Brain, Behavior, and Immunity

Microbes residing in the gastrointestinal tract exert immunomodulatory impacts on the brain through the gut-brain axis. Short-chain fatty acids (SCFAs) produced by bacterial fermentation of dietary fiber can enter the brain parenchyma and are implicated in microglia-mediated inflammation. While the gut microbiome is required to maintain microglial homeostasis, the mechanisms by which microbiota-derived metabolites affect microglia remains unknown. We examined the roles of SCFAs, specifically butyrate, propionate and acetate, on microglial function in response to SCFAs both in vitro using BV2 cells and in vivo in mice. We observed in vivo that SCFAs impact microglial transcriptional responses to LPS in a sex- and metabolite-specific manner with butyrate having the strongest effect. Enriched gene sets included signatures associated with LPS responsive microglia, Arg1 positive microglia, microglial cell cycle related genes and genes affiliated with changes in microglial morphology. We observed a similar effect in vitro, where metabolite administration enhanced phagocytosis, blunted proliferation and nitric oxide production. We then evaluated global histone modification levels following metabolite treatment and detected an enhancement of H3K9ac, H3K27ac, and H3K4me3 both in vivo and in BV2 cells treated with butyrate. Finally, we showed that butyrate is a potent HDAC inhibitor possibly contributing to enhanced acetylation. Hence, our findings suggest that SCFAs impact microglial function in a metabolite- and sex-specific manner, and that butyrate blunts inflammation by regulating microglial histone acetylation. Our results provide a more in-depth understanding of gut microbiome-microglia crosstalk, opening the door for new microbiome- and microglia-targeted therapies.

Decreases in social behavior are a hallmark aspect of acute "sickness behavior" in response to infection. However, immune insults that occur during the perinatal period may have long-lasting consequences for adult social behavior by impacting the developmental organization of underlying neural circuits. Microglia, the resident immune cells of the central nervous system, are sensitive to immune stimulation and play a critical role in the developmental sculpting of neural circuits, making them likely mediators of this process. Here, we investigated the impact of a postnatal day (PND) 4 lipopolysaccharide (LPS) challenge on social behavior in adult mice. Somewhat surprisingly, neonatal LPS treatment decreased sociability in adult female, but not male mice. LPS-treated females also displayed reduced social interaction and social memory in a social discrimination task as compared to saline-treated females. Somatostatin (SST) interneurons within the anterior cingulate cortex (ACC) have recently been suggested to modulate a variety of social behaviors. Interestingly, the female-specific changes in social behavior observed here were accompanied by an increase in SST interneuron number in the ACC. Finally, these changes in social behavior and SST cell number do not appear to depend on microglial inflammatory signaling, because microglia-specific genetic knock-down of myeloid differentiation response protein 88 (MyD88; the removal of which prevents LPS from increasing proinflammatory cytokines such as TNF and IL-1{beta}) did not prevent these LPS-induced changes. This study provides novel evidence for enduring effects of neonatal immune activation on social behavior and SST interneurons in females, independent of microglial inflammatory signaling.

Chronic stress is a significant risk factor for the development and recurrence of anxiety disorders. Chronic stress impacts the immune system, causing microglial functional alterations in the medial prefrontal cortex (mPFC), a brain region involved in the pathogenesis of anxiety. High mobility group box 1 protein (HMGB1) is an established modulator of neuronal firing and a potent pro-inflammatory stimulus released from neuronal and non-neuronal cells following stress. HMGB1, in the context of stress, acts as a danger-associated molecular pattern (DAMP), instigating robust proinflammatory responses throughout the brain, so much so that localized drug delivery of HMGB1 alters behavior in the absence of any other forms of stress, i.e., social isolation, or behavioral stress models. Few studies have investigated the molecular mechanisms that underlie HMGB1-associated behavioral effects in a cell-specific manner. The aim of this study is to investigate cellular and molecular mechanisms underlying HMGB1-induced behavioral dysfunction with regard to cell-type specificity and potential sex differences. Here, we report that both male and female mice exhibited anxiety-like behavior following increased HMGB1 in the mPFC as well as changes in microglial morphology. Interestingly, our results demonstrate that HMGB1-induced anxiety may be mediated by distinct microglial MyD88-dependent mechanisms in females compared to males. This study supports the hypothesis that MyD88 signaling in microglia may be a crucial mediator of the stress response in adult female mice.

