Background
Chronic mental and emotional stress are detrimental to physical and mental well-being and often cause affective disorders such as depression and anxiety [
1]. It has been shown that chronic stress challenges are associated with an inflammatory response in brain that is characterized by toll-like receptor-4 (TLR4)/nuclear factor kappa B (NF-κB) activation and release of proinflammatory cytokines [
2,
3].
TLRs are pattern recognition receptors thought to mediate the innate immune response [
4,
5]. Their expression is modulated rapidly in response to pathogens, a variety of cytokines, and environmental stress. Specifically, TLR4 regulates the adrenal response to stress and inflammatory stimuli as well as the brain’s response to stress [
6]. TLR4 recruits adapter proteins, such as myeloid differentiation factor 88 (MyD88), and activates downstream signaling molecules, such as transcription factor nuclear factor kappa B (NF-κB), to elicit production of multiple proinflammatory cytokines. Activation of TLR4 complex may underlie the pathophysiology of many inflammatory diseases, such as depression, diabetes, obesity, chronic fatigue syndrome, and neuroinflammatory disorders [
7]. Drugs and compounds that attenuate the TLR signaling pathway could represent new treatment possibilities for TLR4-mediated inflammatory disorders.
The cholinergic system plays a crucial role in motor, cognitive, and affective processes, and also regulates central and peripheral inflammation [
8,
9]. Acetylcholine interacts with nicotinic acetylcholine receptor α7 subunit (α7nAChR) in tissue macrophages and other immune cells and inhibits the synthesis/release of tumor necrosis factor-α (TNF-α) and other inflammatory cytokines. This neural anti-inflammatory response, known as the cholinergic anti-inflammatory pathway, is fast and integrated through the central nervous system (CNS) [
8]. α7nAChRs, composed of five identical α7 subunits, mainly regulate the cholinergic anti-inflammatory pathway by mediating peripheral macrophage activity [
10]. In the CNS, both microglia and neurons express functional α7nAChRs. Indeed, stimulation of microglial α7nAChRs was shown to inhibit glial activation and decrease proinflammatory mediator expression and reactive oxygen species production [
11,
12]. Dysregulation of cholinergic signaling has been implicated in multiple inflammatory disorders, including sepsis, myocardial or cerebral ischemia, cerebral hemorrhage, and Alzheimer’s disease [
12‐
16]. Activation of the cholinergic system after brain injury might repair the CNS capacity to control the exaggerated inflammation.
Neuronal cell damage after chronic stress is observed predominantly in the hippocampus, a structure densely populated with α7nAChRs. Although the TLR4 proinflammatory pathway has been implicated in the development of chronic stress [
2,
17], the roles of cholinergic signaling and α7nAChR-associated anti-inflammatory properties in chronic stress have not yet been examined. We therefore investigated whether chronic immobilization stress disrupts cholinergic function and activates the inflammatory responses, and whether treatment aimed at restoring α7nAChR function offers benefits by regulating inflammation and reducing neuronal death.
Discussion
The CRS model is known to induce psychological stress and is often used to study depression [
37,
38]. Although diverse factors contribute to the development of depression, recent research has suggested that neuroinflammation has a significant role in the pathogenesis of depressive disorders [
39,
40]. In this study, we found that the α7nAChR agonist DMXBA reduced inflammation and alleviated anhedonia and depression-like behavior in mice subjected to CRS.
Proinflammatory cytokines and glial activation are thought to contribute to neuronal damage after various cerebral injuries [
41‐
44]. Some investigators have reported that the TLR4 signaling pathway plays an important role in neuroinflammation after chronic stress exposure [
2,
3,
45], and others have shown that TLR4-deficient mice subjected to subacute stress exhibit only a minor inflammatory response in brain tissue [
17]. Our study shows that repeated restraint induced-psychological stress leads to increased levels of TNF-α and IL-1β, as well as elevated activity of microglia in the hippocampus of mice. The increased inflammatory response in the hippocampus may be partly related to upregulation of the TLR4 pathway, although we observed no obvious stimulation of NF-κB p65 subunit in the brain hippocampus after CRS. Chronic inflammatory stimulation may restrict NF-κB action, as has been reported in a chronic mild stress model in rats [
2]. Furthermore, we found that the selective α7nAChR partial agonist DMXBA mitigated chronic stress-induced activation of the TLR4 signaling pathway while reducing depression-like behaviors in mice. It has been reported that DMXBA antagonizes α4β2 nAChRs in addition to serving as an agonist for α7nAChRs [
46]. Notably, we found that pretreatment with α-BGT, a selective α7nAChR antagonist, abolished the anti-depressive and anti-inflammatory effects of DMXBA, suggesting that the protective effects of DMXBA are α7nAChR-dependent. Although α-BGT at the dose we used did not reverse DMXBA’s inhibitory effect on microglial activation, some studies have reported that microglial α7nAChR does participate in the α7nAChR-mediated anti-inflammatory effect during ischemic events [
15,
47,
48]. To our knowledge, these results provide the first evidence to support a regulatory role of the cholinergic system in chronic stress-induced behavioral deficits and the accompanying neuroinflammatory response in the hippocampus of mice.
