Monika R. Fleshner - US grants

DESCRIPTION (Adapted from the applicant's abstract): Exposure to acute or chronic stress (mental or physical) is associated with an increase in disease susceptibility and severity. Evidence suggests that stress alters disease processes by modulating the immune system. Given the health problems associated with repeated acute and chronic stressor exposure, it is important to develop treatments which could prevent the immunologically deleterious consequences of stressor exposure. A simple pharmacological approach is currently unrealistic because the complex neurohormonal mediators of stress-induced changes in immune function are not understood. Another approach would be to develop a behavioral intervention that would buffer the deleterious consequences of stress, and thereby contribute to an overall improvement in health. Preliminary data from the applicant's laboratory suggest that regular, moderate, physical activity is a successful intervention capable of having such an effect. Regular, moderate exercise is associated with improved overall health. The health benefits of physical exercise are both direct and indirect. Regular, moderate exercise, for example, is associated with a decrease in coronary heart disease and high blood pressure, probably due to direct improvements in cardiovascular function. In addition to a reduction in heart disease, regular, moderate exercise is also associated with a decrease in bacterial and viral illness, which could be due to the indirect health benefits of exercise, i.e., stress reduction. Thus, it is possible that the reported reduction in infectious disease associated with exercise is due to the prevention of stress-induced suppression of the immune system. Using a well established animal model of stress, the applicant investigated the effect of acute stressor exposure on the development of a specific antibody response to a benign protein, keyhole limpet hemocyanin (KLH). Measurement of specific antibody levels in the blood after challenge with KLH provides and excellent measure of the in vivo immune response and the cellular mechanisms are well understood. Thus, using this well characterized stress and immunity paradigm, the applicant presents evidence that regular, moderate, exercise can prevent the effect of stress on anti-KLH antibody. The goal of this research proposal, therefore, is to better characterize the protective effect of regular, moderate physical activity on the detrimental immunological consequences of acute stressor exposure.

There is evidence that exposure to acute stress (mental or physical) modulates immune responses. Although previous research has focused on the immunosuppressive effects of stress, more recently it has become clear that acute stressor exposure can potentiate, as well as suppress, the immune system. Specifically there is evidence that acute stressor exposure stimulates many aspects of innate immunity; such as, resolution of bacterial inflammation, fever, macrophage/neutrophil nitric oxide, proinflammatory cytokines, and acute phase proteins. The stress-induced potentiation of innate immunity is an adaptive component of the "stress response". Interestingly, physical activity or exercise has also been reported to potentiate the same aspects of innate immunity that are stimulated by stress. In addition, physical activity can prevent the immunosuppressive effects of stress. Recently, we have reported that exposure to an established animal model of acute stress facilitates the resolution of an in vivo bacterial challenge. Rats exposed to inescapable tailshock stress, and challenged subcutaneously with E. coli, resolve the inflammatory response to the bacterial challenge nearly 2 days faster than non-stressed controls. Thus, given that physical activity can both stimulate aspects of innate function, and modulate the immunological consequences of acute stressor exposure, we were interested in investigating what effect physical activity would have on the stress-induced increase in the resolution of bacterial inflammation. Preliminary data suggest that physical activity, prior to exposure to an acute stressor, potentiates the stimulatory effect of stress on bacterial inflammation resolution. That is, rats that exercised prior to exposure to acute stress completely resolved the bacterial inflammation 3-4 days faster than rats exposed to either stress or exercise alone. Thus, the goal of this proposal is to characterize whether the decreased time required to resolve bacterial inflammation is due to an increase in bacterial clearance or a decrease in other inflammatory factors.

