Studies in humans and animals suggest that exposure to stressors can trigger or exacerbate anxiety disorders. One biological process increasingly investigated is the inflammatory response, given its major role in the pathophysiology of mental illnesses like depression and anxiety. Immune signaling contributes to the regulation of neurobiological processes modulating anxiety and depression-associated behaviors in response to stressors. Exposure to traumatic and stressful events results in hypothalamic-pituitary-adrenal (HPA) axis reactivity, immune system activation, and the release of proinflammatory cytokines. While extensive research characterizes the role of cytokine/chemokine dysfunction in stress-related mental health disorders, our understanding of inflammation's role in their etiology and maintenance remains limited. To understand inflammation's role in stress-related reactions, the researchers measured levels of multiple immune mediators, cytokines, and chemokines in the peripheral blood during the acute phase of stress. It's been hypothesized that brain activity directly controls adaptive immune responses in lymphoid organs, but evidence of stress-induced brain activity dysfunction leading to immune responses in lymphoid organs is scarce. Previous research showed splenic denervation impaired plasma cell formation during T cell-dependent immune responses in mice, suggesting a connection between the central nucleus of the amygdala (CeA), the paraventricular nucleus (PVN), and spleen activity. However, splenectomy didn't affect behavioral performance in stressed or wild-type mice. Human studies and cross-neuronal viral tracking in animal models hint at the contribution of brain activity and inflammatory signaling to stress-induced anxiety, but the precise neuronal location and identity involved haven't been causally proven. This study aimed to identify these neurons using adult rats and a range of neuroscience technologies, including electrophysiological techniques, fiber optic recording, optogenetics, and chemogenetics, to identify glutamatergic neurons in the red nucleus (RN) crucial in stress-induced anxiety and inflammation.

The existing literature highlights the intricate relationship between stress, inflammation, and anxiety disorders. Studies have consistently shown a link between exposure to stressors and the exacerbation or onset of anxiety. The inflammatory response is increasingly recognized as a significant player in the development and maintenance of these disorders, with immune signaling pathways influencing neurobiological processes that regulate mood and behavior. The role of specific cytokines and chemokines in these disorders has been explored, with some studies reporting alterations in cytokine levels in individuals with anxiety, depression, PTSD, and chronic stress. However, the precise mechanisms linking brain activity, immune responses in lymphoid organs, and the resulting anxiety remain unclear. While prior research has suggested connections between specific brain regions and immune system function (e.g., the amygdala and spleen), the evidence is largely correlational or based on non-specific immune system manipulation. This study aimed to move beyond correlation and address the lack of causal evidence linking specific brain regions to immune responses and resulting behavioral changes in anxiety.

The study involved both a clinical study and animal experiments. **Clinical Study:** Psychiatrists working on the front lines of the COVID-19 pandemic in Wuhan (stressed group) and psychiatrists working in non-COVID-19 settings (control group) were recruited. Participants completed questionnaires assessing depression (PHQ-9), generalized anxiety (GAD-7), PTSD (PCL-5), and general mental disorder symptoms (SRQ-20). Blood samples were collected at baseline, one month, six months, and one year post-stress exposure to measure levels of immune mediators (MCP-1, IL-1β, IL-2, IL-4, IL-6, TGF-α, TNF-α, BDNF, CCL5) and cortisol using multiplex assays and ELISA. fMRI scans were performed on a subset of participants (37 stressed, 35 controls) to assess functional connectivity of brain regions, particularly the red nucleus (RN). **Animal Experiments (Rats):** Male and female rats were subjected to restraint stress (2h/day for 3 days). Behavioral tests (open field test, elevated plus maze test) were used to assess anxiety-like behavior. Blood samples were collected to measure CCL5 and corticosterone levels via ELISA. Cervical lymph nodes (CLNs) were surgically removed in a separate group to determine their role in CCL5 production. Flow cytometry and intracellular cytokine staining were conducted to examine CD4+ and CD8+ lymphocyte counts in blood and CLNs. RNA sequencing (RNA-seq) was used to identify differentially expressed genes in CLNs of stressed rats. To determine the upstream brain regions involved, retrograde transsynaptic PRV tracing was employed. Chemogenetic techniques (using AAVs expressing hM4D(Gi) for inhibition and hM3D(Gq) for activation) were used to manipulate the activity of RN glutamatergic neurons and assess their impact on anxiety-like behavior and CCL5 levels. Fiber photometry was used to monitor calcium signaling in RN glutamatergic neurons in freely behaving rats. Finally, chemogenetic inhibition of the M1-RN pathway was also examined. Anisomycin, a protein synthesis inhibitor, was used to examine the effect of translation processes.

