Progress 10/01/07 to 09/30/12

Outputs
OUTPUTS: The proposed research relates to goal #3 of the USDA national goal areas, a healthy, well-nourished population. Circadian rhythms describe biological phenomena that oscillate within a 24-h cycle. By anticipating both environmental and internal changes, organisms can efficiently program their physiological tasks. Thus, innate immune mechanisms-such as NK cell function-could have developed a circadian regulation in order to optimize their activity when most needed. NK cells are critical for immune surveillance against fungal, bacterial and viral infections. They also play a vital role in cellular resistance to malignancy and tumor metastasis. Circadian systems regulate the immune system by various molecular and physiological pathways. Disruption to the circadian temporality of these pathways is associated with disease formation and progression. Circadian clock genes have been shown to regulate pathways involved in cellular proliferation, apoptosis, and DNA damage response, as aberrant rhythms in these genes are associated with various diseases. However, there is growing evidence that specific circadian genes differentially regulate functional pathways of immunocompetent cells. To extend our previous findings of the role of Period 2 in regulating splenocyte rhythms, we report mice carrying a mutation in the Period 1 gene (Per1-/- mice), involved in the negative limb of the molecular clock, display significantly altered rhythms of cytokine, IFNγ, and cytolytic factors, perforin and granzyme B, in splenic natural-killer (NK) cells. Altered rhythms of NK cell immune factors were accompanied by changes in circadian expression of circadian clock genes, Bmal1 and Per2. In addition, Per1-/- circadian running-wheel activity rhythms remained rhythmic during constant darkness, albeit with a shortened free-running circadian period, suggesting primary involvement of peripheral molecular clocks. These findings indicate the Per1 gene through NK cellular clocks modulates immune pathways. PARTICIPANTS: Ryan W. Logan - a postdoctoral fellow Olivia Wynne - a postdoctoral fellow Dale Levitt - a undergraduate student did George H. Cook project research Debbi Price - a undergraduate student TARGET AUDIENCES: Health professionals and scientists. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Much research attention has been placed on exploring the consequences of aberrant clock gene expression in tumor cells, which has revealed their roles in pathways controlling cell cycle checkpoints, cell proliferation and apoptosis, as well as DNA repair. Altered Per1 expression and mutations in the gene have been found in human gliomas and other types of tumors. However, the role of circadian systems and clock genes in immune functioning is important to understand disease formation and progression. Our study reports novel evidence of Per1 to modulate circadian expression of cytokines and cytolytic factors in splenic NK cells. Attenuated expression of perforin and granzyme B accompanied by changes in IFNγ rhythms could detrimentally affect the ability of NK cells to kill tumor cells. The mechanistic link between Per1, or other clock genes, and cytokine and cytolytic factor transcription is currently unknown, but may involve known transcription factors involved in immune response, such as NF-κB. In-depth molecular studies are required to tease apart differential roles of clock genes in immunocompetent cells, and how disruptions to cellular clocks within the diseased cell and immune cell may create particular vulnerabilities to disease.

Publications

• Ryan W. Logan, Olivia Wynne, Dale Levitt, Debbi Price, and Dipak K. Sarkar. 2012 Altered circadian expression of cytokines and cytolytic factors in splenic natural killer cells of Per1-/- mutant mice. Journal of Interferon and Cytokine Research (in Press).

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: The proposed research relates to goal #3 of the USDA national goal areas, a healthy, well-nourished population. Circadian rhythms describe biological phenomena that oscillate within a 24-h cycle. By anticipating both environmental and internal changes, organisms can efficiently program their physiological tasks. Thus, innate immune mechanisms-such as NK cell function-could have developed a circadian regulation in order to optimize their activity when most needed. NK cells are critical for immune surveillance against fungal, bacterial and viral infections. They also play a vital role in cellular resistance to malignancy and tumor metastasis. Perturbations of daily rhythms caused by external stressors (e.g., changes of weather, and the light dark cycle) and internal stressors (psychological distress) may compromise the first line of defense against infections and cancer by disrupting the circadian rhythm of NK cell function. We have recently initiated a study to determine if the consequences of chronic circadian disruption may also extend to the innate immune system to promote cancer growth, as natural-killer (NK) cell function is modulated by circadian mechanisms and plays a key role in lysis of tumor cells. PARTICIPANTS: Ryan W. Logan, (postdoctoral student) Changqing Zhang (predoctoral student) Sengottuvelan Murugan (postdoctoral student) Stephanie O'Connell (Undergraduate student) Dale Levitt (undergraduate student) Alan M. Rosenwasser (collaborator from University of Maine, Orono, Maine. TARGET AUDIENCES: Our findings indicate circadian disruption may promote tumor growth by altering the circadian rhythms of NK cell function. This information is applicable for both human as well as domestic animals. PROJECT MODIFICATIONS: Nonce

