Cold-stressed Mice Research Articles

Electroacupuncture Reduced Fibromyalgia-Pain-like Behavior through Inactivating Transient Receptor Potential V1 and Interleukin-17 in Intermittent Cold Stress Mice Model.

Fibromyalgia (FM) is a widespread musculoskeletal pain associated with psychological disturbances, the etiopathogenesis of which is still not clear. One hypothesis implicates inflammatory cytokines in increasing central and peripheral sensitization along with neuroinflammation, leading to an elevation in pro-inflammatory cytokines, e.g., interleukin-17A (IL-17A), enhanced in FM patients and animal models. The intermittent cold stress (ICS)-induced FM-like model in C57BL/6 mice has been developed since 2008 and proved to have features which mimic the clinical pattern in FM patients such as mechanical allodynia, hyperalgesia, and female predominance of pain. Electroacupuncture (EA) is an effective treatment for relieving pain in FM patients, but its mechanism is not totally clear. It was reported as attenuating pain-like behaviors in the ICS mice model through the transient receptor potential vanilloid 1 (TRPV1) pathway. Limited information indicates that TRPV1-positive neurons trigger IL-17A-mediated inflammation. Therefore, we hypothesized that the IL-17A would be inactivated by EA and TRPV1 deletion in the ICS-induced FM-like model in mice. We distributed mice into a control (CON) group, ICS-induced FM model (FM) group, FM model with EA treatment (EA) group, FM model with sham EA treatment (Sham) group, and TRPV1 gene deletion (Trpv1-/-) group. In the result, ICS-induced mechanical and thermal hyperalgesia increased pro-inflammatory cytokines including IL-6, IL-17, TNFα, and IFNγ in the plasma, as well as TRPV1, IL-17RA, pPI3K, pAkt, pERK, pp38, pJNK, and NF-κB in the somatosensory cortex (SSC) and cerebellum (CB) lobes V, VI, and VII. Moreover, EA and Trpv1-/- but not sham EA countered these effects significantly. The molecular mechanism may involve the pro-inflammatory cytokines, including IL-6, IL-17, TNFα, and IFNγ. IL-17A-IL-17RA play a crucial role in peripheral and central sensitization as well as neuroinflammation and cannot be activated without TRPV1 in the ICS mice model. EA alleviated FM-pain-like behaviors, possibly by abolishing the TRPV1- and IL-17A-related pathways. It suggests that EA is an effective and potential therapeutic strategy in FM.

Dietary rutin alleviated the damage by cold stress on inflammation reaction, tight junction protein and intestinal microbial flora in the mice intestine

Low temperature is a common stress source for the poultry industry in the north of China. However, the low energy consuming and economical way to reduce the negative effects from cold stress is still limited. Therefore, the aim of this study was to investigate the effect of rutin on intestinal barrier in mice under low temperature. The cold stress model was established at 4°C for 3 h each day and the experiment lasted for 21 days. Forty Balb/c mice were randomly divided into four treatments: CON, normal temperature with the basal diet; RUT, normal temperature with the basal diet +150 mg/kg body weight (BW) of rutin; CS, mice under cold stress with basal diet; CR, 150 mg/kg of BW rutin under cold stress. Rutin supplementation significantly increased the ileum villus-to-crypt ratio compared with these non-supplemented treatments. Rutin attenuated the hypothermia induced morphological damage in the ileum. In addition, rutin improved the antioxidant capacity of mice under cold stress. Rutin supplementation significantly increased the trypsin activity and inhibited the lipase in cold stressed mice. Rutin supplementation significantly inhibited the production of inflammatory factors induced by cold stress. Rutin induced the inhibition of TLR4 and NF-кB, thereby reducing the expression of inflammation-related genes. In addition, rutin improved the reduction of the intestinal claudin-1 and occludin expression in those mice in the cold stress (P < .05) and improved the intestinal ZO-1 expression in cold stressed mice. Finally, rutin alleviated the dysregulation of intestinal microflora in the mice under cold stress.

