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NF‐kB, but not HIF‐1a, Inhibition Gene‐specifically Blocks Hypoxia‐induced Microglial Priming
Author(s) -
Ewald Andrea C,
Kiernan Elizabeth A,
Janke Anna C,
Watters Jyoti J
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.1058.8
Subject(s) - microglia , hypoxia (environmental) , h3k4me3 , immune system , gene expression , biology , lipopolysaccharide , microbiology and biotechnology , priming (agriculture) , intermittent hypoxia , epigenetics , immunology , inflammation , cancer research , chemistry , medicine , gene , promoter , biochemistry , germination , botany , organic chemistry , oxygen , obstructive sleep apnea
The innate immune system has the capacity for “memory,” the ability to change future cellular responses based on prior experiences. Previous studies in our lab have shown that chronic intermittent hypoxia (IH), a hallmark of sleep‐disordered breathing, can induce cellular memory in both adult and neonatal microglia, CNS resident immune cells. When the immune system is challenged with lipopolysaccharide (LPS) the day following the last IH exposure, LPS‐induced microglial gene expression is augmented, indicating that IH pre‐exposure primes the microglial immune response. Hypoxia‐induced priming of microglial inflammatory gene responses are associated with upregulation of histone 3 lysine 4 trimethylation (H3K4me3) at gene promoters/enhancers, supporting an epigenetic role in microglial memory following hypoxia exposure. To begin to address the signaling mechanisms underlying IH‐induced priming, and the subsequent changes in H3K4me3, we developed an in vitro model of hypoxia which mirrors the microglial responses to IH observed in vivo. Using this model, we tested the hypothesis that hypoxia‐induced potentiation of inflammatory gene expression in microglia requires the activation of the hypoxia‐responsive transcription factors HIF‐1a and NF‐kB, key modulators of inflammatory gene expression. Immortalized N9 microglia were treated overnight with echinomycin (HIF‐1a inhibitor, 1 nM) or BOT‐64 (NF‐kB inhibitor, 3 μM) during the hypoxia exposure period (1.5% O2 for 16 hrs), after which time the cells were washed and stimulated with LPS (100 ng/mL; 3 hrs). We used qRT‐PCR to assess the gene expression of two inflammatory molecules that are potentiated by hypoxia pre‐exposure: inducible nitrogen oxide synthase (iNOS) and interleukin‐1 beta (IL‐1B). We found that while HIF‐1a inhibition did not attenuate the hypoxia‐induced potentiation of iNOS nor IL‐1B, NF‐kB inhibition had gene‐specific effects. Specifically, BOT‐64 blocked the potentiation of iNOS, but not IL‐1B. These results suggest that hypoxia‐induced priming of microglia involves the activation of NF‐kB, as well as at least one other unidentified pathway, to influence microglial gene expression. Ongoing studies are examining if NF‐kB inhibition also attenuates upregulation of H3K4me3 in inflammatory gene regulatory regions to provide insights into the mechanism whereby NF‐kB may regulate hypoxia‐induced microglial priming. Because this form of cellular memory augments the microglial inflammatory response, it may be associated with exacerbated CNS damage during sleep disordered breathing. Support or Funding Information This work was supported by NIH R01 NS085226.