DESCRIPTION (Applicant's abstract): The literature indicate that physically active organisms are less susceptible to the deleterious consequences of stress on illness and immune function. Using an established animal model of stress, my laboratory has investigated the effect of acute stressor exposure on the development of a specific antibody response to a benign protein, keyhole limpet hemocyanin (KLH) in physically active versus sedentary rats. Measurement of specific antibody levels in the blood after challenge with KLH provides an excellent measure of the in vivo immune response, and a reduction in the specific antibody response to a bacteria, virus or soluble toxin, could render the organism more susceptible to disease caused by that pathogen. Sedentary rats that are immunized with KLH, and exposed to a single session of tail shock stress, have a reduction in the antibody response to KLH (anti-KLH Ig). In contrast, rats that are allowed to live with a running wheel before exposure to an acute stressor, do not suffer the immunologically deleterious consequences of stress. The central theme of this proposal is to determine the immune-neuroendocrine-brain mechanism(s) of the stress-buffering effect of physical activity. Preliminary data suggest that physical activity modulates the brain and neuroendocrine responses to stressor exposure. Compared to sedentary stressed rats, physically active rats exposed to tail shock stress have a smaller increase in c-Fos immunoreactivity (neuronal activation marker) in several stress reactive brain areas that are important for activation of sympathetic descending pathways. These changes may be responsible for the reduction in stress-induced sympathetic nervous system output (plasma and splenic norepinephrine (NE)) found in physically active versus sedentary animals. Importantly, there is evidence that exposure to stress levels of NE elevates nitric oxide (NO) which leads to a reduction in anti-KLH Ig. Blockade of stress-induced sympathetic output prevents these effects. It is reasonable to hypothesize, therefore, that physically active rats are resistant to the negative effects of stress on immune function because they have a reduction in sympathetic nervous system output leading to a reduction in immunosuppressive NO, and prevention of suppressed anti-KLH Ig.

DESCRIPTION (provided by applicant): Exposure to an acute (<4 hrs) mental or physical stressor stimulates a cascade of behavioral & physiological responses that function to facilitate fight/flight responses and improve an organism's chances of survival. At the cellular level, one important and highly conserved response to stress is stimulation of heat shock proteins, specifically heat-shock protein 72 (Hsp 72). Although the mechanisms of induction and functions of intracellular Hsp72 have been thoroughly studied, only recently has it been discovered that extracellular Hsp72 (crisp72) can be rapidly released into the blood in high concentrations after exposure to either mental or physical stressors. The release of eHsp72 appears to be a highly conserved response across species and stressors, leading us to propose that crisp72 release may be a previously unrecognized feature of the acute stress response. The gender, species & stressor generalizability, cellular source(s), signal(s) and function(s) of extracellular Hsp72 released after acute stressor exposure remain largely unexplored. We present preliminary data that concentrations of crisp72 increase in the blood of males & females, and rodents & humans after exposure to a variety of stressors, i.e., conditioned contextual fear, predatory stress, tailshock stress, restraint stress, exhaustive exercise stress, and handshock stress (humans). In addition, we have evidence that eHsp72 is rapidly (<15min) released via an a1-adrenergic receptor mediated mechanism from brown adipose tissue (BAT) because a1-adrenergic blockade (prazosin) prevents stress-induced increases of eHsp72 in the blood; and an adrenergic agonist (NE) releases crisp72 from BAT. Furthermore, we report that crisp72 in vitro stimulates NO and inflammatory cytokines from innate immune cells, and this effect is potentiatied in the presence of LPS. Finally, crisp72 at the site of bacterial challenge facilitates inflammation recovery, and both blockade of stress-induced crisp72 release (prazosin) and immunoneutralization of crisp72 at the site of inflammation, prevents the positive effects of stress. We propose, therefore, that crisp72 released after exposure to acute stress may function as an endogenous "danger signal" for the immune system. Hence, in presence of bacterial challenge, eHsp72 potentiates macrophage/neutrophil release of nitric oxide (NO) & inflammatory cytokines resulting in facilitated killing & recovery from in vivo bacterial challenge. We hypothesize, therefore, that exposure to an acute stressor stimulates the release of crisp72 into the blood via an alpha1-adrenergic receptor-mediated mechanism from brown adipose tissue and elevated crisp72 functions to facilitate innate immunity in the presence of bacterial challenge (Escherichia coli).

DESCRIPTION (provided by applicant): Exposure to stressful events can precipitate and/or exacerbate various types of affective disorders such as depression and anxiety. Physical activity increases stress resistance and reduces depression and anxiety. For example, voluntary freewheel running increases stress resistance and conveys antidepressant/anxiolytic effects in various animal models of affective dysregulation, including "learned helplessness" (LH). LH is an acute behavioral state produced after exposure to uncontrollable, but not controllable, stress. Evidence suggests that LH is due to activation of a neural circuit that produces intense stimulation of serotonin (5HT) neurons of the dorsal raphe nucleus (DRN). This intense 5HT drive results in sensitization of the DRN 5HT system. The neurobiological mechanisms for the protective effect of physical activity on LH remain unknown. Results from our preliminary studies suggest that physical activity (voluntary freewheel running) prevents LH by changing the DRN 5HT response to uncontrollable stress. In the DRN, rats that voluntary freewheel run compared to sedentary (standard housing) controls have an increase in 5HT/1A somatodendritic autoreceptor expression (mRNA & protein), a decrease in 5HT/1B axonal autoreceptor mRNA, and a decrease in serortonin transporter expression (SERT, mRNA & protein). Freewheel running also conveys resistance to stress-induced decreases in DRN 5HT/1A protein expression and results in an attenuation of DRN 5HT neural responses (cFos expression) to uncontrollable stress. The reduction in stress-induced cFos expression is mirrored in a subset of areas that interact with the DRN and are involved with LH, namely the bed nucleus of the stria terminalis (BNST), the locus coeruleus (LC)-A5, and the amygdala (AMG). The changes in neural responses to stress are more robust after 6wks than after 3wks of freewheel running, and 6wks of freewheel running prevents LH behaviors (escape deficit & exaggerated freezing) after uncontrollable stress. The current proposal will test the hypothesis that the protective effect of physical activity on the development of learned helplessness is due to modulation of the DRN 5HT response. Both intra-DRN alterations and modulation of stress-induced interactions of the DRN with the AMG, BNST and LC-A5 could contribute to this effect. Together, these changes may prevent the intense DRN 5HT activation during uncontrollable stress, and/or the subsequent development & expression of LH.