Impacts
In order to determine if NK cell function is disrupted by a model of human shift-work and jet-lag, Fischer (344) rats were exposed to either a standard 12:12 light-dark cycle, or a chronic shift-lag paradigm, consisting of repeated 6 hour photic advances occurring every 2 days for a total of 20 days, and placed into 5-7 days of constant darkness. This model resulted in considerable circadian disruption as assessed by circadian running wheel activity. NK cells were enriched from control and shifted animals, and gene, protein, and cytolytic activity assays were performed. Chronic shift-lag altered the circadian expression of clock genes, Per2 and Bmal1, and cytolytic factors, perforin and granzyme B, as well as the cytokine, IFNγ. These alterations were correlated with suppressed circadian expression of NK cytolytic activity. Further, chronic shift-lag attenuated NK cell cytolytic activity under stimulated conditions in vivo using intravenous injection of mammary cancer cells, and promoted subsequent lung tumor growth. Collectively, these findings indicate circadian disruption may promote tumor growth by altering the circadian rhythms of NK cell function.

Publications

• Ryan W. Logan, Changqing Zhang, Sengottuvelan Murugan, Stephanie OConnell, Dale Levitt, Alan M. Rosenwasser and Dipak K. Sarkar. 2012 Chronic shift-lag alters the circadian clock of natural killer cells and promotes lung cancer growth in rats. Journal of Imunology (in Press)

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Research pertaining to this grant has evolved toward determining the role of the circadian clock in controlling circadian NK cell function. Also, to evaluate the physiological relevance of circadian NK cell function in tumor clearance and the susceptibility of NK cell function to jet-lag, an animal model for circadian disruption. It tests the hypothesis that NK cell cytolytic activity maintains a circadian rhythm pattern in the spleen. It proposes to identify the contribution of central and peripheral clocks in NK cell circadian function. It determines the susceptibility of NK cell circadian function to simulated jet-lag. In order to assess the role of daily sympathetic input in circadian NK cell function we completed a study to determine whether splenic sympathectomy can be achieved by the local injection of guanethidine. Guanethidine effects are specific for sympathetic neurons and include blockade of norepinephrine release, depletion of neuronal norepinephrine stores, and blockade of norepinephrine reuptake into the neurons, which collectively creates a functional sympathectomy. Ten days after surgery, we determined the norepinephrine content in saline and guanethidine-treated spleens at two representative timepoints: ZT7 and ZT19. These two timepoints correspond with the peak and nadir (or vice versa) of most the rhythms of NK cell cytotoxic factors (i.e., granzyme B, perforin, IFN-γ, TNF-α, Per2, Bmal1, and splenic norepinephrine content). Thus, the variations observed between these two timepoints should be representative of the daily changes in the mentioned variables. Guanethidine treatment abolished the physiological rhythm in splenic NE content. As a consequence, NK cells obtained from sympathectomized spleens lost the daily rhythms of granzyme B and TNF-α levels, as well as the rhythms of the clock proteins PER2 and BMAL1, while maintaining the normal daily oscillations of perforin and IFN-γ. These results demonstrate that sympathetic input to the spleen, as judged by the daily rhythm in splenic NE content, entrains the NK cell molecular clock as well as the daily expression rhythms of granzyme B and TNF-α in NK cells. In addition, the data obtained in this study suggest that non-neural circadian cues may be controlling the daily rhythms of perforin and IFN-γ in splenic NK cells. PARTICIPANTS: Graduate Student Maria Agapito and five undergraduates Dale Levitt, Jacklin Barreira, Garima Arora, Greeshma Hedge and Stephanie J. O'Connell. TARGET AUDIENCES: Researchers of human health issues relevant to Cancers, AIDS and other immune diseases. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Our previous studies identified that natural killer cells of the immune system maintain circadian rhythm in their cytolytic function. However, how the central and peripheral clock systems entrain the rhythmic cytolytic function of NK cells via altering the expression cytolytic factors granzyme-B and perforin and cytokines TNF-α and IFN-γ are not known. We now provide evidence that sympathetic input from the brain entrains the NK cell molecular clock as well as the daily expression rhythms of granzyme B and TNF-α in NK cells. In addition, the data obtained in this study suggest that non-neural circadian cues may be controlling the daily rhythms of perforin and IFN-γ in splenic NK cells. These findings began to unfold the mechanism by which central and peripheral clocks interact to entrain the cellular molecules governing the circadian cytolytic activity of NK cells.

