Elsevier

Brain, Behavior, and Immunity

Volume 84, February 2020, Pages 180-199
Brain, Behavior, and Immunity

Repeated stress induces a pro-inflammatory state, increases amygdala neuronal and microglial activation, and causes anxiety in adult male rats

https://doi.org/10.1016/j.bbi.2019.11.023Get rights and content

Highlights

  • Repeated social defeat stress (RSDS) induces anxiety-like behavior.

  • RSDS increases dual negative but decreases dual positive, CD4+ and regulatory T-cells.

  • RSDS increases CD4+ and CD8+ T-cells positive for type 2-like profile.

  • RSDS shifts the balance towards a specific set of T-cells and cytokines.

  • RSDS activates microglia and increases neuronal firing in the basolateral amygdala.

Abstract

A link exists between immune function and psychiatric conditions, particularly depressive and anxiety disorders. Psychological stress is a powerful trigger for these disorders and stress influences immune state. However, the nature of peripheral immune changes after stress conflicts across studies, perhaps due to the focus on few measures of pro-inflammatory or anti-inflammatory processes. The basolateral amygdala (BLA) is critical for emotion, and plays an important role in the effects of stress on anxiety. As such, it may be a primary central nervous system (CNS) mediator for the effects of peripheral immune changes on anxiety after stress. Therefore, this study aimed to delineate the influence of stress on peripheral pro-inflammatory and anti-inflammatory aspects, BLA immune activation, and its impact on BLA neuronal activity. To produce a more encompassing view of peripheral immune changes, this study used a less restrictive approach to categorize and group peripheral immune changes. We found that repeated social defeat stress in adult male Sprague-Dawley rats increased the frequencies of mature T-cells positive for intracellular type 2-like cytokine and serum pro-inflammatory cytokines. Principal component analysis and hierarchical clustering was used to guide grouping of T-cells and cytokines, producing unique profiles. Stress shifted the balance towards a specific set that included mostly type 2-like T-cells and pro-inflammatory cytokines. Within the CNS component, repeated stress caused an increase of activated microglia in the BLA, increased anxiety-like behaviors across several assays, and increased BLA neuronal firing in vivo that was prevented by blockade of microglia activation. Because repeated stress can trigger anxiety states by actions in the BLA, and altered immune function can trigger anxiety, these results suggest that repeated stress may trigger anxiety-like behaviors by inducing a pro-inflammatory state in the periphery and the BLA. These results begin to uncover how stress may recruit the immune system to alter the function of brain regions critical to emotion.

Introduction

Chronic psychological stress is one of the most common triggers of clinical depression, causing lasting anxiety, depressed mood and other symptoms (van Praag, 2005, Bartolomucci and Leopardi, 2009). These psychiatric symptoms and experience of stress are both associated with alterations of the immune system, suggesting that, in some cases, stress may trigger depression and anxiety via effects on the immune system. The immune system may exert this effect through white blood cells, particularly T-cell populations of lymphocytes and monocytes, as well as cytokines that mediate cell-to-cell communication (Dantzer, 2001, Anisman and Merali, 2002, Konsman et al., 2002, Kiecolt-Glaser et al., 2003, Wohleb and Delpech, 2017). Mature T-cells are characterized as either CD4+ helper (Th) or CD8+ cytotoxic (Tc) T-cells. T-cells are either pro-inflammatory (type 1) or anti-inflammatory (type 2) depending on the predominant types of cytokines secreted. T-helper (Th)-1 cells secrete mainly pro-inflammatory cytokines whereas the Th-2 cells mainly secrete anti-inflammatory cytokines (Mosmann and Sad, 1996). Regulatory T-cells (Treg cells) are a specialized subset of CD4+ T-cells that suppress potentially deleterious effects of Th cells (Corthay, 2009). The relative balance of different types of T-cells greatly influence the inflammatory state.

