Prior Hypoxia Exposure Enhances Murine Microglial Inflammatory Gene Expression in vitro Without Concomitant H3K4me3 Enrichment.

Elizabeth A Kiernan,Abiye Agbeh,Avtar S Roopra,Jyoti J Watters,Andrew O Knutson,Andrea C Ewald,Tao Wang,Jonathan N Ouellette

doi:10.3389/fncel.2020.535549

Abstract

Hypoxia (Hx) is a component of multiple disorders, including stroke and sleep-disordered breathing, which often precede or are comorbid with neurodegenerative diseases. However, little is known about how hypoxia affects the ability of microglia, resident CNS macrophages, to respond to subsequent inflammatory challenges that are often present during neurodegenerative processes. We, therefore, tested the hypothesis that hypoxia would enhance or “prime” microglial pro-inflammatory gene expression in response to a later inflammatory challenge without programmatically increasing basal levels of pro-inflammatory cytokine expression. To test this, we pre-exposed immortalized N9 and primary microglia to hypoxia (1% O2) for 16 h and then challenged them with pro-inflammatory lipopolysaccharide (LPS) either immediately or 3–6 days following hypoxic exposure. We used RNA sequencing coupled with chromatin immunoprecipitation sequencing to analyze primed microglial inflammatory gene expression and modifications to histone H3 lysine 4 trimethylation (H3K4me3) at the promoters of primed genes. We found that microglia exhibited enhanced responses to LPS 3 days and 6 days post-hypoxia. Surprisingly, however, the majority of primed genes were not enriched for H3K4me3 acutely following hypoxia exposure. Using the bioinformatics tool MAGICTRICKS and reversible pharmacological inhibition, we found that primed genes required the transcriptional activities of NF-κB. These findings provide evidence that hypoxia pre-exposure could lead to persistent and aberrant inflammatory responses in the context of CNS disorders.

Highlights

Hypoxia (Hx) is a component of multiple disorders affecting the central nervous system (CNS), including stroke, cancer, sleep-disordered breathing, and apneas of prematurity (Rowat et al, 2001; Martin et al, 2011; Wei et al, 2011; Joseph et al, 2015; Lavie, 2015)
To identify the effects of Hx pre-exposure on subsequent microglial responses to an inflammatory challenge, we first tested the global effects of Hx alone on microglial gene transcription
Similar to the effects of Hx reported in other cell types (Batie et al, 2019), KEGG Pathway analysis demonstrated that genes upregulated by Hx were primarily involved in the immune system and metabolic function

Summary

INTRODUCTION

Hypoxia (Hx) is a component of multiple disorders affecting the central nervous system (CNS), including stroke, cancer, sleep-disordered breathing, and apneas of prematurity (Rowat et al, 2001; Martin et al, 2011; Wei et al, 2011; Joseph et al, 2015; Lavie, 2015). These simultaneous exposures do not elucidate the effects of Hx pre-exposure in the absence of other stimuli on macrophage priming, nor the underlying molecular mechanisms contributing to long-term effects Both Hx and macrophage pathogen priming shift cell metabolism to glycolysis (Roiniotis et al, 2009; Chiba et al, 2017; Lachmandas et al, 2017), impacting the availability of cofactors necessary for histone modifications that regulate inflammatory gene programs (Cheng et al, 2014; Arts et al, 2016b; Netea et al, 2016). The application MAGICTRICKS identified NF-κB as a common DNA binding factor regulating genes primed by Hx, and its pharmacological inhibition confirmed an important role for this transcription factor in Hx-induced gene priming

MATERIALS AND METHODS

RESULTS

DISCUSSION

DATA AVAILABILITY STATEMENT