Circulating myeloid-derived MMP8 in stress susceptibility and depression.

Psychosocial stress has profound effects on the body, including the immune system and the brain1,2. Although a large number of pre-clinical and clinical studies have linked peripheral immune system alterations to stress-related disorders such as major depressive disorder (MDD)3, the underlying mechanisms are not well understood. Here we show that expression of a circulating myeloid cell-specific proteinase, matrix metalloproteinase 8 (MMP8), is increased in the serum of humans with MDD as well as in stress-susceptible mice following chronic social defeat stress (CSDS). In mice, we show that this increase leads to alterations in extracellular space and neurophysiological changes in the nucleus accumbens (NAc), as well as altered social behaviour. Using a combination of mass cytometry and single-cell RNA sequencing, we performed high-dimensional phenotyping of immune cells in circulation and in the brain and demonstrate that peripheral monocytes are strongly affected by stress. In stress-susceptible mice, both circulating monocytes and monocytes that traffic to the brain showed increased Mmp8 expression following chronic social defeat stress. We further demonstrate that circulating MMP8 directly infiltrates the NAc parenchyma and controls the ultrastructure of the extracellular space. Depleting MMP8 prevented stress-induced social avoidance behaviour and alterations in NAc neurophysiology and extracellular space. Collectively, these data establish a mechanism by which peripheral immune factors can affect central nervous system function and behaviour in the context of stress. Targeting specific peripheral immune cell-derived matrix metalloproteinases could constitute novel therapeutic targets for stress-related neuropsychiatric disorders.

Social stress induces autoimmune responses against the brain.

Clinical studies have revealed a high comorbidity between autoimmune diseases and psychiatric disorders, including major depressive disorder (MDD). However, the mechanisms connecting autoimmunity and depression remain unclear. Here, we aim to identify the processes by which stress impacts the adaptive immune system and the implications of such responses to depression. To examine this relationship, we analyzed antibody responses and autoimmunity in the chronic social defeat stress (CSDS) model in mice, and in clinical samples from patients with MDD. We show that socially stressed mice have elevated serum antibody concentrations. We also confirm that social stress leads to the expansion of specific T and B cell populations within the cervical lymph nodes, where brain-derived antigens are preferentially delivered. Sera from stress-susceptible (SUS) mice exhibited high reactivity against brain tissue, and brain-reactive immunoglobulin G (IgG) antibody levels positively correlated with social avoidance behavior. IgG antibody concentrations in the brain were significantly higher in SUS mice than in unstressed mice, and positively correlated with social avoidance. Similarly, in humans, increased peripheral levels of brain-reactive IgG antibodies were associated with increased anhedonia. In vivo assessment of IgG antibodies showed they largely accumulate around blood vessels in the brain only in SUS mice. B cell-depleted mice exhibited stress resilience following CSDS, confirming the contribution of antibody-producing cells to social avoidance behavior. This study provides mechanistic insights connecting stress-induced autoimmune reactions against the brain and stress susceptibility. Therapeutic strategies targeting autoimmune responses might aid in the treatment of patients with MDD featuring immune abnormalities.

Chemokine receptor 5 signaling in PFC mediates stress susceptibility in female mice.

Chronic stress induces changes in the periphery and the central nervous system (CNS) that contribute to neuropathology and behavioral abnormalities associated with psychiatric disorders. In this study, we examined the impact of peripheral and central inflammation during chronic social defeat stress (CSDS) in female mice. Compared to male mice, we found that female mice exhibited heightened peripheral inflammatory response and identified C-C motif chemokine ligand 5 (CCL5), as a stress-susceptibility marker in females. Blocking CCL5 signaling in the periphery promoted resilience to CSDS. In the brain, stress-susceptible mice displayed increased expression of C-C chemokine receptor 5 (CCR5), a receptor for CCL5, in microglia in the prefrontal cortex (PFC). This upregulation was associated with microglia morphological changes, their increased migration to the blood vessels, and enhanced phagocytosis of synaptic components and vascular material. These changes coincided with neurophysiological alterations and impaired blood-brain barrier (BBB) integrity. By blocking CCR5 signaling specifically in the PFC were able to prevent stress-induced physiological changes and rescue social avoidance behavior. Our findings are the first to demonstrate that stress-mediated dysregulation of the CCL5-CCR5 axis triggers excessive phagocytosis of synaptic materials and neurovascular components by microglia, resulting in disruptions in neurotransmission, reduced BBB integrity, and increased stress susceptibility. Our study provides new insights into the role of cortical microglia in female stress susceptibility and suggests that the CCL5-CCR5 axis may serve as a novel sex-specific therapeutic target for treating psychiatric disorders in females.