Effects of Chronic Cold Exposure on Proteomics of Lung Tissue in Mice

Background: Cold exposure can induce inflammation-related injury in lung tissue, but the exact mechanism is still unclear. Objective: The study aimed to clarify the proteomic characteristics of lung tissue under cold exposure. Methods: Forty mice were randomly equally divided into a control group and a model group. The model group was exposed to - 20°C for two weeks (4 hours per day), while the control group was maintained at 22 ± 2°C. H&amp;E staining and ELISA were used to verify the injury of lung tissue. Furthermore, a quantitative analysis of the overall proteome in the lung of mice exposed to cold stress was conducted by using LC-MS/MS. 15 differentially expressed proteins were selected for PRM validation. Results: According to our results, cold exposure induced lung injury, and the expressions of 151 proteins were upregulated and those of 95 proteins were downregulated. Bioinformatics analysis showed that differentially expressed proteins were associated with tricarboxylic acid cycle, fat metabolism, glycolysis, and oxidative phosphorylation. The expression of gabra2, Klkb1, and complement- related proteins was significantly upregulated. The results of PRM validation were consistent with those of proteomics. Conclusion: We found changes in glycolysis, gabra2, Klkb1, and the complement system in the lung tissue of cold-stressed mice, which may play an important role in cold stress-induced lung injury.

Integrated metabolism and epigenetic modifications in the macrophages of mice in responses to cold stress.

The negative effects of low temperature can readily induce a variety of diseases. We sought to understand the reasons why cold stress induces disease by studying the mechanisms of fine-tuning in macrophages following cold exposure. We found that cold stress triggers increased macrophage activation accompanied by metabolic reprogramming of aerobic glycolysis. The discovery, by genome-wide RNA sequencing, of defective mitochondria in mice macrophages following cold exposure indicated that mitochondrial defects may contribute to this process. In addition, changes in metabolism drive the differentiation of macrophages by affecting histone modifications. Finally, we showed that histone acetylation and lactylation are modulators of macrophage differentiation following cold exposure. Collectively, metabolism-related epigenetic modifications are essential for the differentiation of macrophages in cold-stressed mice, and the regulation of metabolism may be crucial for alleviating the harm induced by cold stress.

Comprehensive Transcriptional Profiling and Mouse Phenotyping Reveals Dispensable Role for Adipose Tissue Selective Long Noncoding RNA Gm15551.

Cold and nutrient-activated brown adipose tissue (BAT) is capable of increasing systemic energy expenditure via the uncoupled respiration and secretion of endocrine factors, thereby protecting mice against diet-induced obesity and improving insulin response and glucose tolerance in men. Long non-coding RNAs (lncRNAs) have recently been identified as fine-tuning regulators of cellular function. While certain lncRNAs have been functionally characterised in adipose tissue, their overall contribution in the activation of BAT remains elusive. We identified lncRNAs correlating to interscapular brown adipose tissue (iBAT) function in a high fat diet (HFD) and cold stressed mice. We focused on Gm15551, which has an adipose tissue specific expression profile, is highly upregulated during adipogenesis, and downregulated by β-adrenergic activation in mature adipocytes. Although we performed comprehensive transcriptional and adipocyte physiology profiling in vitro and in vivo, we could not detect an effect of gain or loss of function of Gm15551.

Adipose Lipolysis Regulates Cardiac Glucose Uptake and Function in Mice under Cold Stress.

The heart primarily uses fatty acids as energy substrates. Adipose lipolysis is a major source of fatty acids, particularly under stress conditions. In this study, we showed that mice with selective inactivation of the lipolytic coactivator comparative gene identification-58 (CGI-58) in adipose tissue (FAT-KO mice), relative to their littermate controls, had lower circulating FA levels in the fed and fasted states due to impaired adipose lipolysis. They preferentially utilized carbohydrates as energy fuels and were more insulin sensitive and glucose tolerant. Under cold stress, FAT-KO versus control mice had >10-fold increases in glucose uptake in the hearts but no increases in other tissues examined. Plasma concentrations of atrial natriuretic peptide and cardiac mRNAs for atrial and brain-type natriuretic peptides, two sensitive markers of cardiac remodeling, were also elevated. After one week of cold exposure, FAT-KO mice showed reduced cardiac expression of several mitochondrial oxidative phosphorylation proteins. After one month of cold exposure, hearts of these animals showed depressed functions, reduced SERCA2 protein, and increased proteins for MHC-β, collagen I proteins, Glut1, Glut4 and phospho-AMPK. Thus, CGI-58-dependent adipose lipolysis critically regulates cardiac metabolism and function, especially during cold adaptation. The adipose-heart axis may be targeted for the management of cardiac dysfunction.

Plasma levels of corticosterone, tumor necrosis factor receptor 1 and interleukin 6 are influenced by age, sex and chronic inflammation in mice treated with acute temperature stress.