The physiological stress response facilitates flight or fight responses and potentiates chances of survival by dilating pupils and increasing energy mobilization, heart rate, respiration, and blood flow to skeletal muscles. Current research indicates that potentiated innate immunity due to the release of danger-associated-molecular-patterns (DAMPs) should be added to this list.

This project focuses on the role of the stress-inducible member of the 70-kDa Hsp (heat-shock) family of proteins Hsp72, as an important systemic DAMP. The discovery of Hsp72 as a DAMP would have implications for all organisms that generate adaptive acute stress responses. Using a systems biology approach, this project examines the nature of eHsp72 release and its function in host defense following bacterial challenge. The results of this work will enrich the interplay of research between the fields of stress physiology and immunology and could yield ways of capitalizing on the adaptive aspect of stress. In addition to specific scientific advancements, this research will sustain recruitment of undergraduate students from underrepresented minority groups in the biological sciences, with the goal of preparing them to enter graduate school.

The PI has historically trained and mentored undergraduate students from underrepresented groups. Students involved in the project will be mentored and trained in research design, critical thinking, oral and written communication. Undergraduate students will present their work at scientific meetings and in peer-reviewed journals as co-authors. The project outcomes will be disseminated to the general public through television, radio, and mainstream periodicals.

DESCRIPTION (provided by applicant): While there has been increasing recognition of the importance of microglial and astrocyte activation in the creation & maintenance of diverse enhanced pain states, an important aspect of glial functioning that has not yet been explored in the context of pain enhancement is the effect of a sensitized, or primed, microglial response. Evidence has accrued from outside of the pain field that the past history of microglial activation can dramatically alter their response to new challenges. Microglia can reach a primed state via a variety of challenges, including peripheral or central trauma/inflammation, stress, prior pain, and exposure to opioids, which strikingly are known co-morbidities for the transition of acute to chronic pain, including neuropathic pain. While in such a primed state, microglia now dramatically over-respond to new challenges, stronger and longer than before. We believe such prior challenges that result in glial priming can set the stage for the transition of acute to chronic pain following peripheral & central neural damage, resulting in chronic neuropathic pain. Re-activation of primed microglia may lead to a transition from acute pain to chronic pain as a result of a neuroinflammatory response that is greatly amplified in both magnitude and duration. Goals. (1) In accordance with the specified goals of this RFA, develop new rat models to study the transition from acute to chronic neuropathic pain, based on the premise that a first challenge (Hit 1: peripheral or central trauma/inflammation, stress, prior pain, exposure to opioids) will markedly enhance pain induced by a subsequent (second) challenge (Hit 2: peripheral or central neural inflammation/injury). (2) Utilize the refined robust models to test the potential of non-opioid, non-addictive blood-brain barrier permeable glial activation inhibitors & resolvins to prevent the transition of acute to chronic pain. (3) Given the remarkably powerful positive effects produced by chronic voluntary exercise in creating resiliency to a multitude of negative outcomes (including constraining glial/immune reactivity), chronic voluntary exercise will also be tested for its ability to prevent the transition from acute to chronic pain, an approach enabled by teaming with an expert from outside the pain field (M. Fleshner). (4) Discover intracellular changes that differentiate rats which do vs. do not transition from acute to chronic neuropathic pain, & define how these potential cellular markers of impending chronic pain are affected by successful interventions (glial inhibitors, voluntary exercise). This will lay the groundwork for identifying and targeting changes reliably predictive of the transition of acute to chronic neuropathic pain.