Acetate supplementation rescues social deficits and alters transcriptional regulation in prefrontal cortex of Shank3 deficient mice.

BACKGROUND: The pathophysiology of autism spectrum disorder (ASD) involves genetic and environmental factors. Mounting evidence demonstrates a role for the gut microbiome in ASD, with signaling via short-chain fatty acids (SCFA) as one mechanism. Here, we utilize mice carrying deletion to exons 4-22 of Shank3 (Shank3KO) to model gene by microbiome interactions in ASD. We identify SCFA acetate as a mediator of gut-brain interactions and show acetate supplementation reverses social deficits concomitant with alterations to medial prefrontal cortex (mPFC) transcriptional regulation independent of microbiome status. METHODS: Shank3KO and wild-type (Wt) littermates were divided into control, Antibiotic (Abx), Acetate and Abx + Acetate groups upon weaning. After six weeks, animals underwent behavioral testing. Molecular analysis including 16S and metagenomic sequencing, metabolomic and transcriptional profiling were conducted. Additionally, targeted serum metabolomic data from Phelan McDermid Syndrome (PMS) patients (who are heterozygous for the Shank3 gene) were leveraged to assess levels of SCFA's relative to ASD clinical measures. RESULTS: Shank3KO mice were found to display social deficits, dysregulated gut microbiome and decreased cecal levels of acetate - effects exacerbated by Abx treatment. RNA-sequencing of mPFC showed unique gene expression signature induced by microbiome depletion in the Shank3KO mice. Oral treatment with acetate reverses social deficits and results in marked changes in gene expression enriched for synaptic signaling, pathways among others, even in Abx treated mice. Clinical data showed sex specific correlations between levels of acetate and hyperactivity scores. CONCLUSION: These results suggest a key role for the gut microbiome and the neuroactive metabolite acetate in regulating ASD-like behaviors.

Central regulation of stress-evoked peripheral immune responses.

Stress-linked psychiatric disorders, including anxiety and major depressive disorder, are associated with systemic inflammation. Recent studies have reported stress-induced alterations in haematopoiesis that result in monocytosis, neutrophilia, lymphocytopenia and, consequently, in the upregulation of pro-inflammatory processes in immunologically relevant peripheral tissues. There is now evidence that this peripheral inflammation contributes to the development of psychiatric symptoms as well as to common co-morbidities of psychiatric disorders such as metabolic syndrome and immunosuppression. Here, we review the specific brain and spinal regions, and the neuronal populations within them, that respond to stress and transmit signals to peripheral tissues via the autonomic nervous system or neuroendocrine pathways to influence immunological function. We comprehensively summarize studies that have employed retrograde tracing to define neurocircuits linking the brain to the bone marrow, spleen, gut, adipose tissue and liver. Moreover, we highlight studies that have used chemogenetic or optogenetic manipulation or intracerebroventricular administration of peptide hormones to control somatic immune responses. Collectively, this growing body of literature illustrates potential mechanisms through which stress signals are conveyed from the CNS to immune cells to regulate stress-relevant behaviours and comorbid pathophysiology.

Peripheral immune-derived matrix metalloproteinase promotes stress susceptibility.

Psychosocial stress has profound effects on the body, including the peripheral immune system and the brain1,2. Although a large number of pre-clinical and clinical studies have linked peripheral immune system alterations to stress-related disorders such as major depressive disorder (MDD)3,4,5, the underlying mechanisms are not well understood. Here we show that a peripheral myeloid cell-specific proteinase, matrix metalloproteinase 8 (MMP8), is elevated in serum of subjects with MDD as well as in stress-susceptible (SUS) mice following chronic social defeat stress (CSDS). In mice, we show that this increase leads to alterations in extracellular space and neurophysiological changes in the nucleus accumbens (NAc), thereby altering social behaviour. Using a combination of mass cytometry and single-cell RNA-sequencing, we performed high-dimensional phenotyping of immune cells in circulation and brain and demonstrate that peripheral monocytes are strongly affected by stress. Both peripheral and brain-infiltrating monocytes of SUS mice showed increased Mmp8 expression following CSDS. We further demonstrate that peripheral MMP8 directly infiltrates the NAc parenchyma to control the ultrastructure of the extracellular space. Depleting MMP8 prevented stress-induced social avoidance behaviour and alterations in NAc neurophysiology and extracellular space. Collectively, these data establish a novel mechanism by which peripheral immune factors can affect central nervous system function and behaviour in the context of stress. Targeting specific peripheral immune cell-derived matrix metalloproteinases could constitute novel therapeutic targets for stress-related neuropsychiatric disorders.