Resiliency is the ability to respond to, adapt to and recover from stressors. Deterioration of resiliency in older adults has been hypothesized to be regulated by age-related changes in stress response systems, including the Hypothalamic Pituitary Adrenal (HPA) axis and the innate immune system response. Although age-related chronic inflammation is strongly related to lack of resiliency, the impact of chronic inflammation on acute stress response is unclear. Here we describe the impact of a five-hour exposure to cold temperature acute stressor, on immune and corticosterone response using older and younger IL-10tm/tm mice, a mouse model with chronic inflammatory pathway activation, and age and gender matched C57/Bl6 background control (WT) mice. Overall, mice exposed to 4°C for 5h had significantly higher plasma corticosterone levels compared to those that remained at room temperature (25°C), with the exception of the WT females. Cold stressed mice had lower plasma tumor necrosis factor receptor 1 (TNFR1) levels with varying significance, in all ages and phenotypes, with the exception of the old female WT mice. In contrast, the effects of cold stress on pro-inflammatory cytokine interleukin 6 (IL-6) levels were inconsistent and not significant, with the exception of the female IL-10tm/tm mice. In conclusion, these findings demonstrate that sex, age and chronic inflammatory pathway activation all influence corticosterone secretion and inflammatory processes in the face of acute cold stress.

Zinc-α2-glycoprotein promotes skeletal muscle lipid metabolism in cold-stressed mice.

Skeletal muscle is the most abundant tissue in the adult body and plays an essential role in maintaining heat production for the entire body. Recently, muscle-derived non-shivering thermogenesis under cold conditions has received much attention. Zinc-α2-glycoprotein (ZAG) is an adipokine that was shown to influence energy metabolism in the adipose tissue. We used ZAG knock-out (ZAG KO) and wild-type (WT) mice to investigate the effect of ZAG on the lipid metabolism of skeletal muscle upon exposure to a low temperature (6°C) for one week. The results show that cold stress significantly increases the level of lipolysis, energy metabolism, and fat browning-related proteins in the gastrocnemius muscle of WT mice. In contrast, ZAG KO mice did not show any corresponding changes. Increased expression of β3-adrenoceptor (β3-AR) and protein kinase A (PKA) might be involved in the ZAG pathway in mice exposed cold stress. Furthermore, expression of lipolysis-related proteins (ATGL and p-HSL) and energy metabolism-related protein (PGC1α, UCP2, UCP3 and COX1) was significantly enhanced in ZAG KO mice after injection of ZAG-recombinant plasmids. These results indicate that ZAG promotes lipid-related metabolism in the skeletal muscle when the animals are exposed to low temperatures. This finding provides a promising target for the development of new therapeutic approaches to improve skeletal muscle energy metabolism.

Zinc-α2-glycoprotein promotes browning of white adipose tissue in cold-exposed male mice

The promotion of white adipose tissue (WAT) browning has emerged as a promising therapeutic target to increase energy expenditure and decrease weight gain. Zinc-α2-glycoprotein (ZAG) is a newly identified adipokine that regulates lipid metabolism. It shows high expression in brown adipose tissue (BAT), but whether ZAG plays a key role in the browning of white adipose tissue is still largely unclear. In the present study, we explored the relationship between ZAG and the browning of WAT in cold-exposed ZAG knockout (KO) mice and 3T3-L1 adipocytes with overexpressed ZAG. The results showed that cold stress induced marked accumulation of ZAG in wild type (WT) mice. Additionally, ZAG deficiency inhibited the loss of body weight and adipose tissue weight in cold stressed mice. ZAG KO mice were resistant to cold-induced expression of browning markers and energy metabolism in WAT. Furthermore, replenishment ZAG plasmid improved the reduction in cold-induced browning of WAT in ZAG KO mice. In vitro, ZAG overexpression promoted browning and mitochondrial biogenesis and increased the expression of β3-AR and P–P38 in 3T3-L1 adipocytes. These findings demonstrate that ZAG can promote the browning of white adipose tissue and can serve as a potential therapeutic target for treating metabolic diseases such as obesity.

Oxytocin/Osteocalcin/IL-6 and NGF/BDNF mRNA Levels in Response to Cold Stress Challenge in Mice: Possible Oxytonic Brain-Bone-Muscle-Interaction.

