Six weeks of voluntary wheel running modulates inflammatory protein (MCP-1, IL-6, and IL-10) and DAMP (Hsp72) responses to acute stress in white adipose tissue of lean rats.

To prime local tissues for dealing with potential infection or injury, exposure to an acute, intense stressor evokes increases in circulating and local tissue inflammatory proteins. Regular physical activity facilitates stress-evoked innate reactivity and modulates the expression of inflammatory proteins in immuno-metabolic tissues such as white adipose tissue (WAT). The impact of regular physical activity on stress-evoked inflammatory protein expression in WAT, however, remains unclear. To investigate this question, lean male F344 rats (150-175g) were allowed voluntary access to a running wheel for 6weeks followed by exposure to an acute stressor (100, 1.5mA-5s inescapable tail shocks). Using ELISAs, corticosterone, heat shock protein 72 (Hsp72), macrophage chemoattractant protein (MCP-1), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1ß, IL-6, and IL-10 concentrations were measured in plasma and subcutaneous, intraperitoneal (epididymal and retroperitoneal WAT depots) and visceral (omental and mesenteric WAT depots) WAT compartments. Acute stress increased plasma concentrations of all proteins except TNF-α and, depending upon the compartment examined, WAT concentrations of MCP-1, IL-1ß, IL-6, and IL-10. Exercise ubiquitously increased IL-1ß within WAT, potentiated stress-evoked Hsp72 in plasma and WAT, and differentially increased stress-evoked MCP-1, IL-6, and IL-10 within WAT. These data suggest: (a) inflammatory proteins in non-obese WAT may serve compartment-specific immune and metabolic roles important to the acute stress response and; (b) voluntary habitual exercise may optimize stress-induced augmentation of innate immune function through increases in stress-evoked Hsp72, MCP-1, IL-6, and IL-10 and decreases in IL-1ß/IL10 and TNF-α/IL10 ratios within white adipose tissue.

The inflammasome and danger associated molecular patterns (DAMPs) are implicated in cytokine and chemokine responses following stressor exposure.

Exposure to stressors or trauma in the absence of pathogenic challenge can stimulate a systemic sterile inflammatory response characterized by high concentrations of blood and tissue cytokines, chemokines, and danger associated molecular patterns (DAMPs) such as heat shock protein-72 (Hsp72), and uric acid. The signaling pathways responsible for these responses remain unclear, however, the inflammasome may play a role. In vitro, DAMPs are known to stimulate the inflammasome in the presence of LPS to activate caspase-1 which cleaves immature precursors of interleukin (IL)-1ß and IL-18 into their mature releasable forms. Furthermore, in vivo neutralization of the LPS selectively attenuates the stress-induced increase in the inflammasome-dependent cytokines IL-1ß and IL-18. Thus, the current experiments tested the hypothesis that inflammasome-mediated processes are necessary for a systemic stress-induced inflammatory response to an acute stressor. The data presented (1) establish that male F344 rats exposed to an acute severe stressor (100 tail shocks) have elevated plasma concentrations of inflammatory proteins (IL-1ß, IL-18, IL-6, IL-10, and monocyte chemotactic protein (MCP)-1), and DAMPs (uric acid and Hsp72); (2) demonstrate that inhibiting caspase-1 in vivo, using the caspase-1 inhibitor ac-YVAD-cmk, attenuates stress-induced production of IL-1ß, IL-18, and IL-6 in both the circulation and peripheral tissues; and (3) implicates the DAMPs uric acid and Hsp72 as important signals contributing to inflammasome-dependent inflammatory responses using a stepwise multiple regression. The results increase our mechanistic understanding of systemic sterile inflammatory responses, and provide novel evidence that the inflammasome may be an important pharmacological target for treatment of these conditions.

The impact of acute-stressor exposure on splenic innate immunity: a gene expression analysis.

Exposure to intense, acute-stressors modulates immune function. We have previously reported, for example, that exposure to a single session of inescapable tailshock suppresses acquired and potentiates innate immune responses mediated by the spleen. The mechanisms for these changes remain unknown, however, they likely involve stress-induced modulation of cytokines. Cytokines operate in coordinated networks that include other immunoregulatory factors. Broad-scoped analyses are required to gain an understanding of the net-impact of stress on these immunoregulatory factors and the immune system. The goal of this study, therefore, is to examine the impact of acute-stressor exposure on network-wide changes in splenic immunoregulatory factor expression. One hundred and sixty-one genes linked to innate immune responses were quantified in the spleen following exposure to tailshock using an RT-PCR based gene array. Expression changes in 17 of the measured genes were confirmed using individual RT-PCR reactions. Further assessment of the expression changes using Exploratory Gene Association Networks (EGAN) identified important ontologies, processes and pathways that are indicative of a broader impact of stress on the immune system. Interestingly, EGAN identified several linkages between immunoregulatory factors that may be important in explaining previous results concerning the functional consequences of stress on splenic immunity. Additional processes, some of which are novel to this study, were also uncovered that may be important in directing future studies examining the impact of stress on the immune system. In this way, these analyses provide a better understanding of how acute stressor exposure modulates splenic immunity and may function as predictive tool for future related studies.

Stress-induced facilitation of host response to bacterial challenge in F344 rats is dependent on extracellular heat shock protein 72 and independent of alpha beta T cells.