Social trauma engages lateral septum circuitry to occlude social reward.

In humans, traumatic social experiences can contribute to psychiatric disorders1. It is suggested that social trauma impairs brain reward function such that social behaviour is no longer rewarding, leading to severe social avoidance2,3. In rodents, the chronic social defeat stress (CSDS) model has been used to understand the neurobiology underlying stress susceptibility versus resilience following social trauma, yet little is known regarding its impact on social reward4,5. Here we show that, following CSDS, a subset of male and female mice, termed susceptible (SUS), avoid social interaction with non-aggressive, same-sex juvenile C57BL/6J mice and do not develop context-dependent social reward following encounters with them. Non-social stressors have no effect on social reward in either sex. Next, using whole-brain Fos mapping, in vivo Ca2+ imaging and whole-cell recordings, we identified a population of stress/threat-responsive lateral septum neurotensin (NTLS) neurons that are activated by juvenile social interactions only in SUS mice, but not in resilient or unstressed control mice. Optogenetic or chemogenetic manipulation of NTLS neurons and their downstream connections modulates social interaction and social reward. Together, these data suggest that previously rewarding social targets are possibly perceived as social threats in SUS mice, resulting from hyperactive NTLS neurons that occlude social reward processing.

Whole blood transcriptional signatures associated with rapid antidepressant response to ketamine in patients with treatment resistant depression.

Ketamine has rapid and sustained antidepressant effects in patients with treatment-resistant depression (TRD). However, the underlying mechanisms of action are not well understood. There is increasing evidence that TRD is associated with a pro-inflammatory state and that ketamine may inhibit inflammatory processes. We thus investigated whole blood transcriptional profiles related to TRD and gene expression changes associated with treatment response to ketamine. Whole blood was collected at baseline (21 healthy controls [HC], 26 patients with TRD) and then again in patients with TRD 24 hours following a single intravenous infusion of ketamine (0.5 mg/kg). We performed RNA-sequencing and analyzed (a) baseline transcriptional profiles between patients with TRD and HC, (b) responders vs. non-responders before ketamine treatment, and (c) gene expression signatures associated with clinical improvement. At baseline, patients with TRD compared to HC showed a gene expression signature indicative of interferon signaling pathway activation. Prior to ketamine administration, the metabotropic glutamate receptor gene GRM2 and the ionotropic glutamate receptor gene GRIN2D were upregulated in responders compared to non-responders. Response to ketamine was associated with a distinct transcriptional signature, however, we did not observe gene expression changes indicative of an anti-inflammatory effect. Future studies are needed to determine the role of the peripheral immune system in the antidepressant effect of ketamine.

Individual history of winning and hierarchy landscape influence stress susceptibility in mice.

Social hierarchy formation is strongly evolutionarily conserved. Across species, rank within social hierarchy has large effects on health and behavior. To investigate the relationship between social rank and stress susceptibility, we exposed ranked male and female mice to social and non-social stressors and manipulated social hierarchy position. We found that rank predicts same sex social stress outcomes: dominance in males and females confers resilience while subordination confers susceptibility. Pre-existing rank does not predict non-social stress outcomes in females and weakly does so in males, but rank emerging under stress conditions reveals social interaction deficits in male and female subordinates. Both history of winning and rank of cage mates affect stress susceptibility in males: rising to the top rank through high mobility confers resilience and mice that lose dominance lose stress resilience, although gaining dominance over a subordinate animal does not confer resilience. Overall, we have demonstrated a relationship between social status and stress susceptibility, particularly when taking into account individual history of winning and the overall hierarchy landscape in male and female mice.