Oxytocin (Oxt), osteocalcin (Ost), and NGF/BDNF have a role in bone homeostasis, reproduction, and cognition. Oxt/Ost is required for muscle repair. We investigated gene response of muscle and the inter-organ communication following cold stress (CS). The mRNA quantity of Ngf, Ost, Oxt, Bdnf, p75ntr, Ntrk1, Gprc6a, Oxtr, Ntrk2, UCP1, and Il-6 genes in bone, brain, soleus (SOL), and tibialis anterior (TA) muscles from adult mice following CS were investigated. The myosin heavy-chain Mhc2b, Mhc1, Mhc2x, and Mhc2a gene expression were investigated. Mice were maintained at T = 23°C or 4°C for 6 h and 5-days (5d). CS mice did not show signs of muscle degeneration. An upregulation of Ucp1 and Ngf genes by 2 and 1.5 folds, respectively, in TA after 6 h CS and Ntrk1 by 4 and 22 folds in SOL muscle after 6 h and 5d CS, respectively, was observed; while after 6 h CS p75Ntr was downregulated in either muscle. Bdnf was unaffected, while after 5d CS Ntrk2 was upregulated in TA. Ost was downregulated in SOL by 0.9-folds at 5d. Following 5d CS, Oxtr and Il-6 genes were upregulated, respectively, by 1 and 1.5 folds in SOL. A downregulation of Mhc2b, respectively, by 0.96 and 0.88-folds after 6 h and 5d CS in SOL and Mhc2a was also downregulated by 0.88-fold after 5d CS in TA. Mhc1 and Mhc2x were not affected. Changes in the expression levels of genes in TA and SOL muscles, bone, and brain following CS were regulated by IL6 and Oxt. CS potentiates the slow-twitch phenotype of SOL which is in line with the metabolic need of this muscle, and the potentiation of the slow-twitch phenotype in TA. Oxt and IL6 coordinate a phenotype-dependent tonic effect of slow-twitch muscle and Oxt regulates the inter-organ interaction between brain and SOL muscle. Muscle tropism is maintained by NGF signaling following CS.

Activation of the MAPK signaling pathway induces upregulation of pro-apoptotic proteins in the hippocampi of cold stressed adolescent mice

Stress induces many non-specific responses in the hippocampus, especially during adolescence. Low environmental temperature is known to induce stress, but its influence on the hippocampus, especially in adolescent mice is not clear. We compared apoptotic-related protein levels and MAPK signaling pathway activation in hippocampal neurons of adolescent mice under low temperature conditions (4 °C for 12 h) with western blotting and immunohistochemistry. Western bolt results demonstrated that the levels of phospho-JNK, phospho-p38, and cleaved-caspase 3 significantly increased, while the ratio of Bcl-XL/Bax decreased, in the cold stress group. The results of immunohistochemistry (IHC) and Nissl staining demonstrated that the protein optical density of caspase 3 increased and Nissl bodies decreased in the cold stress group compared with controls. Thus, we conclude that cold exposure initiates activation of the MAPK signaling pathway and subsequently induces the upregulation of pro-apoptotic proteins in the hippocampi of adolescent mice. Overall our study reveals the relationship between cold stress and apoptosis in adolescent mice.

Evaluation of Short and Long Term Cold Stress Challenge of Nerve Grow Factor, Brain-Derived Neurotrophic Factor, Osteocalcin and Oxytocin mRNA Expression in BAT, Brain, Bone and Reproductive Tissue of Male Mice Using Real-Time PCR and Linear Correlation Analysis.

The correlation between the Ngf/p75ntr-Ntrk1 and Bdnf, Osteocalcin-Ost/Gprc6a and Oxytocin-Oxt/Oxtr genes, was challenged investigating their mRNA levels in 3 months-old mice after cold-stress (CS). Uncoupling protein-1 (Ucp-1) was used as positive control. Control mice were maintained at room temperature T = 25°C, CS mice were maintained at T = 4°C for 6 h and 5-days (N = 15 mice). RT-PCR experiments showed that Ucp-1 and Ngf genes were up-regulated after 6 h CS in brown adipose tissues (BAT), respectively, by 2 and 1.5-folds; Ucp-1 was upregulated also after 5-days, while Ngfr (p75ntr) and Ntrk1 genes were downregulated after 6 h and 5-days CS in BAT. NGF and P75NTR were upregulated in bone and testis following 5-days, and P75NTR in testis after 6 h CS. Bdnf was instead up-regulated in bone following 5-days CS and down-regulated in testis. OST was upregulated by 16 and 3-fold in bone and BAT, respectively, following 5-days CS. Gprc6a was upregulated after 6 h in brain, while Bglap (Ost) gene was downregulated. Oxt gene was upregulated by 5-fold following 5-days CS in bone. Oxtr was upregulated by 0.5 and 0.3-fold, respectively, following 6 h and 5-days CS in brain. Oxtr and Oxt were downregulated in testis and in BAT. The changes in the expression levels of control genes vs. genes following 6 h and 5-days CS were correlated in all tissues, but not in BAT. Correlation in BAT was improved eliminating Ngfr (p75ntr) data. The correlation in brain was lost eliminating Oxtr data. In sum, Ucp-1 potentiation in BAT after cold stress is associated with early Ngf-response in the same tissue and trophic action in bone and testis. In contrast, BDNF exerts bone and neuroprotective effects. Similarly to Ucp-1, Bglap (Ost) signaling is enhanced in bone and BAT while it may exert local neuroprotective effects thought its receptor. Ngfr (p75ntr) regulates the adaptation to CS through a feed-back loop in BAT. Oxtr regulates the gene-response to CS through a feed-forward loop in brain. Overall these results expand the understanding of the physiology of these molecules under metabolic thermogenesis.

