2, 4</italic>, and <italic>6</italic>. Microglial calreticulin/LRP genes implicated in microglia-neuronal communication relevant for the neuronal development were up regulated in second hit microglia.</p><p><bold>Discussion:</bold> We identified a unique <italic>HMOX1</italic><sub>down</sub> and <italic>FBP</italic><sup>up</sup> 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.</p>