Sex-specific peripheral and central responses to stress-induced depression and treatment in a mouse model.

Major depressive disorder affects ~20% of the world population and is characterized by strong sexual dimorphism with females being two to three times more likely to develop this disorder. Previously, we demonstrated that a combination therapy with dihydrocaffeic acid and malvidin-glucoside to synergistically target peripheral inflammation and stress-induced synaptic maladaptation in the brain was effective in alleviating chronic social defeat stress (CSDS)-induced depression-like phenotype in male mice. Here, we test the combination therapy in a female CSDS model for depression and compared sex-specific responses to stress in the periphery and the central nervous system. Similar to male mice, the combination treatment is also effective in promoting resilience against the CSDS-induced depression-like behavior in female mice. However, there are sex-specific differences in peripheral immune responses and differential gene regulation in the prefrontal cortex to chronic stress and to the treatment. These data indicate that while therapeutic approaches to combat stress-related disorders may be effective in both sexes, the mechanisms underlying these effects differ, emphasizing the need for inclusion of both sexes in preclinical studies using animal models.

Orexin signaling in GABAergic lateral habenula neurons modulates aggressive behavior in male mice.

Heightened aggression is characteristic of multiple neuropsychiatric disorders and can have various negative effects on patients, their families and the public. Recent studies in humans and animals have implicated brain reward circuits in aggression and suggest that, in subsets of aggressive individuals, domination of subordinate social targets is reinforcing. In this study, we showed that, in male mice, orexin neurons in the lateral hypothalamus activated a small population of glutamic acid decarboxylase 2 (GAD2)-expressing neurons in the lateral habenula (LHb) via orexin receptor 2 (OxR2) and that activation of these GAD2 neurons promoted male-male aggression and conditioned place preference for aggression-paired contexts. Moreover, LHb GAD2 neurons were inhibitory within the LHb and dampened the activity of the LHb as a whole. These results suggest that the orexin system is important for the regulation of inter-male aggressive behavior and provide the first functional evidence of a local inhibitory circuit within the LHb.

Nucleotides released from palmitate-activated murine macrophages attract neutrophils.

Obesity and elevation of circulating free fatty acids are associated with an accumulation and proinflammatory polarization of macrophages within metabolically active tissues, such as adipose tissue, muscle, liver, and pancreas. Beyond macrophages, neutrophils also accumulate in adipose and muscle tissues during high-fat diets and contribute to a state of local inflammation and insulin resistance. However, the mechanisms by which neutrophils are recruited to these tissues are largely unknown. Here we used a cell culture system as proof of concept to show that, upon exposure to a saturated fatty acid, palmitate, macrophages release nucleotides that attract neutrophils. Moreover, we found that palmitate up-regulates pannexin-1 channels in macrophages that mediate the attraction of neutrophils, shown previously to allow transfer of nucleotides across membranes. These findings suggest that proinflammatory macrophages release nucleotides through pannexin-1, a process that may facilitate neutrophil recruitment into metabolic tissues during obesity.

Role of Monocyte-Derived MicroRNA106b∼25 in Resilience to Social Stress.

BACKGROUND: Clinical studies suggest that heightened peripheral inflammation contributes to the pathogenesis of stress-related disorders, including major depressive disorder. However, the molecular mechanisms within peripheral immune cells that mediate enhanced stress vulnerability are not well known. Because microRNAs (miRs) are important regulators of immune response, we sought to examine their role in mediating inflammatory and behavioral responses to repeated social defeat stress (RSDS), a mouse model of stress vulnerability that produces susceptible and resilient phenotypes. METHODS: We isolated Ly6chigh monocytes via fluorescence-activated cell sorting in the blood of susceptible and resilient mice following RSDS and profiled miR expression via quantitative real-time polymerase chain reaction. Bone marrow chimeric mice were generated to confirm a causal role of the miR-106b∼25 cluster in bone marrow-derived leukocytes in mediating stress resilience versus susceptibility. RESULTS: We found that RSDS produces an increase in circulating Ly6chigh inflammatory monocytes in both susceptible and resilient mice. We next investigated whether intrinsic leukocyte posttranscriptional mechanisms contribute to individual differences in stress response and the resilient phenotype. Of the miRs profiled in our panel, eight were significantly regulated by RSDS within Ly6chigh monocytes, including miR-25-3p, a member of the miR-106b∼25 cluster. Selective knockout of the miR-106b∼25 cluster in peripheral leukocytes promoted behavioral resilience to RSDS. CONCLUSIONS: Our results identify the miR-106b∼25 cluster as a key regulator of stress-induced inflammation and depression that may represent a novel therapeutic target for drug development.