Fibromyalgia syndrome: metabolic and autophagic processes in intermittent cold stress mice.

Fibromyalgia is characterized by widespread musculoskeletal pain, fatigue, and depression. The aim was to analyze potential mitochondrial dysfunction or autophagy in mice after exposure to intermittent cold stress (ICS). Muscle and liver specimens were obtained from 36 mice. Lactate dehydrogenase (LDH) activity was measured. Microtubule‐associated protein light chain 3 (MAP1LC3B) and glycogen content were determined histologically; muscle ultrastructure by electron microscopy. Mitochondrial‐ and autophagy‐related markers were analyzed by RT‐qPCR and Western blotting. ATP level, cytotoxicity, and caspase 3 activity were measured in murine C2C12 myoblasts after ICS exposure. Coenzyme Q10B (COQ10B) transcript was up‐regulated in limb muscle of ICS mice, whereas its protein content was stable. Cytochrome C oxidase 4 (COX4I1) and LDH activity increased in limb muscle of male ICS mice. Glycogen content was lower in muscle and liver tissue of male ICS mice. Electron micrographs of ICS mice specimens showed mitochondrial damage and autophagic vesicles. A significant up‐regulation of autophagic transcripts of MAP1LC3B and BECLIN 1 (BECN1) was observed. Map1lc3b protein showed an aggregated distribution in ICS mice and SqSTM1/p62 (p62) protein level was stable. Furthermore, ATP level and caspase activity, detected as apoptotic marker, were significantly lowered after ICS exposure in differentiated C2C12 myoblasts. The present study shows that ICS mice are characterized by mitochondrial dysfunction, autophagic processes, and metabolic alterations. Further investigations could dissect autophagy process in the proposed model and link these mechanisms to potential therapeutic options for fibromyalgia.

Thermoneutral Housing Accelerates Metabolic Inflammation to Potentiate Atherosclerosis but Not Insulin Resistance

Chronic, low-grade inflammation triggered by excess intake of dietary lipids has been proposed to contribute to the pathogenesis of metabolic disorders, such as obesity, insulin resistance, type 2 diabetes, and atherosclerosis. Although considerable evidence supports a causal association between inflammation and metabolic diseases, most tests of this link have been performed in cold-stressed mice that are housed below their thermoneutral zone. We report here that thermoneutral housing of mice has a profound effect on the development of metabolic inflammation, insulin resistance, and atherosclerosis. Mice housed at thermoneutrality develop metabolic inflammation in adipose tissue and in the vasculature at an accelerated rate. Unexpectedly, this increased inflammatory response contributes to the progression of atherosclerosis but not insulin resistance. These findings not only suggest that metabolic inflammation can be uncoupled from obesity-associated insulin resistance, but also point to how thermal stress might limit our ability to faithfully model human diseases in mice.