Microglia cells are increasingly recognized to contribute to brain health and disease. Preclinical studies using laboratory rodents are essential to advance our understanding of the physiological and pathophysiological functions of these cells in the central nervous system. Rodents are nocturnal animals, and they are mostly maintained in a defined light-dark cycle within animal facilities, with many laboratories investigating microglial molecular and functional profiles during the animals light (sleep) phase. However, only a few studies have considered possible differences in microglial functions between the active and sleep phases. Based on initial evidence suggesting that microglial intrinsic clock genes can affect their phenotype, we sought to investigate differences in transcriptional, proteotype and functional profiles of microglia between light (sleep) and dark (active) phases, and how these changes are affected in pathological models. We found marked transcriptional and proteotype differences between microglia harvested during the light or dark phase. Amongst others, these differences related to genes and proteins associated with immune responses, motility, and phagocytosis, which were reflected by functional alterations in microglial synaptic pruning and response to bacterial stimuli. Possibly accounting for such circadian changes, we found RNA and protein regulation in SWI/SNF and NuRD chromatin remodeling complexes between light and dark phases. Importantly, we show that microglial circadian transcriptional changes are impaired in a model of immune-mediated neurodevelopmental disorders. Our findings emphasize the importance of considering circadian factors in studying microglial cells and indicate that implementing a circadian perspective is pivotal for advancing our understanding of their physiological and pathophysiological roles in brain health and disease. This may also open novel avenues towards therapeutic strategies for modulating microglial functions during specific windows of the active or sleep phase.

Ulcerative colitis (UC) is a chronic inflammatory disease characterized by colonic inflammation and bloody diarrhoea. Accumulating evidence suggests that UC not only affects the intestinal tract, but also distant organs including the brain. Environmental factors are key determinants of the disease course, yet the impact and potential disease modifying effects of living environment complexity on microbiota-gut-brain axis signalling during colitis remain unclear. To address this gap, we investigated how enhanced environmental complexity (EC) affects the disease course and gut-brain axis signalling during experimental colitis in mice. Our results show that EC exacerbates dextran sulphate sodium (DSS)-induced colitis in female mice, but not in male mice, as evidenced by greater weight loss and higher disease activity. Immune cell profiling across the gut-brain axis reveals strong effects of DSS treatment on colonic, circulating and brain immune cell populations and a restriction of central nervous system (CNS) T cell infiltration due to EC. In addition, female EC/DSS mice have higher circulating corticosterone levels than controls indicating chronic stress. Metabolomics across the gut-brain axis revealed that EC exacerbates colitis-induced metabolite perturbations in plasma, brain tissue, brain interstitial and cerebrospinal fluid. Notably, microbiota-derived metabolites, including deoxycholic acid and trimethylamine-N-oxide (TMAO), are increased in EC/DSS mice, concordant with EC-associated microbiome changes and anxiety-like behaviour. Overall, this study indicates that EC worsens experimental colitis in female mice and directs microbiota-gut-brain axis signalling during colitis towards a less favourable state. From a translational perspective, this study highlights the importance of environmental factors for a sex-specific disease course of UC and associated neurobehavioral comorbidities.

