Immunosuppression is associated with epigenetic remodelling in a murine model of exertional heat stroke

Exertional heat stroke (EHS) is prevalent among athletes and military personnel, especially when training in hot and humid conditions. As their ability to regulate their core body temperature becomes impaired due to excessive endogenous and exogenous heat accumulation, severe hyperthermia and neurological dysfunction occurs. EHS is a true medical emergency, as risk of fatality is high if immediate treatment is not available. Even with appropriate cooling, individuals with EHS have a heightened risk of infection and future chronic health impairments such as cardiovascular disease (Murray et al. 2021). Furthermore, due to the often seasonal nature of this condition (i.e. higher in summer months), the risk of recurrence exists. Increased risk of infection poses a serious health risk to patients recovering from EHS, but little is known as to why this occurs. A recent review described the role of immune cell dysfunction in the systemic inflammation observed during EHS; however, the mechanisms of heat shock on immune dysfunction remain unclear (Lim, 2018). Of greater pertinence to recovering patients is the question of why immune responses remain disabled long after the resolution of EHS. Addressing this knowledge gap is a critical stepping point in increasing our understanding of EHS and eventually improving outcomes for patients. The recent work by Murray et al. (2021), published in The Journal of Physiology, addressed this gap in knowledge and postulated that EHS may influence long-term immunity by remodelling the epigenome. Epigenetic variations influence phenotype by altering the structure of DNA without changing the actual nucleotide sequence. For example, methylation and demethylation of cytosine residues in CpG dinucleotides results in the repression or promotion of gene expression, respectively. Such epigenetic modifications have previously been demonstrated to alter immune function (Kim et al. 2016), though whether EHS produces immunomodulation via this mechanism was unknown before this research. Briefly, the authors induced EHS in young female C57BL/6J mice by placing them in an enclosed running wheel in an environmental chamber set to 37.5˚C and 30–38% relative humidity and forcing them to run at a speed at which their core temperature remained 41˚C until they lost consciousness. Each EHS mouse was paired with a matched exercise control (EXC) mouse that ran at the same speed and for the same duration as the EHS mouse, but at 22.5˚C. Following killing after either 4 or 30 days of recovery, whole blood was collected for assessment of immune reaction to lipopolysaccharide (LPS), and spines were removed for isolation of bone marrow-derived monocytes for analysis of DNA methylation. Consistent with their hypothesis, the epigenome of EHS monocytes was significantly altered compared with those from EXC animals at 4 days post-exercise and even more so after 30 days, indicating sustained epigenetic remodelling after the acute stimulus. At both time points, the majority of differentially methylated CpGs (DMC) were hypermethylated and a significant number were located in promoter regions, suggesting suppression of gene expression. A total of 3522 genes were affected by methylation after 4 days and 4212 after 30 days, with 1715 of these DMCs occurring at both time points. Using KEGG pathway analysis, the authors discovered various inflammatory pathways to be significantly affected at both time points, indicating that the promoter regions of genes related to inflammation may be chronically epigenetically modified due to EHS. Significant alterations in methylation status were seen in genes related to NFκB, a family of transcription factors central to immune and inflammatory responses. Specifically, the promoters of NFκB-associated genes, RelA, RelB, c-Rel and Bcl3, were hypermethylated, while genes that inhibit NFκB, such as Nfkbia, were hypomethylated, in EHS versus EXC mice. This suggests a reduction in NFκB-mediated transcription that could underlie the immunosuppression seen with EHS. Indeed, TNFα and IL-6 secretion was attenuated in EHS versus EXC mice administered LPS. Given that the expression of these cytokines is stimulated by NFκB, these findings provide a potential genomic mechanism for the immunosuppression related to EHS. By demonstrating that EHS induces epigenetic remodelling in monocytes that may be associated with an impaired immune response, Murray and colleagues laid the foundations for future research in the area of epigenetics, immunity, and EHS. Their focused analysis of DMCs in gene promoters related to immune pathways enabled significant detail to be elucidated, especially pertaining to NFκB. But in their methylome analysis they also discovered that a significant number of DMCs were located in other areas of the genome such as introns and intergenic regions; at both 4 and 30 days recovery, these regions were the genomic regions with the most DMCs. Many non-coding RNA molecules