The impact of β-adrenergic signaling on radioresistance and anti-tumor immunity

Our laboratory has demonstrated that mildly cool housing temperatures, (a stress known to be mediated by sympathetic nerve activity and β-adrenergic receptors) can significantly enhance the incidence, growth, and metastasis of tumors in murine models by suppressing anti-tumor immunity [1]. Furthermore, we have shown that mild cool stress also decreases the sensitivity of cancer cells to several types of chemo- and targeted therapeutics [2]. These data strongly suggest that baseline responses of mouse models of tumor growth and tumor immunology may be significantly influenced by housing temperature and the degree of adrenergic stress experienced by tumor bearing mice. We have now become interested in dissecting the mechanisms by which increased adrenergic stress signaling resulting from cool housing conditions impacts anti-tumor immunity and the response of tumors to therapeutics. To investigate the effects of increased adrenergic signaling on anti-tumor immunity, we used β-AR antagonists (β-blockers) to block β-AR signaling resulting from mild cold stress induced by housing temperatures. The addition of a β-blocker to these mice significantly delayed tumor growth, and recapitulated the increased tumor growth control observed in mice housed at thermoneutrality where cold stress is alleviated. However, β-AR blockade did not alter tumor growth in mice housed at thermoneutrality compared to vehicle controls, suggesting that β-AR signaling is responsible for the impairment of anti-tumor immunity in chronically cold stressed mice. To further investigate the effects of β-AR signaling on suppressing anti-tumor immunity, we repeated these experiments in immunodeficient SCID mice and demonstrated that β-blockers had no effect on tumor growth in mice regardless of housing temperature. We also examined the effects of adrenergic stress signaling on radio-resistance. We addressed this question by stimulating β-ARs on Pan02 (murine) and Mia-PACA2 (human) pancreatic tumor cells and performed clonogenic assays. Those cells treated with a β-agonist exhibited improved survival and proliferation at varying doses of radiation when compared to untreated controls similar to our earlier studies using chemotherapy. In summary, β-adrenergic receptor activity appears to play a fundamental role in the regulation of both anti-tumor immunity as well as intrinsic therapeutic sensitivity. Therefore, strategic combinations of β-receptor antagonists with chemo-radiation therapy, or immunotherapy may provide significantly improved tumor control. This work was supported by the Peter T. Rowley Breast Cancer Research Grant and the Harry J. Lloyd Charitable Trust.

Abstract B43: The degree of adrenergic stress signaling regulates the severity of graft versus host disease following allogeneic hematopoietic cell transplantation

Abstract Adrenergic stress is recognized to influence multiple arms of the immune system. Recent work in our lab supports the observation that conditions which cause physiologic stress (i.e., increase adrenergic stress) reduce T cell function. T cells are crucial mediators of graft versus host disease (GVHD), a common and often lethal side effect of allogeneic hematopoietic cell transplantation (allo-HCT). As many patients succumb to GVHD, being able to understand underlying factors that regulate its severity is paramount. Our recent work led us to hypothesize that stress induced β-adrenergic receptor (AR) agonists such as norepinephrine may dampen GVHD following allo-HCT. To investigate the role of β-AR signaling on GVHD we performed MHC-mismatched allo-HCT in which recipient mice were treated with PBS (β-AR signaling intact) or a β-AR antagonist, propranolol, (i.e., a β-blocker) to inhibit β-AR signaling. Following allo-HCT, GVHD was significantly increased in mice treated with propranolol as evidenced by more weight loss (P&amp;lt;0.01) and decreased survival (P&amp;lt;0.01) compared to mice treated with PBS. To confirm that the weight loss and decreased survival were indicative of T cell-mediated GVHD, we performed allo-HCT with T cell depleted bone marrow. No GVHD was observed in PBS- or propranolol-treated groups when the graft did not contain allogeneic T cells. Furthermore, syngeneic transplants also yielded no GVHD in PBS- or propranolol-treated groups. We next evaluated pathology in the liver, small and large intestine. We found that liver pathology was significantly increased (P&amp;lt;0.05) in mice treated with propranolol compared to the PBS treated group. Thus, for the first time we have found that the level of systemic β-AR-mediated stress signaling regulates the severity of GVHD. We next asked whether we could regulate GVHD by manipulating endogenous levels of the β-AR agonist, norepinephrine, by exposing mice to physiological stress. To test this we used mildly cool housing temperature to activate the adrenergic stress response as temperature manipulation is a classic means to study norepinephrine-driven stress signaling. We housed mice at 22°C, a temperature in which mice are cold stressed, and 30°C, a temperature in which the adrenergic stress response is significantly reduced. Therefore in this model, we predicted that GVHD would be reduced in severity in mice housed at 22°C compared to those housed at 30°C. We first confirmed that mice housed at 22°C were more stressed by measuring norepinephrine levels, finding that norepinephrine was significantly elevated both pre- and 13 days post-allo-HCT (P&amp;lt;0.05). As predicted, cold stressed mice housed at 22°C had significantly less GVHD as evidenced by reduced weight loss (P&amp;lt;0.01), improved survival (P&amp;lt;0.01), and less liver pathology (P&amp;lt;0.05) when compared to mice housed at 30°C. This strongly supports our results obtained using pharmacological inhibition of β-AR signaling. At 30°C, there is minimal norepinephrine-driven stress, and therefore is not surprising that propranolol treatment of these mice had no effect on the severity of GVHD. Thus, these findings suggest that allogeneic graft vs. host responses were responsible for weight loss and decreased survival in conditions in which adrenergic stress was alleviated (housing at 30°C) or the stress response was blocked (treatment with propranolol). Therefore β-AR signaling plays a vital and previously unrecognized role in modulating the severity of GVHD. Clinically, using β-blockers to achieve pharmacologic inhibition of β-AR signaling is commonly used to alleviate symptoms of hypertension or other age-related diseases in elderly patients. Of note, β-blockers are also routinely given post allo-HCT to ameliorate symptoms such as hypertension and tachycardia. Thus, following allo-HCT, it may be important to reconsider the use of β-blockers as our findings suggest that the degree of β-AR signaling regulates the severity of GVHD. Citation Format: Nicholas D. Leigh, Kathleen M. Kokolus, Jason W-L Eng, Jingxin Qiu, George L. Chen, Philip L. McCarthy, Xuefang Cao, Elizabeth A. Repasky. The degree of adrenergic stress signaling regulates the severity of graft versus host disease following allogeneic hematopoietic cell transplantation. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B43.