Social animals are faced with a critical decision when sick: to affiliate or withdraw. The behavioural response during an immune challenge varies by individual and species. However, the social and biological factors leading to variation in this decision during an immune challenge are unknown. Here, we explore the affiliative behavioural phenotype, "huddlers" and "non-huddlers," as a social predictor, and microglial activation and peripheral cytokine expression as biological predictors of social-decision making during an immune challenge. To measure social-decision making, we recorded social approach and time spent in proximity to a familiar same-sex conspecific versus a novel object in the social California mouse following lipopolysaccharide (LPS) treatment. Behavioural phenotype predicted both the social response to LPS and the relationship between cytokine activity and social behaviour, suggesting that social experience regulates social decision-making during sick versus healthy conditions. Additionally, we found that elevated microglia activity in the dentate gyrus (DG) and medial prefrontal cortex (mPFC) negatively correlated with social behaviour, positioning these regions for future exploration for the role of microglia on social decision-making. Our results identify the predictive power of behavioural phenotypes on social response to sickness and a link between microglia and decision-making during sickness.

Neuroinflammation contributes to psychiatric disorders, but preventive strategies targeting brain immune cells remain unexplored. Here we demonstrate that low-dose lipopolysaccharide (LPS) preconditioning prevents systemic inflammation-induced behavioral abnormalities through microglia-dependent mechanisms in male mice. Mice received preconditioning with 0.2 mg/kg LPS or saline for two consecutive days, followed by high-dose LPS challenge (5 mg/kg) or saline seven days later. Behavioral assessment revealed that preconditioning specifically prevented social preference deficits induced by systemic inflammation (preference score: -0.49{+/-}0.19 vs 0.14{+/-}0.10, p<0.01), while showing limited effects on locomotor activity and depression-like behaviors. Additionally, LPS preconditioning prevented anxiety-like behavior in a chronic corticosterone model and attenuated hippocampal inflammatory gene expression. Immunohistochemical analysis demonstrated that preconditioning suppressed microglial activation in hippocampal CA1 region, particularly reducing PBR/IBA1 ratio (37.5{+/-}2.4% vs 27.6{+/-}2.8%, p<0.01), with less pronounced effects in CA3. Critically, pharmacological microglial depletion using PLX3397 during the preconditioning period completely abolished these protective effects, establishing the causal role of microglia. Flow cytometric analysis revealed preconditioning-induced shifts in brain macrophage subpopulations defined by TMEM119 and CD45 expression patterns. Transcriptomic profiling identified subpopulation-specific responses, with one subset showing LPS-response pathway enrichment despite minimal gene expression changes, while another displayed extensive but functionally non-specific transcriptional alterations. These findings establish microglial preconditioning as a novel preventive strategy for neuroinflammation-induced social behavioral deficits and suggest potential therapeutic applications for psychiatric disorders involving neuroinflammatory components.

The ability to detect and respond to sickness in others promotes survival. Here we show that mouse dams respond to immune challenged pups by mirroring their inflammatory response. Thus, dams with pups subjected to immune challenge displayed a marked induction of inflammatory mediators in both the brain and the periphery, accompanied by an increase in maternal behaviors and corticosterone levels. This social transmission of inflammation did not require physical contact, and it contributed to the stress hormone response in the dams. In adult dyads, interaction with an immune challenged cagemate did not elicit robust inflammatory signaling but induced an increased responsiveness to a subsequent immune challenge. The identification of social transmission of inflammation, or inflammatory responsiveness, may open new avenues for research on social behavior, just like the description of similar phenomena such as observational fear and transmitted pain have done.

There are multiple reports of elevated inflammation in patients with major depression. It is, however, unclear whether these reported perturbations in immune functions in depression affect the general functionality of the peripheral immune system. Here, using single-cell RNAsequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) extracted from blood samples collected prior to a flu vaccination administration/immunization (at baseline), we found downregulated T cell and B-cell-associated gene expression repertoire coupled with a selectively upregulated plasmablast, CD14 classical monocytes, and natural killer (NK) proliferating cell-associated gene expression in 9 depressed patients (6 females) compared to 5 healthy controls (5 females), as well as in association with depression ratings. Our cell type proportion analysis revealed shifts in immune cell populations, specifically reduced numbers of CD4+ T and CD8+ T proliferating cells, plasmablasts, and NK cells in individuals with depression compared with controls. In contrast, we found increased numbers of CD14 classical and CD16 monocytes as well as doublet cells in depressed individuals compared with controls. Although our baseline and flu vaccine challenge did not show marked differences in peripheral immune markers measured by a multiplex cytokine assay, our results suggest impaired innate and adaptive immune responses at the transcriptomic and cellular population levels in depressed patients.

