Suppressed inflammatory phenotype following exertional heat stroke in mice: an epigenetic link?
Authors: Murray Kevin O. , Iwaniec John , Brant Jason , de Carvalho Lucas , Robinson Gerard P , Garcia Christian K , Alzahrani Jamal , Laitano Orlando , Kladde Michael P. , Clanton Thomas L. 
Published: April 2020

Exertional heat stroke (EHS) is a life‐threatening medical event that requires immediate care. Current guidelines recommend return‐to‐physical activity following EHS in only a few weeks, with full return‐to‐physical activity around three months. We have previously observed that a single episode of EHS in mice leads to a long‐lasting, altered epigenetic signature in progenitor monocytes and in skeletal muscle DNA, as well as marked alterations to the heart’s metabolomic profile. These observations occur with no outward indications of underlying disorders in normal mouse behavior or appearance. Therefore, the physiologic manifestations of the epigenetic changes remain unknown.PURPOSETo establish a link between the epigenetic changes we observe and an inflammatory cell phenotype. To do this we tested whether a single EHS exposure leads to a sustained and aberrant inflammatory response in blood immune cells, one month after EHS exposure.METHODSSixteen mice were either subjected to a standardized EHS protocol using a forced running wheel (environmental temp: 37.5°C, 40% humidity) or a matched exercise control trial (EXC) (22.5°C). The EHS mice achieved peak core temps of ~42.2°C, accompanied by transient loss of consciousness. Mice were sacrificed after 30 days of recovery. Blood was collected via cardiac stick. One aliquot of collected blood was exposed to 1 ng/mL of lipopolysaccharide (LPS) in solution of RPMI and Pen/Strep and placed in a 5% CO2 incubator at 37.0°C for 18 hours; the other aliquot served as a control (no LPS). Thereafter, blood was centrifuged, and plasma was collected and frozen in −80°C until cytokine analysis by Luminex. Cytokine concentration in both the LPS and no LPS blood conditions were normalized per 100,000 cells in each individual mouse. Reported as mean ± SD.RESULTSIn EXC group, LPS increased [IL‐6] by 30.01 ± 14.5 pg whereas in the EHS group LPS increased [IL‐6] by only 7.0 ± 17.4 pg (P=0.003). In EXC group, LPS increased [TNF‐a] by 0.33 ± 0.22 pg, whereas in the EHS group LPS increased [TNF‐a] by only 0.11 ± 0.11 pg (P=0.001). Genes integral to TLR‐4‐mediated secretion of IL‐6 and TNF‐a that have differentially methylated CpG sites include: TLR4 (59 sites), Nfkb1 (38), Foxo1 (81), IL‐6 (4), Myd88 (7) and a variety of JAK/Stat and MAPK proteins; some of these CpG sites reside within the promoter region of their respective gene.CONCLUSIONWe have shown a blunted inflammatory response 30 days after a single episode of EHS. This abnormal inflammatory response in mature blood‐residing immune cells may be a manifestation of the unique EHS‐induced DNA methylation pattern we observed within immature progenitor monocytes. Our findings suggest that responsiveness of the immune system could be considered as a potential biomarker for both return‐to‐physical activity and long‐term care, post‐EHS.Support or Funding InformationDOD Grant BA180078. The opinions or assertions contained herein are the private views of the author(s) and are not to be construed as official or reflecting the views of the Army or the DoD.