Central and Peripheral Inflammation Link Metabolic Syndrome and Major Depressive Disorder.

Metabolic syndrome and major depression are two of the most common and debilitating disorders worldwide, occurring with significant rates of comorbidity. Recent studies have uncovered that each of these conditions is associated with chronic, low-grade inflammation. This is characterized by increased circulating pro-inflammatory cytokines, altered leukocyte population frequencies in blood, accumulation of immune cells in tissues including the brain, and activation of these immune cells. Cytokines that become elevated during obesity can contribute to the progression of metabolic syndrome by directly causing insulin resistance. During chronic stress, there is evidence that these cytokines promote depression-like behavior by disrupting neurotransmitter synthesis and signal transduction. Animal models of obesity and depression have revealed a bi-directional relationship whereby high-fat feeding and chronic stress synergize and exacerbate metabolic dysregulation and behavioral abnormalities. Although far from conclusive, emerging evidence suggests that inflammation in the central and peripheral immune system may link metabolic syndrome to major depressive disorder. In this review, we will synthesize available data supporting this view and identify critical areas for future investigation.

A diet enriched with curcumin promotes resilience to chronic social defeat stress.

Chronic exposure to stress is a well-known risk factor for the development of mood and anxiety disorders. Promoting resilience to stress may prevent the development of these disorders, but resilience-enhancing compounds are not yet clinically available. One compound that has shown promise in the clinical setting is curcumin, a polyphenol compound found in the rhizome of the turmeric plant (Curcuma longa) with known anti-inflammatory and antidepressant properties. Here, we tested the efficacy of 1.5% dietary curcumin at promoting resilience to chronic social defeat stress (CSDS) in 129/SvEv mice, a strain that we show is highly susceptible to this type of stress. We found that administration of curcumin during CSDS produced a 4.5-fold increase in stress resilience, as measured by the social interaction test. Although the overall effects of curcumin were striking, we identified two distinct responses to curcumin. While 64% of defeated mice on curcumin were resilient (responders), the remaining 36% of mice were susceptible to the effects of stress (non-responders). Interestingly, responders released less corticosterone following acute restraint stress and had lower levels of peripheral IL-6 than nonresponders, implicating a role for the NF-κB pathway in treatment response. Importantly, curcumin also prevented anxiety-like behavior in both responders and non-responders in the elevated-plus maze and open field test. Collectively, our findings provide the first preclinical evidence that curcumin promotes resilience to CSDS and suggest that curcumin may prevent the emergence of a range of anxiety-like symptoms when given to individuals during exposure to chronic social stress.

Interesterified soybean oil promotes weight gain, impaired glucose tolerance and increased liver cellular stress markers.

Interesterified fats have largely replaced hydrogenated vegetable fat, which is rich in trans fatty acids, in the food industry as an economically viable alternative, generating interest to study their health effects. The aim of this study was to evaluate the effect that interesterification of oils and fat has on lipid-induced metabolic dysfunction, hepatic inflammation and ER stress. Five week-old male Wistar rats were randomly divided into three experimental groups, submitted to either normocaloric and normolipidic diet containing 10% of lipids from unmodified soybean oil (SO) or from interesterified soybean oil (ISO), and one more group submitted to a high fat diet (HFD) containing 60% of fat from lard as a positive control, for 8 or 16 weeks. Metabolic parameters and hepatic gene expression were evaluated. The HFD consumption led to increased body mass, adiposity and impaired glucose tolerance compared to SO and ISO at both time points of diet. However, the ISO group showed an increased body mass gain, retroperitoneal WAT mass, fasting glucose, and impaired glucose tolerance during ipGTT at 16 weeks compared to SO. Moreover, at 8 weeks, hepatic gene expression of Atf3 and Tnf were increased in the ISO group compared to the SO group. Thus, replacement of natural fat with interesterified fat on a normocaloric and normolipidic diet negatively modulated metabolic parameters and resulted in impaired glucose tolerance in rats.