BackgroundAdverse life events and psychosocial stressors contribute to a range of neuropsychiatric disorders. However, the role of inflammatory dynamics around stress exposure remains unclear. Using TriNetX, a large international electronic health records database, we examined how systemic inflammatory activity and its temporal dynamics relate to subsequent risk of mental illness and somatic symptoms. MethodsWe compared 36,772 individuals with records of adverse life events and leukocytosis in the surrounding period with matched individuals with normal leukocyte counts, and performed an analogous comparison for socioeconomic and psychosocial stressors in cohorts of 87,936 individuals with leukocytosis and matched controls. To contrast dynamic with static inflammatory responses, we compared cohorts exhibiting leukocyte count changes with those maintaining persistently normal or persistently elevated leukocyte counts around stressor exposure. Outcomes, including new mental health and somatic symptom presentations, were evaluated within two years of the stressor. ResultsFollowing acute stressors, leukocytosis (compared with normal leukocyte counts) was associated with lower rates of subsequent anxiety disorders, cognitive symptoms and several somatic symptom diagnoses, with similar reductions in anxiety observed after chronic stressors. A dynamic inflammatory response was associated with the most favourable outcomes, with lower rates of anxiety, depression, functional neurological disorder, cognitive and sleep difficulties, fatigue, and multiple pain-related and other somatic symptoms than persistently low or high inflammation. ConclusionOur findings suggest that a well-regulated inflammatory response to stressors is associated with a reduced risk of diverse mental health and somatic outcomes, and that a transient immune activation may support recovery rather than confer risk.

The SARS-CoV-2 S1 protein is associated with immune cell activation and persistent neurological symptoms, yet the underlying mechanisms remain unclear, posing a major challenge in elucidating Long COVID pathophysiology. To investigate how circulating S1 contributes to long-term neurological alterations, we intravenously injected hACE2 mice with varying doses of S1 (5, 10, and 20 {micro}g) and observed temporally dysregulated systemic inflammatory responses accompanied by sub-acute CD4+ T cell infiltration into central limbic regions. This immune response induced mild sustained increases in cFos+ cells in the amygdala and mild neuroinflammation in the hippocampal CA1 region, resulting in both acute and long-term anxiety-like behaviors, while working memory remained unaffected. Together, these findings suggest that systemic S1 protein induces a sustained proinflammatory response that promotes lasting neurological alterations through immune-to-brain signaling pathways. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/733010v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@10982caorg.highwire.dtl.DTLVardef@169a275org.highwire.dtl.DTLVardef@28e66aorg.highwire.dtl.DTLVardef@12f6c85_HPS_FORMAT_FIGEXP M_FIG C_FIG

Increased production of cytokines in the in the brain during illness or injury modulates physiological processes, behavior, and cognitive function. It is likely that the pattern of cytokines, rather than the activation of any individual cytokine, determines the functional outcome of neuroimmune signaling. Cytokine networks may thus be particularly useful for understanding sex differences in immune and neuroimmune activation and outcomes. In this project, we aimed to determine the activation and resolution of hippocampal cytokine networks in both male and female mice. We measured 32 cytokines in the hippocampus and periphery of male and female mice at rest, 2, 6, 24, 48, and 168 hours after an acute systemic injection of lipopolysaccharide (LPS; 250g/kg). We hypothesized that males and females would exhibit both differences in individual cytokine levels and differences in network dynamics of hippocampal cytokines. Cytokines with sex-specific activation by LPS included male-specific elevations of IFN{gamma}, CSF1, CSF2, and IL-10; and female-specific activation of the IL-2 family and IL-4. We also observed differences in time course, where females showed more rapid elevations, and faster resolution of cytokine activity compared with males. Network analysis using ARACNE and Cytoscape demonstrated markedly different hippocampal cytokine networks across sex even at baseline, and sex differences in cytokine network activation states in response to LPS. Analysis of global shifts in cytokine concentrations further identified a period of cytokine and chemokine downregulation at 48 hours that was more pronounced in females compared with males. Together, these findings demonstrate that sex differences in neuroimmune responses include both differences in intensity of the cytokine response, and importantly differences in cytokine networks activated. Such sex differences in cytokine networks in the brain are likely critical for short and long-term functional outcomes associated with neuroimmune activation.