There is strong evidence that stress can impact T-cell populations in humans, a major source of peripheral cytokines. Psychological stress in humans is associated with an increased accumulation of peripherally primed monocytes with higher potential for inflammatory signaling as evidenced by increased pro-inflammatory-related gene expression (Miller et al., 2008, Cole et al., 2011, Powell et al., 2013), with subsequent increase of cytokines (Kiecolt-Glaser et al., 2003, Hou et al., 2017), particularly those categorized as pro-inflammatory interleukins (IL), IL-6, IL-1β, and IL-10. Similarly, meta-analysis of patients with stress-related psychiatric disorders, such as depression or post-traumatic stress disorders, indicate a picture of increased pro-inflammatory cytokines (Tursich et al., 2014, Passos et al., 2015, Breen et al., 2018, Renna et al., 2018). However, meta-analyses find variability in the effects of stress on T-cell populations, with some evidence pointing towards a reduction of Th-1 and Th-2 cells and other findings suggesting a shift from Th-1 to Th-2 cells (Segerstrom and Miller, 2004, Nakata, , 2012). Furthermore, the effects of stress on peripheral cytokines does not follow a pattern that would be predicted for a simple general increase in Th1 or Th2 cell function (e.g. Maes et al., 1998, Kamezaki et al., 2012). Indeed, not all studies find increased type 1 pro-inflammatory profiles, some studies find a decrease, and others highlight the importance of changes in type 2 profiles (Nakano et al., 1998, Kaufmann et al., 2007). While meta-analyses support an effect of stressors on several pro-inflammatory cytokines in healthy individuals (Segerstrom and Miller, 2004, Marsland et al., 2017), there is also strong evidence for increased anti-inflammatory cytokines (Marsland et al., 2017) or decreased pro-inflammatory cytokines (Eddy et al., 2016) in healthy individuals, and there is some heterogeneity in the exact pattern of changes across individual studies.

One way to understand discrepencies across studies is recognition that a clear picture might not emerge upon examination of a single cytokine, or small group of cytokines. Instead, better insight might be gained by examining the balance between different groups of cytokines. However, even studies that assess a balance between pro- and anti-inflammatory cytokines, measured as the ratio between a chosen pro- and anti-inflammatory cytokine, have produced variable results (Hashizume et al., 2005, Rehm et al., 2013, Karlsson et al., 2017). The differences in these effects of stress on cytokine profiles could be due to differences in the populations studied, stressor examined, and measures used. This highlights the need for a comprehensive set of measures from the same stressor under the same conditions. Another likely factor is that the immune response includes a number of cytokines that interact dynamically. A broader understanding of the effects of stress may be achieved by examination of a wider number of cytokines and how they co-vary.

Substantially more is known about immune system states following stress in animal models. Chronic stress increases neutrophils and monocytes output in circulation (Powell et al., 2013, Heidt et al., 2014), consistent with greater capacity for inflammatory responses. While more studies have supported an increase of pro-inflammatory cytokines after acute or repeated stress, similar to studies in humans, animal models show a variable shift from type 1 and type 2 cytokine profiles after stress, with findings of increased Th1 cytokines or reduced Th2 cytokines (Bartolomucci et al., 2001, Chida et al., 2005, Savignac et al., 2011, Azzinnari et al., 2014, Stewart et al., 2015) or even decreased Th1 or increased Th2 cytokines (Mormède et al., 2002, Hou et al., 2013, Ahmad et al., 2015, Hu et al., 2016) perhaps depending partly upon which of the diversity of stressors were used (Deak et al., 2003, Deak et al., 2005, Bowers et al., 2008, Hueston et al., 2011, Shaashua et al., 2012, Budiu et al., 2017), or on which cytokines are measured. Therefore, it is important to examine a range of both pro-inflammatory and anti-inflammatory cytokines, along with T-cell populations to fully understand the effects of a specific stressor on the peripheral immune system, and to understand how a balance between cytokines might shift. However, the categorization of cytokines into pro-inflammatory or anti-inflammatory is somewhat artificial, with now established evidence for overlap in the inflammatory effects of type 1 and type 2 cytokines. Therefore, even if using classical pro-inflammatory and anti-inflammatory cytokines as a starting point for analysis, an unbiased characterization of the balance between different cytokine groups is important to test. This is expected to produce a more accurate reflection of the effect of stress on the peripheral immune environment, and may spark exploration of new avenues to interfere with the harmful effects of stress. Thus, one goal of this study is to use a rodent model to test if repeated stress causes an imbalance of the peripheral inflammatory state using an approach that does not focus on any single cytokine, or any pre-determined group of cytokines, but instead determines how the balance between groups of covarying cytokines changes.