The study yielded several key findings: 1. **Elevated CCL5 in Stressed Individuals:** Clinically stressed individuals showed significantly higher CCL5 levels in their peripheral blood one month after exposure to stress compared to controls. This elevation subsided after six months. 2. **CCL5 Production in CLNs:** The researchers found that CLNs were the primary source of stress-induced CCL5, as evidenced by the effect of lymphadenectomy. 3. **Red Nucleus (RN) Involvement:** Retrograde tracing identified glutamatergic neurons in the RN projecting to CLNs. The activity of these neurons was significantly correlated with CCL5 levels and anxiety-like behavior in male rats. 4. **RN Manipulation Affects Anxiety and CCL5:** Chemogenetic inhibition of RN glutamatergic neurons increased anxiety-like behavior and serum/CLN CCL5 levels in rats. Conversely, chemogenetic activation of these neurons reduced anxiety and CCL5 production after restraint stress. 5. **M1-RN Circuit's Role:** Inhibition of the projection from the primary motor cortex (M1) to the RN also induced anxiety-like behavior and increased CCL5 synthesis, highlighting the importance of this neural circuit. 6. **Weakened RN Functional Connectivity:** fMRI analysis of stressed individuals revealed significantly decreased functional connectivity of the RN compared to controls. 7. **Translation's Role:** RNA sequencing showed upregulation of translation initiation factors in CLNs from stressed rats. Inhibition of translation by anisomycin reduced CCL5 levels in CLNs and attenuated anxiety-like behavior. 8. **Norepinephrine Increase:** Norepinephrine levels in CLNs were significantly increased in both stressed rats and rats with RN inhibition. Systemic neutralization of CCL5 reversed anxiety induced by RN inhibition.

This study demonstrates a novel brain-lymph node axis involving the red nucleus (RN) that influences stress-induced anxiety and inflammation. The findings directly address the gap in knowledge regarding the specific brain regions and neuronal types involved in the neuroimmune interaction impacting anxiety. The causal demonstration of the RN's role in modulating CLN activity and anxiety-like behavior is a significant contribution. The correlation between RN activity and CCL5 levels, the effects of manipulating RN neuronal activity on both anxiety and inflammation, and the supplementary fMRI data showing altered functional connectivity in stressed humans all strongly support this central role. The involvement of the M1-RN pathway further expands our understanding of the neural circuits underlying anxiety. These findings suggest potential therapeutic targets for interventions in stress-induced anxiety disorders, particularly focusing on the RN glutamatergic neurons and the translation processes within CLNs.

The study provides compelling evidence for a crucial role of the red nucleus (RN) in mediating stress-induced anxiety-like behavior and inflammation. The RN glutamatergic neurons identified are key regulators of CCL5 production in cervical lymph nodes (CLNs). Manipulating RN activity directly influences both anxiety and the inflammatory response. This work opens up exciting avenues for developing targeted therapies for stress-related anxiety disorders. Future research should investigate the precise mechanisms of communication between the RN and CLNs and explore sex-specific differences in this pathway.

The study has some limitations. The blood samples from stressed psychiatrists were collected one month after their return from stressful environments, potentially missing some early-phase changes. The observed decrease in cortisol levels in the stressed group requires further investigation. The study primarily focused on male rats, requiring further investigation to understand sex-specific differences. The fMRI analysis was conducted on a relatively small sample size, and the effects observed were subtle. The exact mechanism of communication between the RN and CLNs remains unclear.