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