While many immune changes might be initiated in the periphery, immune changes in the central nervous system (CNS) likely produce some of the behavioral outcomes caused by stress (Wohleb et al., 2013, Wohleb et al., 2018, McKim et al., 2018). Microglia are the resident immune cells of the CNS (del Rio-Hortega, 1932, Ginhoux et al., 2010, Ginhoux et al., 2013) with density ranging from 5% to 12% of the total number of cells in different brain regions (Lawson et al., 1990). Acute and chronic stress produce microglia activation in the prefrontal cortex, amygdala, and the hippocampus (Sugama et al., 2009, Tynan et al., 2010, Wohleb et al., 2012), which can contribute to the development of anxiety and depressive behavior after chronic stress (Wohleb et al., 2013, McKim et al., 2018). The basolateral amygdala (BLA) is a primary region involved in stress-induced anxiety. Chronic stress strongly increases BLA neuronal activity and impacts morphology (Vyas et al., 2002, Rosenkranz et al., 2010, Zhang and Rosenkranz, 2012). The BLA may be impacted by the effects of stress on immune function, and activation of BLA microglial cells would be a fairly direct route. Indeed, chronic social defeat stress in mice altered immune signaling genes in the amygdala (Azzinnari et al., 2014). However, the effects of chronic stress on BLA microglia are not clear, with evidence that repeated restraint stress in male rats reduces the proportion of primed to surveillant microglia (Bollinger et al., 2017) or has no effect on microglia number and morphology (Tynan et al., 2010), with differences perhaps related to duration of the stress exposure and related habituation to the repeated stress. Social stressors exert a powerful, lasting impact on immune function in humans (Shimamiya et al., 1985, Gerritsen et al., 1996, Caserta et al., 2008, Miller et al., 2009, Slopen et al., 2015) and rodents (Stefanski and Engler, 1998, Stefanski et al., 2001, Dunphy-Doherty et al., 2018). One way to model this in rats is with repeated social defeat stress (RSDS). RSDS causes alterations that parallel psychiatric symptoms after stress, characterized by increased anxiety, social-avoidance, and anhedonia (Rygula et al., 2005, Berton et al., 2006, Rygula et al., 2006). Additionally, RSDS is known to have a robust effect on immune parameters in rodents (Wohleb et al., 2013), and these immune changes are required to produce RSDS effects on anxiety (Wohleb et al., 2011, Wohleb et al., 2013). Despite robust effects of social stress on immune function, it remains untested if social stress impacts BLA microglia activation. Thus, one goal of this study is to test the effect of repeated stress on microglia and neuronal activity in the BLA, and whether disruption of microglia activation can prevent the effects of stress on BLA neuronal activity. The BLA is comprised of multiple nuclei that have distinct roles in behavior, most notably the lateral (LAT) and basal (BA) nuclei, and it is important to consider both nuclei of the BLA.

In this study we used RSDS to explore how repeated social stress impacts the peripheral immune balance by measuring how peripheral immune precursor and mature T-cells and serum cytokines co-vary. To achieve this, we used an unbiased approach to group T-cell profiles and cytokines, and measured the effects of stress on the balance between these groups. We also examined parallel effects of repeated social stress on a CNS site important in anxiety, by measuring BLA-sensitive anxiety behaviors, BLA microglia number and activation, BLA neuronal activity, and whether blockade of microglia activation alters the effects of stress on BLA neuronal activity.

Section snippets

Ethical approval and animal subjects

All experiments were performed in compliance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011) and were approved by the Institutional Animal Care and Use Committee (IACUC) of Rosalind Franklin University of Medicine and Science. Care was taken to reduce the total number of animals used for the study. Adult male Sprague-Dawley rats (Envigo, Indianapolis, IN) were obtained at post-natal day 59–63 and were housed 2–3 per cage in the climate-controlled

Results

RSDS measures were obtained on each day of the stress exposure. Overall, rats were consistently attacked and submissions were displayed. The mean ± S.E.M. of the number of attacks encountered by the defeated rats and the latency to submission are shown in Fig. 1.

