Cure Alveolar Soft Part Sarcoma International (iCureASPS)

Manipulation of ambient housing temperature to study the impact of chronic stress on immunity and cancer in mice


Abstract

Mice are the pre-eminent research organism in which to model human diseases and study the involvement of the immune response. Rapidly accumulating evidence indicates a significant involvement of stress hormones in cancer progression, resistance to therapies, and suppression of immune responses. As a result, there has been a concerted effort to model human stress in mice. Here, we discuss recent literature showing how mice in research facilities are chronically stressed at baseline due to environmental factors. Focusing on housing temperature, we suggest that the stress of cool housing temperatures contributes to the impact of other imposed experimental stressors and therefore has a confounding effect on mouse stress models. Furthermore, we propose that manipulation of housing temperature is a useful approach for studying the impact of chronic stress on disease and the immune response and for testing therapeutic methods of reducing the negative effects of chronic stress.

Keywords: adrenergic stress, thermoneutral housing, destressing mouse models

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6352311/

Introduction:

For decades, it has been recognized, largely through epidemiological observations, that certain forms of chronic stress resulting from psychological conditions such as depression, lack of social support, and anxiety, suppress immunity and may serve as a risk factor for cancer progression (1–3). Research into the interrelationships between stress, the nervous system, and the immune system have given rise to the field of “psychoneuroimmunology”. Recent laboratory research in this area has begun to provide a mechanistic understanding of the pathways that mediate the negative impact of stress on cancer (4). Moreover, studies are now identifying behavioral (1) or pharmacological (5) interventions that reduce stress and improve cancer outcomes. As is the case for the study of other important human diseases, mouse models have been developed to carry out preclinical investigations into these relationships. However, recent reports have raised concerns that the physiology of control mice housed under standard vivarium conditions reflects the adverse effects of choices that have been made regarding several housing parameters (6–9); density, cage tops, cage color, bedding, temp, cage environment, husbandry, noise (10, 11); and light intensity (12). This concern is forcing researchers to re-examine presumptions that we have held about the physiology of mouse used for preclinical experiments and to consider how these factors affect experimental outcomes. In this brief review, we focus on evidence that mice are chronically stressed at baseline due to housing temperatures and discuss how this inherent stress may affect disease models and our efforts to understand how stress impacts these models. This is particularly important for any disease or therapy with an immune component considering that this baseline stress is known to be immunosuppressive (13–16). We also highlight the utility of manipulating housing temperature to model the impact of stress in murine models of cancer and other diseases.

Housing temperatures for mice affect chronic adrenergic stress levels and experimental outcomes:

There are a myriad of factors that can affect the outcomes of pre-clinical studies. Many of these factors are specifics of the experimental design (including mouse strain, age, and sex as well as the source of mice and reagents) that are choices investigators make and report, enabling others to assess how these factors may affect outcomes. However, there are many other environmental factors that impact outcomes but are not reported because they are mandated by the Guide for Care and Use of Laboratory Animals (34) and implemented in animal facilities by the staff. Environmental factors such as the type of light, the type of cage, room temperature, humidity, diet, and noise levels are somewhat hidden variables that are seldom reported but are known to influence mouse physiology (10, 12, 35–38). Investigators naturally presume that these housing decisions are made based on optimizing the biology of the mice, but this is not always the case; many of these decisions are based, with good reason, on convenience and comfort of the people who work long hours in these facilities. However, it was pointed out almost a decade ago, that standard housing conditions provide a lifestyle for mice where they are “sedentary, have continuous access to food, and have virtually no environmental stimulation” and consequently are “metabolically morbid” being “overweight, insulin resistant, hypertensive” and at risk for premature death (6). These authors raised the alarm that presuming that these mice represent healthy baseline controls is problematic and could bias the outcomes of experiments. Soon after this report, Feldman et al(36) reported in a pivotal study that the outcomes of experiments studying obesity in UPC1 knockout mice differed depending on whether mice were housed at standard temperatures (~22˚C) or thermoneutrality (~30˚C) and that these mice demonstrated the expected obesity only when housed at 30˚C. Thus, the role for UCP1 in adaptive adrenergic thermogenesis, which had been questioned on the basis of negative results obtained in mice housed at 22˚C, was confirmed when chronic cold stress was alleviated by housing at 30˚C, clearly demonstrating the significance of considering ambient housing temperatures when planning and interpreting experiments. Our group first reported that mouse models of cancer and anti-tumor immunity (39–41), immune responses in graft vs host disease (42), dendritic cell biology (43), and radiosensitivity of hematopoietic stem cells (44) are each significantly influenced by room temperature. These are representative of a growing number of papers reporting how choice of housing temperature impacts experimental outcomes and reproducibility in several mouse models of disease and we have recently reviewed this topic (45–47). Since these reviews were published, similar effects on mouse models of Alzheimer’s (48), osteoporosis (49), fatty liver disease (50), and asthma (51) have also been reported.