Abstract IA21: Impact of thermoregulatory metabolism on immunosuppression and therapeutic responsiveness of tumors

Abstract An important, but underappreciated, aspect of using mouse models in cancer research concerns the impact of basic housing conditions on mouse physiology and how this influences what we have come to consider “baseline” responses. In fact, we recently published data (Kokolus et al., PNAS, 2013; Kokolus et al., Front. Imm. 2014) demonstrating the surprising influence of mild cold stress (resulting from standard, IACUC-enforced housing temperatures) on tumor growth rates, CD8+T cell and dendritic cell function, and the degree of immunosuppressive cell activity which together show that the anti-tumor immune response in control mice is significantly suppressed. Newer data (N. Leigh/K. Kokolus et al., see separate abstract at this meeting) has revealed that mild cold stress also significantly influences the severity of graft vs. host disease (GVHD) in major mouse models of bone marrow transplantation, a condition which also depends upon the balance between effector T cell function and immunosuppressive activity which apparently can be easily shifted by thermoregulatory stress. These and other data, including our observations that intrinsic therapeutic sensitivity of tumor cells is significantly influenced by housing temperature (J. Eng et al., submitted; see separate abstract at this meeting), are disconcerting in terms of revealing the degree to which the outcomes of mouse experimentation can be influenced by these variables which are rarely recognized. However, it has also been fortuitous as we have begun to appreciate intriguing relationships between thermoregulatory metabolism and immune cell activity which, if understood more completely, could be exploited to improve immunotherapy for cancer or other diseases. Mild cold stress stimulates the production of norepinephrine by the sympathetic nervous system which in turn stimulates metabolic heat production via thermogenesis in brown fat and other tissues. This talk will focus on our recognition that increased β-adrenergic signaling by norepinephrine in mildly cold-stressed mice is a key event, driving not only changes in anti-tumor immunity, but also but therapeutic responsiveness in mouse tumor models, as well as the observed differences in GVHD. Excitingly, treatment of mice with common β−blockers largely reverses the phenotypes associated with mild cold stress in each of these models. Overall, these data support the idea that systemic stress is a significant variable that should be considered when evaluating therapies in mouse tumor models or for evaluating the impact of metabolism on the immune response. Alleviating this stress may identify novel targets for improving cancer therapies, and in particular, immunotherapies, in patients. Citation Format: Elizabeth A. Repasky. Impact of thermoregulatory metabolism on immunosuppression and therapeutic responsiveness of tumors. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr IA21.

Standard sub-thermoneutral caging temperature influences radiosensitivity of hematopoietic stem and progenitor cells.