Discussion

In this study, we examined the effects of repeated social defeat stress on immune system parameters in rats. Although earlier studies have shown an association between altered immune status and stress and stress-associated disorders, few studies provide a holistic assessment of the effects of repeated stress on the immune system, as performed here. The underlying rationale was that better understanding of changes can be achieved by clustering immune parameters based on how they co-vary, in

Conclusions

Chronic stress, which is an important factor contributing to the development of depression, has significant effects on the immune system parameters. Here we examined the effects of chronic stress on the circulating immune parameters using the resident-intruder model of repeated social defeat stress (RSDS) in adult male rats. We have also examined the effect of the stress on the resident immune cells of the amygdala (BLA), the microglia, as well as the BLA neuronal firing in vivo. The findings

Funding

This study was supported by the National Institutes of Health grants MH084970 and MH109484. The funding body had no role in the design of the study, collection and analysis of data and decision to publish.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors gratefully acknowledge Drs. Anthony R. West, Gloria E. Meredith, Grace E. Stutzmann and Robert A. Marr for scientific guidance, discussion, and assistance. The authors thank Dr. Robert J. Bridges for helpful discussion and guidance on ELISA experiments. The authors also thank Robert Dickinson of the flow-cytometry core facility and Matthew Anagnostopoulos of the Biological Resource Facility at Rosalind Franklin University of Medicine and Science for technical support. A portion of

References (168)

  • S.L. Bowers et al.

    Stressor-specific alterations in corticosterone and immune responses in mice

    Brain Behav. Immun.

    (2008)
  • G.C. Brown et al.

    How microglia kill neurons

    Brain Res.

    (2015)
  • A. Brynskikh et al.

    Adaptive immunity affects learning behavior in mice

    Brain Behav. Immun. (Netherlands)

    (2008)
  • M.T. Caserta et al.

    The associations between psychosocial stress and the frequency of illness, and innate and adaptive immune function in children

    Brain Behav. Immun.

    (2008)
  • Y. Chida et al.

    Social isolation stress exacerbates autoimmune disease in MRL/lpr mice

    J. Neuroimmunol.

    (2005)
  • R. Dantzer et al.

    Neural and humoral pathways of communication from the immune system to the brain: parallel or convergent?

    Auton. Neurosci.

    (2000)
  • T. Deak et al.

    Exposure to forced swim stress does not alter central production of IL-1

    Brain Res.

    (2003)
  • T. Deak et al.

    Stress-induced increases in hypothalamic IL-1: a systematic analysis of multiple stressor paradigms

    Brain Res. Bull.

    (2005)
  • F. Dunphy-Doherty et al.

    Post-weaning social isolation of rats leads to long-term disruption of the gut microbiota-immune-brain axis

    Brain Behav. Immun.

    (2018)
  • P. Eddy et al.

    A systematic review and meta-analysis of the effort-reward imbalance model of workplace stress with indicators of immune function

    J. Psychosom. Res.

    (2016)
  • E. Ellwardt et al.

    Understanding the role of T cells in CNS homeostasis

    Trends Immunol. (England)

    (2016)
  • B. Engelhardt et al.

    The ins and outs of T-lymphocyte trafficking to the CNS: Anatomical sites and molecular mechanisms

    Trends Immunol. (England)

    (2005)
  • H. Engler et al.

    Acute amygdaloid response to systemic inflammation

    Brain Behav. Immun.

    (2011)
  • J.C. Felger et al.

    Inflammatory cytokines in depression: neurobiological mechanisms and therapeutic implications

    Neuroscience

    (2013)
  • A. Gadek-Michalska et al.

    Cytokines, prostaglandins and nitric oxide in the regulation of stress-response systems

    Pharmacol. Rep. (Poland)

    (2013)
  • M. Girotti et al.

    Chronic intermittent cold stress sensitizes neuro-immune reactivity in the rat brain

    Psychoneuroendocrinology (England)

    (2011)
  • J. Herz et al.

    Myeloid cells in the central nervous system

    Immunity

    (2017)
  • N. Hou et al.