With respect to the study of stress and cancer and anti-tumor immunity, we discovered that tumor growth is accelerated by chronic (mild) cold stress by standard room temperature of ~ 22˚C as compared to a thermoneutral 30˚; thus, the efficacy of the anti-tumor immune response differs significantly depending on the housing temperature (41). In this model, we observed a significant increase in anti-tumor effector CD8+ T-cells in the tumor microenvironment and in draining lymph nodes, and a decrease in both regulatory T cells and MDCS (immunosuppressive cells) at 30˚C, demonstrating that housing mice at 22˚C alone results in significant suppression of the anti-tumor immune response. We also observed that this effect is lost if tumors are grown in immunodeficient mice, implicating a role for the adaptive immune response (39, 41). We went on to show that this difference is also lost when mice (housed at 22˚C) were treated with β-adrenergic receptor antagonists (β-blockers) confirming that the degree of adrenergic stress is a function of room temperature (39, 41). These results suggest that trying to study the efficacy of immunotherapy when mice are housed at 22˚C is extremely problematic. In fact, we found that the effect of housing temperature on the efficacy of immunotherapy (the checkpoint inhibitor anti-PD-1) was dramatic. Both mammary and melanoma tumors showed little to no response at 22˚C but had a significant response at 30˚C (39). We also demonstrated that the increased adrenergic signaling at 22˚C has a direct effect on tumor cells, engaging survival mechanisms such as upregulation of anti-apoptotic molecules that increase tumor cell resistance to cytotoxic therapies(40) and could possibly increase resistance to cytotoxic immune cells. These issues have critical implications for interpreting the results of experiments studying the effects of adrenergic stress and the development of strategies for overcoming stress to improve response to immune or cytotoxic therapies in mice.

Housing temperatures for mice affect chronic adrenergic stress levels and experimental outcomes:

“There are a myriad of factors that can affect the outcomes of pre-clinical studies ...”( of mice )

“With respect to the study of stress and cancer and anti-tumor immunity, we discovered that tumor growth is accelerated by chronic (mild) cold stress by standard room temperature of ~ 22˚C as compared to a thermoneutral 30˚; thus, the efficacy of the anti-tumor immune response differs significantly depending on the housing temperature (41). In this model, we observed a significant increase in anti-tumor effector CD8+ T-cells in the tumor microenvironment and in draining lymph nodes, and a decrease in both regulatory T cells and MDCS (immunosuppressive cells) at 30˚C, demonstrating that housing mice at 22˚C alone results in significant suppression of the anti-tumor immune response. We also observed that this effect is lost if tumors are grown in immunodeficient mice, implicating a role for the adaptive immune response (39, 41). We went on to show that this difference is also lost when mice (housed at 22˚C) were treated with β-adrenergic receptor antagonists (β-blockers) confirming that the degree of adrenergic stress is a function of room temperature (39, 41). These results suggest that trying to study the efficacy of immunotherapy when mice are housed at 22˚C is extremely problematic. In fact, we found that the effect of housing temperature on the efficacy of immunotherapy (the checkpoint inhibitor anti-PD-1) was dramatic. Both mammary and melanoma tumors showed little to no response at 22˚C but had a significant response at 30˚C (39). We also demonstrated that the increased adrenergic signaling at 22˚C has a direct effect on tumor cells, engaging survival mechanisms such as upregulation of anti-apoptotic molecules that increase tumor cell resistance to cytotoxic therapies(40) and could possibly increase resistance to cytotoxic immune cells. These issues have critical implications for interpreting the results of experiments studying the effects of adrenergic stress and the development of strategies for overcoming stress to improve response to immune or cytotoxic therapies in mice.”