The production of new blood cells relies on a hierarchical network of hematopoietic stem and progenitor cells (HSPCs). To maintain lifelong hematopoiesis, HSPCs must be protected from ionizing radiation or other cytotoxic agents. For many years, murine models have been a valuable source of information regarding factors that either enhance or reduce the survival of HSPCs after exposure of marrow to ionizing radiation. In a recent series of studies, however, it has become clear that housing-related factors such as the cool room temperature required for laboratory mice can exert a surprising influence on the outcome of experiments. Here we report that the mild, but chronic cold-stress endured by mice housed under these conditions exerts a protective effect on HSPCs after both non-lethal and lethal doses of total body irradiation (TBI). Alleviation of this cold-stress by housing mice at a thermoneutral temperature (30°C) resulted in significantly greater baseline radiosensitivity to a lethal dose of TBI with more HSPCs from mice housed at thermoneutral temperature undergoing apoptosis following non-lethal TBI. Cold-stressed mice have elevated levels of norepinephrine, a key molecule of the sympathetic nervous system that binds to β-adrenergic receptors. We show that blocking this signaling pathway in vivo through use of the β-blocker propanolol completely mitigates the protective effect of cold-stress on HSPC apoptosis. Collectively this study demonstrates that chronic stress endured by the standard housing conditions of laboratory mice increases the resistance of HSPCs to TBI-induced apoptosis through a mechanism that depends upon β-adrenergic signaling. Since β-blockers are commonly prescribed to a wide variety of patients, this information could be important when predicting the clinical impact of HSPC sensitivity to TBI.

Mild cold stress resulting from standard housing conditions for laboratory mice masks the severity of graft vs. host disease in mouse models of bone marrow transplantation (TRAN3P.887)

Abstract Clinical bone marrow transplants (BMT) yield lethal graft vs. host disease (GVHD) after transplant of un-manipulated bone marrow (BM). This differs from murine GVHD models that typically require addition of spleen or lymph node derived T cells. Recent work has shown that mice housed at mandated sub-thermoneutral ambient temperatures (22°C) are cold stressed, resulting in heat generation activated by the sympathetic nervous system. Since cold stressed mice exhibit weakened immune responses, we hypothesize that baseline GVHD is reduced compared to that seen in mice at thermoneutral temperatures (30°C). We compared GVHD in mice receiving MHC-mismatched un-manipulated BM while housed at 22 vs. 30°C at which cold stress is alleviated. In this BMT model, mice housed at 22°C show no signs of GVHD, however, we found weight loss and lethal GVHD in mice housed at 30°C. The same trend was observed with MHC-matched allogeneic BMT. As housing at 22°C activates the sympathetic nervous system, we blocked its activation by administering propranolol, a commonly prescribed β-blocker. Following BMT, mice at 22°C treated with β-blocker displayed lethal GVHD similar to untreated mice at 30°C. This suggests that T cell responses are dampened by cold stress through a β-adrenergic pathway which is circumvented by thermoneutral housing. This study provides insight into translational discrepancies of clinical and murine BMT and demonstrates lethal GVHD in a mouse model generated by un-manipulated BM.

Myelophil attenuates brain oxidative damage by modulating the hypothalamus-pituitary-adrenal (HPA) axis in a chronic cold-stress mouse model

Ethnopharmacological relevanceMyelophil is composed of Astragali Radix and Salviae Miltiorrhizae Radix, according to the long traditional pharmacological practices, and it has been used for patients with chronic fatigue-associated symptoms including concentration problem or memory loss. Aim of the studyThis study aimed to evaluate the clinical relevance of Myelophil on brain oxidative damage using a chronic cold stress mice model. Material and methodsBalb/c mice were subjected to cold stress (4°C for 4h) six times per week for 2 weeks with or without oral administration of Myelophil (50, 100, or 200mg/kg), or ascorbic acid (50mg/kg). ResultsChronic cold stress induced histopathological hippocampal apoptosis with drastically increased serum levels of total reactive oxygen species and nitric oxide, as well as brain lipid peroxidation levels, protein carbonyl, and caspase-3/7 activity. These alterations were significantly ameliorated by Myelophil treatment. Myelophil administration significantly recovered the depleted glutathione and its enzymes, superoxide dismutase activity, and catalase protein and gene expression levels. Serum levels of corticosterone, dopamine, and adrenaline were notably altered by chronic cold stress but were significantly ameliorated by Myelophil treatment. Myelophil also normalized alterations in tumor necrosis factor-α, interleukin (IL)-1β, and IL-10 gene expression and protein levels. Chronic cold stress up-regulated gene expression levels of phenylethanolamine N-methyltransferase and monoamine oxidase-B, and glucocorticoid receptors in the hypothalamus and hippocampus, respectively, whereas Myelophil treatment completely normalized these levels. ConclusionsThese results suggest that Myelophil has potent pharmaceutical effects against chronic cold-stress-induced brain damage by relieving oxidative stress and inflammation and regulating stress hormones in mice.