Circulating NOD1 Activators and Hematopoietic NOD1 Contribute to Metabolic Inflammation and Insulin Resistance.

Insulin resistance is a chronic inflammatory condition accompanying obesity or high fat diets that leads to type 2 diabetes. It is hypothesized that lipids and gut bacterial compounds in particular contribute to metabolic inflammation by activating the immune system; however, the receptors detecting these "instigators" of inflammation remain largely undefined. Here, we show that circulating activators of NOD1, a receptor for bacterial peptidoglycan, increase with high fat feeding in mice, suggesting that NOD1 could be a critical sensor leading to metabolic inflammation. Hematopoietic depletion of NOD1 did not prevent weight gain but protected chimeric mice against diet-induced glucose and insulin intolerance. Mechanistically, while macrophage infiltration of adipose tissue persisted, notably these cells were less pro-inflammatory, had lower CXCL1 production, and consequently, lower neutrophil chemoattraction into the tissue. These findings reveal macrophage NOD1 as a cell-specific target to combat diet-induced inflammation past the step of macrophage infiltration, leading to insulin resistance.

Deconstructing metabolic inflammation using cellular systems.

Over the past years, we have embarked in a systematic analysis of the effect of obesity or fatty acids on circulating monocytes, microvascular endothelial cells, macrophages, and skeletal muscle cells. With the use of cell culture strategies, we have deconstructed complex physiological systems and then reconstructed "partial equations" to better understand cell-to-cell communication. Through these approaches, we identified that in high saturated fat environments, cell-autonomous proinflammatory pathways are activated in monocytes and endothelial cells, promoting monocyte adhesion and transmigration. We think of this as a paradigm of the conditions promoting immune cell infiltration into tissues during obesity. In concert, it is possible that muscle and adipose tissue secrete immune cell chemoattractants, and indeed, our tissue culture reconstructions reveal that myotubes treated with the saturated fatty acid palmitate, but not the unsaturated fatty acid palmitoleate, release nucleotides that attract monocytes and other compounds that promote proinflammatory classically activated "(M1)-like" polarization in macrophages. In addition, palmitate directly triggers an M1-like macrophage phenotype, and secretions from these activated macrophages confer insulin resistance to target muscle cells. Together, these studies suggest that in pathophysiological conditions of excess fat, the muscle, endothelial and immune cells engage in a synergistic crosstalk that exacerbates tissue inflammation, leukocyte infiltration, polarization, and consequent insulin resistance.

Saturated fatty acids activate caspase-4/5 in human monocytes, triggering IL-1ß and IL-18 release.

Obesity is associated with metabolic tissue infiltration by monocyte-derived macrophages. Saturated fatty acids contribute to proinflammatory gene induction in tissue-embedded immune cells. However, it is unknown how circulating monocytes, the macrophage precursors, react to high-fat environments. In macrophages, saturated fatty acids activate inflammatory pathways and, notably, prime caspase-associated inflammasomes. Inflammasome-activated IL-1ß contributes to type 2 diabetes. We hypothesized that 1) human monocytes from obese patients show caspase activation, and 2) fatty acids trigger this response and consequent release of IL-1ß/IL-18. Human peripheral blood monocytes were sorted by flow cytometry, and caspase activity was measured with a FLICA dye-based assay. Blood monocytes from obese individuals exhibited elevated caspase activity. To explore the nature and consequence of this activity, human THP1 monocytes were exposed to saturated or unsaturated fatty acids. Caspase activity was revealed by isoform-specific cleavage and enzymatic activity; cytokine expression/release was measured by qPCR and ELISA. Palmitate, but not palmitoleate, increased caspase activity in parallel to the release of IL-1ß and IL-18. Palmitate induced eventual monocyte cell death with features of pyroptosis (an inflammation-linked cell death program involving caspase-4/5), scored through LDH release, vital dye influx, cell volume changes, and nuclear morphology. Notably, selective gene silencing or inhibition of caspase-4/5 reduced palmitate-induced release of IL-1ß and IL-18. In summary, monocytes from obese individuals present elevated caspase activity. Mechanistically, palmitate activates a pyroptotic program in monocytes through caspase-4/5, causing inflammatory cytokine release, additional to inflammasomes. These caspases represent potential, novel, therapeutic targets to taper obesity-associated inflammation.