Publications

• Logan RW, Arjona A, Sarkar DK. Role of sympathetic nervous system in the entrainment of circadian natural-killer cell function. Brain Behav Immun. 2011 Jan;25(1):101-9.

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: The proposed research relates to goal #3 of the USDA national goal areas, a healthy, well-nourished population. Circadian rhythms describe biological phenomena that oscillate within a 24-h cycle. By anticipating both environmental and internal changes, organisms can efficiently program their physiological tasks. Thus, innate immune mechanisms-such as NK cell function-could have developed a circadian regulation in order to optimize their activity when most needed. NK cells are critical for immune surveillance against fungal, bacterial and viral infections. They also play a vital role in cellular resistance to malignancy and tumor metastasis. Perturbations of daily rhythms caused by external stressors (e.g., changes of weather, and the light dark cycle) and internal stressors (psychological distress) may compromise the first line of defense against infections and cancer by disrupting the circadian rhythm of NK cell function. In accordance with the currently proposed hierarchical model for the modulation of mammalian rhythms, we anticipate that the hypothalamic suprachiasmatic nucleus (SCN) will coordinate splenic NK cell circadian function via humoral and/or neural cues. We have recently initiated study to determine how SCN regulate circadian rhythm of NK cell regulatory beta-endorphin neuronal function using an animal model where sleep-wake disturbances and circadian immune function is disrupted by fetal alcohol exposure. We have previously shown that fetal alcohol exposure altered the circadian expression of the beta-endorphin precursor proopiomelanocortin (POMC) gene in the arcuate nucleus (ARN) and the clock-governing rPer1 and rPer2 genes in the SCN and ARN. Per2 mutant (mPer2Brdml) and wild type (C57BL/6J) mice were used to determine the effect of Per2 mutation on ethanol-regulated β-endorphin neuronal activity during neonatal period using an in vitro mediobasal hypothalamic (MBH) cell culture model and an in vivo milk formula feeding animal model. The β-endorphin neuronal activity following acute and chronic ethanol treatments, was evaluated by measuring the peptide released from cultured cells or peptide levels in the MBH tissues, using enzyme-linked immunosorbent assay (ELISA). Per2 mutant mice showed a higher basal level of β-endorphin release from cultured MBH cells and a moderate increase in the peptide content in the MBH in comparison to control mice. However, unlike wild type mice, Per2 mutant mice showed no stimulatory or inhibitory β-endorphin secretory responses to acute and chronic ethanol challenges in vitro. Furthermore, Per2 mutant mice, but not wild type mice, failed to show the stimulatory and inhibitory responses of MBH β-endorphin levels to acute and chronic ethanol challenges in vivo.These results suggest for the first time that the Per2 gene may be critically involved in regulating β-endorphin neuronal function. Furthermore, the data revealed an involvement of the Per2 gene in regulating β-endorphin neuronal responses to ethanol. PARTICIPANTS: Graduate Student Maria Agapito and an undergraduate Nadia Milan. TARGET AUDIENCES: Researchers of human health issues relevant to Cancers, AIDS and other immune diseases. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
As described previously, identifying the mechanisms that orchestrate the circadian rhythm of NK cell function could lead to better designed chronomodulated treatments for cancer, viral infections, and other immune-related diseases. The study conducted during the last funding period identified some basic biological mechanisms relevant to a neuronal signal (beta-endorphin activation) that regulate the circadian rhythm of NK cell function. These data revealed new information about how the clock gene alter the cellular function of NK cell regulatory β-endorphin neurons.