    A novel chronic stress-induced shift in the Th1 to Th2 response promotes colon cancer growth

    Biochem. Biophys. Res. Commun.

    (2013)
  • R. Hou et al.

    Peripheral inflammatory cytokines and immune balance in Generalised Anxiety Disorder: case-controlled study

    Brain Behav. Immun.

    (2017)
  • C.M. Hueston et al.

    Stress-dependent changes in neuroinflammatory markers observed after common laboratory stressors are not seen following acute social defeat of the Sprague Dawley rat

    Physiol. Behav.

    (2011)
  • T.K. Inagaki et al.

    Inflammation selectively enhances amygdala activity to socially threatening images

    Neuroimage

    (2012)
  • H. Kettenmann et al.

    Microglia: new roles for the synaptic stripper

    Neuron (United States)

    (2013)
  • J.P. Konsman et al.

    Cytokine-induced sickness behaviour: Mechanisms and implications

    Trends Neurosci.

    (2002)
  • T.E. Kraynak et al.

    Functional neuroanatomy of peripheral inflammatory physiology: a meta-analysis of human neuroimaging studies

    Neurosci. Biobehav. Res.

    (2018)
  • L.J. Lawson et al.

    Heterogeneity in the distribution and morphology of microglia in the normal adult mouse brain

    Neuroscience

    (1990)
  • S.K. Lutgendorf et al.

    Depressed and anxious mood and T-cell cytokine expressing populations in ovarian cancer patients

    Brain Behav. Immun.

    (2008)
  • M. Maes

    Depression is an inflammatory disease, but cell-mediated immune activation is the key component of depression

    Prog. Neuropsychopharmacol. Biol. Psychiatry (England)

    (2011)
  • M. Maes et al.

    The new '5-HT' hypothesis of depression: Cell-mediated immune activation induces indoleamine 2,3-dioxygenase, which leads to lower plasma tryptophan and an increased synthesis of detrimental tryptophan catabolites (TRYCATs), both of which contribute to the onset of depression

    Prog. Neuropsychopharmacol. Biol. Psychiatry (England)

    (2011)
  • M. Maes et al.

    The effects of psychological stress on humans: increased production of pro-inflammatory cytokines and a Th1-like response in stress-induced anxiety

    Cytokine

    (1998)
  • A.L. Marsland et al.

    The effects of acute psychological stress on circulating and stimulated inflammatory markers: A systematic review and meta-analysis

    Brain Behav. Immun.

    (2017)
  • A.H. Miller et al.

    Inflammation and its discontents: the role of cytokines in the pathophysiology of major depression

    Biol. Psychiatry

    (2009)
  • G.E. Miller et al.

    A functional genomic fingerprint of chronic stress in humans: blunted glucocorticoid and increased NF-kappaB signaling

    Biol. Psychiatry

    (2008)
  • T.R. Mosmann et al.

    The expanding universe of T-cell subsets: Th1, Th2 and more

    Immunol. Today

    (1996)
  • S. Munshi et al.

    Effects of peripheral immune challenge on in vivo firing of basolateral amygdala neurons in adult male rats

    Neuroscience

    (2018)
  • S. Acharjee et al.

    Reduced microglial activity and enhanced glutamate transmission in the basolateral amygdala in early CNS autoimmunity

    J. Neurosci.

    (2018)
  • M. Bajo et al.

    IL-1 interacts with ethanol effects on GABAergic transmission in the mouse central amygdala

    Front. Pharmacol. (Switzerland)

    (2015)
  • W.A. Banks

    Blood-brain barrier transport of cytokines: a mechanism for neuropathology

    Curr. Pharm. Des. (United Arab Emirates)

    (2005)
  • W.A. Banks et al.

    Passage of cytokines across the blood-brain barrier

    Neuroimmunomodulation (Switzerland)

    (1995)
  • A. Bartolomucci et al.

    Stress and depression: preclinical research and clinical implications

    PLoS ONE

    (2009)
  • B. Berner et al.

    Analysis of Th1 and Th2 cytokines expressing CD4+ and CD8+ T cells in rheumatoid arthritis by flow cytometry

    J. Rheumatol.

    (2000)
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