including miRNAs, which are short RNA fragments that repress gene expression, are located in introns and have been established as targets of epigenetic modifications including methylation. Genes involved in the immune system are influenced by miRNAs; for example, a variety of miRNAs directly and indirectly regulate NFκB. It is possible that methylation of these sites could contribute to the immunosuppressive phenotype observed in EHS mice. For instance, miRNA-199a has numerous roles including the repression of IKKβ, which inhibits NFκB activity. In one study, methylation of the intronic region in which miRNA-199a resides was found to reduce miRNA-199a activity (Cheung et al. 2011). Thus, analysing the methylation of other genomic regions is a potentially important but uninvestigated avenue in EHS-induced epigenome remodelling. By focusing on NFκB, the researchers were able to gain a detailed understanding of this pathway. But NFκB is only one, albeit important, component of innate responses. The complexity of the immune system suggests that it is unlikely that the immunosuppressive effects of EHS were the result of alterations to NFκB alone, despite it being a central player in immune and inflammatory responses. For instance, TLR4 is a key receptor in innate responses that can also enhance adaptive immunity. Despite playing a role in the response to LPS, methylation of this protein was not reported. In human cancer cell lines, both hyper- and hypomethylation of TLR4 have been shown to affect NFκB activation (Kim et al. 2016); therefore, this would be a valuable target for further investigation. Furthermore, inflammation in response to LPS is only a small part of defending an organism from an infection; thus, an intriguing next step would be to challenge EHS mice with a pathogen to assess more components of the innate response. Given that humans are generally at risk of bacterial infections rather than being exposed to LPS alone, conducting a pathogenic challenge would be very applicable to the end goal of improving healthcare and identifying susceptibility to illness and infection in those recovering from EHS. The authors also showed that the epigenome was modified for at least 30 days post-protocol, along with alterations in cytokine secretion. This prompts the question of whether epigenetic remodelling due to EHS could be seen beyond this period. Given that the incidence of EHS increases during the summer, if epigenetic changes due to EHS can be observed beyond 30 days, previous heat shock experiences could affect immunogenic outcomes of subsequent episodes (i.e. yearly in the summer). A recent review by Bošković & Rando (2018) on the topic of transgenerational epigenetics pointed to heat shock as a potential source of epigenetic remodelling in germ cells and discussed research in plants, flies and worms that showed multigenerational phenotypic changes as a result of this environmental stressor. If EHS can produce epigenetic modifications in gametes, then there could be a transgenerational impairment of immune function. Similarly, examining the epigenetic consequences of EHS during pregnancy could yield important findings. Given this, gametic and transgenerational epigenetic analyses are warranted to further elucidate the effects of EHS. On a related note, Murray and colleagues used female mice in their study, in accordance with previous findings on the effects of EHS in males versus females. But repeating this research in male mice is important, as their epigenome may be modified from EHS, despite not developing the same metabolic disorder (Murray et al. 2021). Elevated risk of infection following EHS is a serious, but relatively unstudied, health risk. Increasing our understanding of this process will eventually lead to improvements in health management and reductions in the mortality and morbidity of EHS. This seminal work by Murray et al. (2021) is an important stepping stone in the understanding of the connections between EHS and increased risk of infection. Their findings on the methylome and NFκB, in combination with future mechanistic research, may lead to more important conclusions. Though not discussed in this article, the risks for longer-term health problems in patients who experience EHS, including cardiovascular disease, may also be under epigenetic control. As cardiovascular disease is one of the leading killers worldwide, it is possible that an increased understanding of EHS will be translatable to this disease. Finally, the authors have generated an immense dataset that, for the most part, remains relatively unexplored to date but will be instrumental to advance our understanding of EHS and help direct future studies in the area. Important next steps include investigating methylation of other components of the immune system, other tissues, and the germline, to determine the magnitude by which EHS influences the epigenome. None. Sole author. None. The author thanks Dr. David Mutch for critically reviewing this manuscript.

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