Publications

• Maria Agapito, Nadia Mian, Dipak K. Sarkar. 2010 Period 2 gene deletion abolishes beta-endorphin neuronal response to ethanol. Alcoholism: Clinical and Experimental Research (submitted).

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: The proposed research relates to goal #3 of the USDA national goal areas, a healthy, well-nourished population. Circadian rhythms describe biological phenomena that oscillate within a 24-h cycle. By anticipating both environmental and internal changes, organisms can efficiently program their physiological tasks. Thus, innate immune mechanisms-such as natural killer (NK) cell function-could have developed a circadian regulation in order to optimize their activity when most needed. NK cells are critical for immune surveillance against fungal, bacterial and viral infections. They also play a vital role in cellular resistance to malignancy and tumor metastasis. Perturbations of daily rhythms caused by external stressors (e.g., changes of weather, and the light dark cycle) and internal stressors (psychological distress) may compromise the first line of defense against infections and cancer by disrupting the circadian rhythm of NK cell function. In accordance with the currently proposed hierarchical model for the modulation of mammalian rhythms, we anticipate that the hypothalamic suprachiasmatic nucleus (SCN) will coordinate splenic NK cell circadian function via humoral and/or neural cues. We have recently initiated study to determine how SCN regulate circadian rhythm of NK cell regulatory beta-endorphin neuronal function using an animal model where sleep-wake disturbances and circadian immune function is disrupted by fetal alcohol exposure. We have previously shown that fetal alcohol exposure altered the circadian expression of the beta-endorphin precursor proopiomelanocortin (POMC) gene in the arcuate nucleus (ARN) and the clock-governing rPer1 and rPer2 genes in the SCN and ARN. To determine whether cAMP and melatonin signal pathways are involved in ethanol's modification of circadian POMC functions in FAE offspring, we measured the circadian changes in mRNA levels of cAMP-response element binding protein (CREB) and melatonin receptor 1 (MTR1) in SCN tissues and laser-captured POMC neurons in the hypothalamus. Pregnant dams between Days 7 and 18 of gestation were fed a liquid diet containing ethanol (FAE), pair-fed a control liquid diet (pair-fed) or fed rat chow ad libitum (ad-lib). Male offspring were sacrificed at 90 d of age at 4-h intervals throughout the day. In both the SCN and POMC neurons of ad-lib and pair-fed rats, the mRNA levels of CREB displayed robust circadian variations with a peak during the night, but in FAE rats the levels of CREB mRNA were expressed in a fragmented fashion and without a distinctive peak. In ad-lib and pair-fed offspring, MTR1 mRNA levels showed elevated expression in the SCN during the light-dark transition, but in the POMC neuron they were elevated during the daytime. The circadian expression of MTR1 in FAE offspring was significantly reduced, however. These data suggest that the fetal alcohol exposure-induced changes in circadian POMC neuronal functions might involve melatonin receptors and cAMP signaling. These data will be presented in the Annual Meeting of the Research Society on Alcoholism at San Diego in June 2009. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
In the last few years, the medical practice of timed administration of drugs based on biological rhythms has been extensively developed, especially in immune-related diseases and in cancer therapy. Hence, identifying the mechanisms that orchestrate the circadian rhythm of NK cell function could lead to better designed chronomodulated treatments for cancer, viral infections, and other immune-related diseases. The study conducted during the last funding period identified some basic biological mechanisms relevant to a neuronal signal (beta-endorphin activation) that regulate the circadian rhythm of NK cell function.

Publications

• 1. Arjona A, Sarkar DK. 2008 Are circadian rhythms the code of hypothalamic-immune communication Insight from natural killer cells. Neurochem Research. 33:708-718.
• 2. Sarkar DK, Agapito M, Chen CP (2009) Fetal alcohol exposure alters he circadian rhythms of CREB and MTR1 mRNA in the suprachiasmatic nucleus and POMC-containing cells in the hypothalamus. Alcoholism: Clinical and Experimental Research (supplement; submitted).