Chronic restraint stress produced significant physiological and behavioral changes, notably reduced weight gain and depression-like behaviors. These changes were also accompanied by signs of microglia activation in the brain. This is consistent with past evidence that exposure to stress can trigger a neuroinflammatory response[3]. To our knowledge, neuroinflammation heavily relies on innate immune responses that are primarily mediated by microglia. As a signaling hub in innate immunity, STING is a key adaptor molecule in orchestrating the body’s response to inflammation, cell death and autophagy[19, 20]. Although STING is best known for its role in immune responses to cytoplasmic DNA sensed by cGAS[21], and microglia utilize the cGAS–STING pathway to orchestrate the antiviral response in the brain[22], the molecular mechanisms underlying cGAS–STING pathway contributions to depression-like behaviors remains unknown. Here, an intriguing suggestion that arises from the present study is that activation of the STING-TBK1-IRF3 pathway might be served as a potential mechanism to boost microglia phagocytosis through promoting the production of IFN-β in chronic restraint stress mice, consequently alleviating neuroinflammation and ameliorating depression-like behavior (Figure7).
Of particular interest is that human microglia constitutively express cGAS and its critical downstream adaptor STING[23]. In a Parkinson’s disease mouse model, STING-mediated inflammation has been implicated in promoting microglia activation and neuronal death[19]. Microglia are activated via the cGAS–STING pathway to induce the production of interferons to launch antiviral defense pathways[24]. Moreover, in the multiple sclerosis model[15], the antiviral drug Ganciclovir inhibits inflammation and leads to protection through activation of the STING pathway. It is interesting to note that excessive engagement of the cGAS–STING pathway in the brain can lead to neuroinflammation and neurodegeneration. It is clearly indicated that the number of microglia in certain stress-sensitive brain regions is increased during chronic stress[4]. This result was also confirmed in our study, indicating that microglia are increased in number and more activated towards M1 phenotypes in prefrontal cortex and hippocampus of RST mice. The expressions of M1-polarized microglia markers as well as the production of pro-inflammatory cytokines were increased in RST mice. Interestingly, in BV2 microglia, we observed that M1-polarized microglia have the lower level of STING expression. Thus, we wondered that whether activation of the STING pathway could ameliorate chronic restraint stress-induced depressive symptoms and relieve neuroinflammation.
CDG, CDA, 3’3-cGAMP, and 2’3-cGAMP are the native agonists of STING[25, 26]. 2’3-cGAMP, as a high-affinity ligand of STING, is a non-classical cyclic dinucleotide that activates STING and triggers potent immune responses[14, 27]. The cGAS–STING pathway is a dual-edged sword, which may be activated or inhibited to arrive at the desired outcome. The activation of STING plays a pivotal role in mediating innate immune responses and in removing multiple pathogens including both viruses and bacteria[28, 29]. In contrast, in chronic neurodegenerative states such as the prion disease, activation of the cGAS–STING signaling pathway ameliorated inflammation and disease progression[30]. Supporting these data, other studies showed that STING activation worsens acute pancreatitis severity in experimental models[31], but plays a protective role in chronic pancreatitis models[32]. Although the detrimental and protective roles of STING in regulating immune response have been studied, the precise role of STING signaling pathway in the chronic stress model has not been clarified. We first observed that STING activation reduced levels of brain pro-inflammatory cytokines or chemokines and ameliorated depression-like behaviors in RST mice.
In the brain, STING, which elicits the interferon response, is expressed predominantly in microglia[33]. Microglia plays a major role in the non-autonomous clearance of protein aggregates in the central nervous system[34]. Previous study reported macrophage proteins that are affected by STING and demonstrated the relationship between STING and phagocytosis[35]. In agreement with this finding, we first found that microglia phagocytosis is affected by STING activation in RST mice. A question arising from our work relates to how cGAMP improved microglia phagocytosis in the procession of chronic stress. One possibility is that higher levels of IFN-β caused by activation of STING signaling pathway may have contributed to the enhanced phagocytosis and anti-depressive effect observed in the present study. It should be noted that IFN-β is also central mediator of CNS inflammation during autoimmunity, where depending on the disease, they can be either protective or detrimental[36]. Previously, Toll/interleukin-1 receptor domain-containing adapter inducing IFN-β (TRIF) deficient microglia exhibited an increased threshold for activation of interferon-regulated genes, suggesting that IFN-β may enhance phagocytic activity[37]. In experimental autoimmune encephalomyelitis, IFN-β producing microglia mediated an enhanced removal of myelin debris compared to IFN-β non-producing microglia[38]. Indeed, we observed that STING-activated microglia exhibit a superior phagocytic capacity, and IFN-β might guide positioning of microglia in the inflamed central nervous system during chronic stress. In vitro, we observed the similar results that cGAMP-treated microglia displayed an increase in phagocytotic rates with LPS stimulation. However, without LPS stimulation, STING activation does not affect microglia phagocytosis, although the release of IFN-β is significantly increased. In support of this, other study confirmed that treatment of macrophages with exogenous IFN-β alone is not sufficient to induce the production of pro-inflammatory cytokines, but retreatment with IFN-β could lead to an increase in the anti-inflammatory cytokine and a decrease in pro-inflammatory cytokine expression in response to LPS stimulation[39]. In vivo, we observed that the IFN-β mRNA expression in the prefrontal cortex was significantly decreased in RST mice compared with control mice, However, the serum IFN-β level was enhanced in RST mice. Consistently, recent study reported that serum IFN-β levels was increased in chronic stress mice, and systemic blockade of type Ⅰ interferons signaling attenuated chronic stress-induced infiltration of macrophages into prefrontal cortex and behavioral abnormalities[40]. The reason behind the differing levels in the brain or serum in chronic restraint stress is not clear. It is possible that IFN-β is produced by microglia or peripheral immune cells and has different effects mediating behavioral changes. IFNβ-knock out mice in peripheral macrophages or resident microglia are warranted to test the above possibility. In this study, we concluded that microglia phagocytosis was enhanced via activating STING-dependent IFN-β signaling pathway in chronic restraint stress model, which drives behavioral changes. Besides microglia phagocytosis, neurogenesis impairments[41], dysregulation of tryptophan-kynurenine metabolism[42] and dysfunction of the HPA axis[43] were also contributed to the depression-like behaviors. More detail of mechanism should be given in future study.
In addition, STING pathway activation is linked to multiple inhibitory feedback loops, such as inflammasome activation, autophagy induction, and autocrine IFN signaling. A previous study confirmed that chronic activation of the STING pathway is associated with reduced mTORC1 signaling in metabolically relevant tissues[44]. The STING pathway is also involved in interacting with the autophagy or mitophagy machinery in innate immune responses[45–47]. Moreover, another study revealed that STING knockout could attenuate cardiac injury by inhibiting NLRP3 inflammasome mediated inflammation, apoptosis and pyroptosis[13]. Further investigations will be needed to elucidate the molecular details, as well as the consequences of the interaction between STING and other signaling pathways in different diseases.