Activation of the in vivo stress response can facilitate antibacterial host defenses. One possible mechanism for this effect is stress-induced release of heat shock protein 72 (Hsp72) into the extracellular environment. Hsp72 is a ubiquitous cellular protein that is up-regulated in response to cellular stress, and modulates various aspects of immune function including macrophage inflammatory/bactericidal responses and T-cell function when found in the extracellular environment. The current study tested the hypothesis that in vivo extracellular Hsp72 (eHsp72) at the site of inflammation contributes to stress-induced restricted development of bacteria, and facilitated recovery from bacteria-induced inflammation, and that this effect is independent of alpha beta (αß) T cells. Male F344 rats were exposed to either inescapable electrical tail-shocks or no stress, and subcutaneously injected with Escherichia coli (ATCC 15746). The role of eHsp72 was investigated by Hsp72-immunoneutralization at the inflammatory site. The potential contribution of T cells was examined by testing male athymic (rnu/rnu) nude rats lacking mature αß T cells and heterozygous thymic intact control (rnu/+) rats. The results were that stressor exposure increased plasma concentrations of eHsp72 and facilitated recovery from bacterial inflammation. Immunoneutralization of eHsp72 at the inflammatory site attenuated this effect. Stressor exposure impacted bacterial inflammation and eHsp72 equally in both athymic and intact control rats. These results support the hypothesis that eHsp72 at the site of inflammation, and not αß T cells, contributes to the effect of stressor exposure on subcutaneous bacterial inflammation.

Acute stressor exposure modifies plasma exosome-associated heat shock protein 72 (Hsp72) and microRNA (miR-142-5p and miR-203).

Exosomes, biologically active nanoparticles (40-100 nm) released by hematopoietic and non-hematopoietic cells, contain a variety of proteins and small, non-coding RNA known as microRNA (miRNA). Exposure to various pathogens and disease states modifies the composition and function of exosomes, but there are no studies examining in vivo exosomal changes evoked by the acute stress response. The present study reveals that exposing male Fisher 344 rats to an acute stressor modulates the protein and miRNA profile of circulating plasma exosomes, specifically increasing surface heat shock protein 72 (Hsp72) and decreasing miR-142-5p and -203. The selected miRNAs and Hsp72 are associated with immunomodulatory functions and are likely a critical component of stress-evoked modulation of immunity. Further, we demonstrate that some of these stress-induced modifications in plasma exosomes are mediated by sympathetic nervous system (SNS) activation of alpha-1 adrenergic receptors (ADRs), since drug-mediated blockade of the receptors significantly attenuates the stress-induced modifications of exosomal Hsp72 and miR-142-5p. Together, these findings demonstrate that activation of the acute stress response modifies the proteomic and miRNA profile of exosomes released into the circulation.

Effects of stressor controllability on diurnal physiological rhythms.

Disruptions in circadian and diurnal rhythms are associated with stress-related psychiatric disorders and stressor exposure can disrupt these rhythms. The controllability of the stressor can modulate various behavioral and neurochemical responses to stress. Uncontrollable, but not controllable, stress produces behaviors in rats that resemble symptoms of anxiety and depression. Whether acute stress-induced disruptions in physiological rhythms are sensitive to controllability of the stressor, however, remains unknown. To examine the role of controllability in diurnal rhythm disruption, adult male Sprague-Dawley rats were implanted with Data Sciences International (DSI) biotelemetry devices. Real-time measurements were obtained before, during and after exposure to a controllable or yoked uncontrollable stressor. Controllable and uncontrollable stress equally disrupted diurnal rhythms of locomotor activity and body temperature but not heart rate. The diurnal heart rate the day following stressor exposure was flattened to a greater extent and was significantly higher in rats with control over stress suggesting a relationship between stressor controllability and the heart rate response. Our results are consistent with the conclusion that acute stress-induced disruptions in diurnal physiological rhythms likely contribute little to the behavioral and affective consequences of stress that are sensitive to stressor controllability.

Commensal bacteria and MAMPs are necessary for stress-induced increases in IL-1ß and IL-18 but not IL-6, IL-10 or MCP-1.

Regular interactions between commensal bacteria and the enteric mucosal immune environment are necessary for normal immunity. Alterations of the commensal bacterial communities or mucosal barrier can disrupt immune function. Chronic stress interferes with bacterial community structure (specifically, α-diversity) and the integrity of the intestinal barrier. These interferences can contribute to chronic stress-induced increases in systemic IL-6 and TNF-α. Chronic stress, however, produces many physiological changes that could indirectly influence immune activity. In addition to IL-6 and TNF-α, exposure to acute stressors upregulates a plethora of inflammatory proteins, each having unique synthesis and release mechanisms. We therefore tested the hypothesis that acute stress-induced inflammatory protein responses are dependent on the commensal bacteria, and more specifically, lipopolysaccharide (LPS) shed from Gram-negative intestinal commensal bacteria. We present evidence that both reducing commensal bacteria using antibiotics and neutralizing LPS using endotoxin inhibitor (EI) attenuates increases in some (inflammasome dependent, IL-1 and IL-18), but not all (inflammasome independent, IL-6, IL-10, and MCP-1) inflammatory proteins in the blood of male F344 rats exposed to an acute tail shock stressor. Acute stress did not impact α- or ß- diversity measured using 16S rRNA diversity analyses, but selectively reduced the relative abundance of Prevotella. These findings indicate that commensal bacteria contribute to acute stress-induced inflammatory protein responses, and support the presence of LPS-mediated signaling in stress-evoked cytokine and chemokine production. The selectivity of the commensal bacteria in stress-evoked IL-1ß and IL-18 responses may implicate the inflammasome in this response.