CX3CR1-Cre mouse lines have produced important advancements in our understanding of microglial biology. Recent studies have demonstrated the adverse effects of tamoxifen-induced CX3CR1-Cre expression during development, which include changes in microglial density, phenotype, and DNA damage, as well as anxiety-like behavior. However, the unintended effects of constitutive CX3CR1-BAC-Cre expression remain unexplored. Here, we characterized the effects of CX3CR1-BAC-Cre expression on microglia in CX3CR1-BAC-Cre+/- and CX3CR1-BAC-Cre-/-male and female littermates during early postnatal development and adulthood in multiple brain regions. Additionally, we performed anxiety-like behavior tests to assess changes caused by Cre expression. We found that CX3CR1-BAC-Cre expression causes subtle region- and sex-specific changes in microglial density, volume, and morphology during development, but these changes normalized by adulthood in all brain regions except the hippocampus. No behavioral effects were found. Our findings suggest that the constitutive-Cre model might be less detrimental than the inducible model, and highlight the need for proper controls.

Hippocampal neuroinflammation is present in multiple diseases and disorders that impact motivated behaviour in a sex-specific manner, but whether neuroinflammation alone is sufficient to disrupt such behaviour is unknown. We investigated this question here using mice. First, the application of an endotoxin to primary cultures containing only hippocampal neurons did not affect their activation. However, when the same endotoxin was applied to mixed neuronal/glial cultures it did increase neuronal activation, providing initial indications of how it might be able to effect behavioural change. We next demonstrated neuroinflammatory effects on behaviour directly, demonstrating that intra-hippocampal administration of the same endotoxin increased locomotor activity and accelerated goal-directed learning in both male and female mice. In contrast, hippocampal neuroinflammation caused sex-specific disruptions to the acquisition of instrumental actions and to Pavlovian food-approach memories. Finally, we showed that hippocampal neuroinflammation had a sexually dimorphic effect on neuronal activation: increasing it in females and decreasing it in males.

Communication between gut microbiota and immune cells within the brain is essential for neurotypical development. Specifically, microglia are known to play a key role in regulating and supporting neural progenitor stem cell production during brain development, and are sensitive to changes in the maternal gut microbial composition during perinatal development. Here, we employed a germ-free (GF) porcine paradigm to examine how the absence of the microbiome affects microglial dynamics during a key epoch of brain development. We utilized automated software to evaluate microglial density and morphology across three developmentally significant regions: the ventricular/subventricular zone (VZ/SVZ), the prefrontal subcortical white matter (PFCSWM), and layers II/III of the prefrontal cortex (PFCII-III). We found no significant differences in microglial morphology or density in the VZ/SVZ or PFCSWM. In contrast, the PFCII-III of P16 piglets exhibited an increase in microglia density paired with morphologies indicative of an activated/reactive functional state. Notably, these effects were identified with no overall changes in microglial density in any of the regions assessed. Transcriptomics on RNA isolated from the PFCII-III revealed a significant upregulation of genes related to neuroinflammation, in agreement with a region-specific microglial and immune response in the absence of microbial colonization during postnatal development. Together, these findings build on the limited knowledge available on how microbiota influence brain development in large animal model organisms with high similarities to human brain anatomy and developmental trajectories. Significance StatementThe prefrontal cortex of porcine display unique, ramified microglia which are sensitive to germ-free conditions whereby they display alterations in morphology with a more transcriptionally reactive signature. These findings indicate that microglia are regionally sensitive to stimuli in the periphery, and studies in lissencephalic mammalian models may not be directly correlative to other higher-order species. The neuroanatomical heterogeneity of microglia across species is informative and understudied, but necessary, to draw conclusions on the array of perturbations spanning neurodevelopmental trajectories in health and disease.