The cholinergic system is important for maintaining proper central and peripheral immune and inflammatory responses and therefore is called the cholinergic anti-inflammatory pathway. This pathway modulates immune and inflammatory responses mainly through acetylcholine activation of α7nAChR [
49]. The cholinergic anti-inflammatory pathway was shown to be effective in cerebral ischemia, intracerebral hemorrhage, and Alzheimer’s disease [
14,
15]. In our study, we further demonstrated a role for the cholinergic system during chronic stress.
We found an upward trend of α7nAChR and a downward trend of AChE activity, as well as significant upregulation of ChAT expression and decreased nuclear STAT3 expression in the hippocampus after 21 days of CRS, indicating relative disturbance of the brain’s cholinergic function. Accompanied by significant neuronal loss/damage in the hippocampus after 21 days of CRS, increased expression of ChAT can be attributed to the smaller number of cells remaining. Hence, the trend toward an increase in α7nAChR may indicate a significant increase in the remaining neurons or activated glial cells. Recent human imaging studies have shown that acetylcholine levels are elevated throughout the brain in depressed patients [
50,
51], indicating that hyperactivity of the brain’s cholinergic system might be a compensatory mechanism for controlling neuroinflammation in chronic stress. However, chronic stress-induced changes in the protein levels of ChAT and acetylcholine may cause maladaptive plasticity downstream of acetylcholine, such as dysregulation of JAK/STAT signaling, that can lead to symptoms related to depression. These observations in humans and animals have promoted interest in a proinflammatory/anti-inflammatory signaling balance hypothesis of depression.
It has been reported that α7nAChR activation interferes with other signaling pathways by activating JAK/STAT signaling and modulating gene transcription in immune cells [
49,
52]. In peritoneal macrophages, the α7nAChR can recruit the tyrosine kinase Jak2, which activates STAT3 and subsequent signaling cascades. The Jak2-STAT3 pathway contributes to the anti-inflammatory potential of α7nAChR by inhibiting the release of anti-inflammatory cytokines. In our study, α7nAChR agonist DMXBA treatment significantly reversed CRS-induced downregulation of nuclear STAT3 in the hippocampus, indicating that pharmacologic activation of α7nAChR restores central cholinergic signaling function. In addition, DMXBA treatment inhibited AChE activity in the brain, an effect that might also promote the actions of central cholinergic signaling during CRS.
Tregs, a subpopulation of CD4
+ T cells, play a crucial role in maintaining immune homoeostasis and preventing the development of many inflammatory diseases, such as rheumatoid arthritis, multiple sclerosis, and ischemic stroke. Although an increase in Treg cells in peripheral blood via signals from the sympathetic nervous system may contribute to stroke-induced immunosuppression [
24], recent reports showed that systemic administration of purified Tregs suppressed inflammatory over-activation in the brain and attenuated neurovascular dysfunction after ischemic stroke [
53,
54]. In contrast, Treg depletion exacerbated ischemic brain injury [
55]. The neuroprotective effect of Tregs may relate in part to their ability to protect the blood–brain barrier and inhibit peripheral inflammatory cell infiltration into the brain [
53,
54]. Recently, Kim et al. [
56] reported that chronically stressed mice with Treg depletion exhibited reduced depression-like behaviors, indicating that Tregs might be associated with the pathophysiologic mechanism of chronic stress-induced depression. In our present study, we found that 21 days of CRS produced a decrease in the mouse splenocyte Treg population, consistent with previous reports of low Treg cell counts in the peripheral blood of patients with major depression [
57,
58]. Interestingly, we observed that 7 days of CRS caused a decrease in Treg cells in mouse spleen, whereas 14 days of CRS restored the Treg population. Treg cells declined again after 21 days of CRS, possibly indicating a long-lasting and decompensated immunologic alteration after chronic stress. We found that treatment with α7nAChR agonist DMXBA completely reversed the chronic stress-induced decline in Treg cells and alleviated depression-like behaviors in CRS mice, indicating that α7nAChR activation may modulate depressive behavior by promoting Treg cell function, which in turn mitigates chronic stress-induced neuroinflammation.
Chronic stress has been shown to induce peripheral immunosuppressive effects and is thought to increase susceptibility to many diseases [
59]. In our study, after 7 days of CRS, mice exhibited significantly higher serum IL-1β and TNF-α levels. However, after 14 and 21 days, these levels had declined and returned to the normal range. Interestingly, IL-1β and TNF-α levels were elevated in the hippocampus at 21 days of CRS, indicating unsynchronized changes of cytokines in the periphery and brain. Although the blood–brain barrier might restrict the passage of harmful factors into the brain in the early stage of injury, or the CNS might change from a steady state to an unstable state after long-term stress, the actual reason for temporal and spatial changes of IL-1β and TNF-α is not yet clear. Treatment with the α7nAChR agonist DMXBA significantly increased the Treg population in the circulation and reduced inflammatory over-activation in the brain but had no apparent effect on serum IL-1β or TNF-α level. Thus, it seems that α7nAChR activation restricts inflammatory response in the brain but does not exacerbate immunosuppression after chronic stress. These findings are in line with its regulation of immune homeostasis between the brain and periphery during chronic stress. A single acute stress and chronic repeated stresses may mobilize different pathophysiologic mechanisms, as a recent report showed that activation of the cholinergic system by acetylcholine or AChE inhibitor physostigmine increased anxiety- and mood-related behaviors in the TST and FST [
60]; however, the detailed mechanism is not fully understood.