Long-term intermittent hypoxia in mice induces inflammatory pathways implicated in sleep apnea and steatohepatitis in humans

Obstructive Sleep Apnea (OSA) engenders repetitive desaturation-reoxygenation sequences called intermittent hypoxia (IH), now widely accepted as an independent risk factor for the occurrence and progression of non-alcoholic fatty liver disease (NAFLD). The specific molecular mechanisms linking IH and the development and progression of NAFLD remain elusive. NAFLD pathogenesis is multifactorial including immune cell-driven inflammation as a key mechanism in the transition from fatty liver to steatohepatitis (NASH). The overall goal of this study was to identify the specific impact of IH on liver inflammation in the absence of major confounders frequently encountered in OSA clinical research (i.e., obesity, diabetes, reduced physical activity). We employed a model of lean mice exposed to long-term IH (16 weeks) and a cohort of lean OSA patients free of comorbidities (n=71) coupled with high-throughput hepatic transcriptomics, lipidomics and targeted serum proteomics. For the first time, we have demonstrated that long-term IH is an independent “hit” sufficient by itself to induce the NASH molecular signatures found in human steatohepatitis transcriptomic datasets.We identified a unique set of biomarkers in mice and humans (PPARs, NRFs, arachidonic acid, IL16, IL20, IFNB and TNFa) associated with early hepatic and systemic inflammation. This molecular connection between IH, sleep apnea and steatohepatitis warrants further investigation in clinical trials and support the systematic implementation of sleep apnea diagnosis in liver disease phenotyping. Our original signatures could allow the identification of potential diagnostic and treatment response markers, as well as therapeutic targets in the comorbid association between NAFLD and OSA. Overall design: Sixteen-week-old male C57BL/6JRj mice (Janvier Labs, France) were housed with ad libitum food and water access on a 12 h light/12 h dark cycle (light ON at 8 am and light OFF at 8 pm). Mice were maintained with regular chow diet (LASQC diet, Rod 16, Altromin international) during all the experiment. Mice were randomly assigned to either intermittent hypoxia (IH) or normoxic control (NC) and directly exposed in their housing cages to 16 weeks of NC or IH, 8h per day during their sleeping period (IH from ZT0=8 am to ZT8=4 pm and NC the rest of the day). The IH stimulus consisted of 60 sec cycles alternating 30 sec of hypoxia (hypoxic plateau at 5% FiO2) and 30 sec of NC (normoxic plateau at 21% FiO2). NC mice were exposed to similar air-air cycles in order to avoid bias from noise and turbulence related to gas flow. After 16 weeks of exposure, mice were sacrificed by decapitation, livers were harvested and immediately frozen in liquid nitrogen until further analysis. For RNA extraction, 50mg of frozen liver tissues were homogenized in TRIzol Reagent and RNA was extracted after precipitation with isopropanol and ethanol washes according to the manufacturer's protocol. RNA quality was monitored for quality control using Agilent Bioanalyzer Nano RNA chip and Nanodrop absorbance ratios of 260/280 nm and 260/230 nm. Library construction was performed according to the Illumina kit protocol (TruSeq stranded mRNA sample preparation).The multiplexed libraries were sequenced on one lane using single-end 100 cycles chemistry for the NovaSeq 6000 (Illumina)