While dopamine is a monoamine neurotransmitter best known for its roles in reward, motivation, and motor function in the central nervous system, its actions extend beyond neurons and can influence non-neuronal cells via epigenetic mechanisms. An increasing body of literature corroborates that dopamine signaling is important in immune cells, which express dopamine receptors (DRD1-DRD5) as well as the molecular machinery for dopamine synthesis and metabolism. Dopamine can regulate inflammatory activity, cell trafficking, and disease pathology, yet the epigenetic mechanisms underlying these effects remain poorly understood. Here, we show that in primary human monocyte-derived macrophages, dopamine increases DNA methylation at the IL-1{beta} proximal promoter in a DNMT-dependent manner, while concurrently upregulating IL-1{beta} gene expression. Dopamine also increases the expression of key epigenetic regulators, including TET2, HDAC2, and HDAC6, suggesting coordinated changes in both DNA methylation and histone modifications that shape inflammatory transcription. Importantly, baseline dopamine receptor expression and donor demographics, including sex and age, influence the magnitude of these epigenetic responses, highlighting inter-individual variability in macrophage sensitivity to dopaminergic signaling. These findings establish dopamine as a modulator of macrophage inflammation via epigenetic remodeling and provide a mechanistic framework for understanding how peripheral immune cells respond to dopaminergic cues. By linking dopamine signaling, epigenetic regulation, and innate immunity, this work identifies potential targets for therapeutic intervention and supports the use of accessible human immune cells to investigate dopaminergic dysregulation in neuroimmunological disorders.

Microglia play pivotal roles in central nervous system development, homeostasis, responses to trauma, neurodegenerative and neuropsychiatric disorders with significant sex-bias in their symptoms and prevalence. The discovery that the survival of microglia in adult brains depends on the expression of the colony-stimulating factor 1 receptor (CSF1R), along with the development of the effective brain permeant CSF1R inhibitors PLX5622, has boosted the investigation of the role of microglia in health, disease and in relations to sex-bias. The effectiveness of PLX5622 in examining the role of microglia has mainly been demonstrated in mice. Surprisingly, despite the critical importance of rat models in brain research, there are only 4 publications in which PLX5622 was used to investigate the roles of microglia in adult rats. This has been attributed to the "impression" that PLX5622 is "ineffective in rats". In view of the importance and interest in the role of microglia, the indispensability of rats for in vivo electrophysiological brain studies and behavioral research and the high efficacy of PLX5622-chow in eliminating microglia from adult mice brains, we examined the effects of PLX5622-chow on the elimination of the microglia in adult female and male rats. We found significant differences in microglia elimination by ad libitum PLX5622 feeding in male and female rats in different brain regions with significantly greater effectivity in female brains. Our pragmatic study provides practical information on the use and design of PLX5622 in gender-related and rat brain preclinical microglia research.

The NKCC1 ion transporter contributes to the pathophysiology of common neurological disorders, but its function in microglia, the main inflammatory cells of the brain, has remained unclear to date. Therefore, we generated a novel transgenic mouse line in which microglial NKCC1 was deleted. We show that microglial NKCC1 shapes both baseline and reactive microglia morphology, process recruitment to the site of injury, and adaptation to osmotic stress in a cell-autonomous manner via regulating membrane potential and chloride fluxes. In addition, microglial NKCC1 deficiency results in increased expression of the D subunit of volume regulated anion channel (VRAC), NLRP3 inflammasome priming and production of interleukin-1{beta} (IL-1{beta}), rendering microglia prone to exaggerated inflammatory responses. In line with this, central (intracortical) administration of the NKCC1 blocker, bumetanide, potentiated intracortical lipopolysaccharide (LPS)-induced cytokine levels, whereas systemic bumetanide application decreased inflammation in the brain. Microglial NKCC1 KO animals exposed to experimental stroke showed significantly increased brain injury, inflammation, cerebral edema and worse neurological outcome. Thus, NKCC1 emerges as an important player in controlling microglial ion homeostasis and inflammatory responses through which microglia modulate brain injury. The contribution of microglia to central NKCC1 actions is likely to be relevant for common neurological disorders.

BackgroundAround 80% of individuals with neurodevelopmental disorders (NDDs) such as schizophrenia and autism spectrum disorders experience disruptions in sleep/circadian rhythms. We explored whether prenatal infection, an established risk factor for NDDs, and environmental circadian disruption synergistically induced sex-specific deficits in mice. MethodsA maternal immune activation (MIA) protocol was used by injecting pregnant mice (at E9.5) with a viral mimic poly IC or saline. Then, juvenile/adolescent offspring (3-7 weeks old) were subjected to either standard lighting (12:12LD) or constant light (LL). ResultsWe found interactions of the two factors on behaviors related to cognition, anxiety, and sociability. Also, poly IC exposure led to a more activated profile of hippocampal microglia in males only, while LL diminished these effects. Using RNA sequencing in the dorsal hippocampus, we found that poly IC exposure led to many differentially expressed genes in males (but not females), and fewer differentially expressed genes were observed after LL exposure. Using the WGCNA analysis, we found several significant gene modules positively associated with poly IC (in comparison to saline exposure) and LL (in comparison to LD exposure) in males, and less so in females. Interestingly, many of the identified hub bottleneck genes were homologous to human genes associated with both sleep/circadian rhythms and neurodevelopmental disorders as identified by GWA studies. ConclusionsOur work demonstrates that in a mouse model of prenatal infection, disruptions in circadian rhythms induced by LL play a role in modulating the effects of MIA at behavioral, cellular, and molecular levels.

BackgroundThe "social transfer of pain" is a phenomenon where a mouse experiencing injury-induced hyperalgesia can trigger hyperalgesia in a mouse briefly housed in the same environment ( bystander). The peripheral mechanisms underlying social transfer of pain in mice are not yet well described. As gut microbes are associated with social interactions, pain states, and pain attenuation via the gut-brain axis, we hypothesized that the bystander gut microbiome may respond to the social transfer of pain. MethodsTo induce the social transfer of pain, complete Freunds adjuvant (CFA) was injected into the hindpaw of a mouse which then underwent social interaction with a bystander for one hour. Mechanical sensitivity was assessed using the Von Frey mechanical sensitivity test. Stool samples and mechanical thresholds were taken prior to social interaction, 4 hours post-social interaction, and 24 hours post-social interaction. Metagenomic sequencing characterized the taxonomic and predicted functional gene response of the gut microbiome to CFA-induced pain, social transfer of pain and control groups. ResultsAt 4 hours post-social interaction, bystander animals experienced increased mechanical sensitivity comparable to CFA-injected animals and significantly lower than controls, indicating enhanced hyperalgesia. Compared to baseline, fecal community composition analyses at 4 hours and 24 hours post-interaction showed significant differences in Unweighted Unifrac in both CFA-injected and bystander animals but not in controls. Differential abundance analyses using Maaslin2 identified significant increases in the relative abundances of short chain fatty acid producing taxa like Lachnospiraceae, Ruminococcaceae, Oscillospiraceae and decreases in commensal mouse gut microbes like Muribaculaceae from baseline to 4 hours and 24 hours post-interaction in both bystanders and CFA-injected animals but not in controls. Functional analysis revealed increased abundance of pathways related to short-chain fatty acid production including pyruvate to butanoate and L-lysine fermentation to acetate and butanoate. The altered gut microbiome of bystanders strongly resembles that observed in CFA-injected animals at 4 hours and 24 hours post-injection, with the addition of a unique bystander bloom in several species of Lachnospiraceae. ConclusionsThe changes in the gut microbiome of bystander animals suggest that the social transfer of pain alters bystander peripheral physiology. These results are the first evidence of the potential for such a link.