High-salt diet induces microbiome dysregulation, neuroinflammation and anxiety in the chronic period after mild repetitive closed head injury in adolescent mice.

The associations between human concussions and subsequent sequelae of chronic neuropsychiatric and cardiovascular diseases such as hypertension have been reported; however, little is known about the underlying biological processes. We hypothesized that dietary changes, including a high-salt diet, disrupt the bidirectional gut-brain axis, resulting in worsening neuroinflammation and emergence of cardiovascular and behavioural phenotypes in the chronic period after repetitive closed head injury in adolescent mice. Adolescent mice were subjected to three daily closed head injuries, recovered for 12 weeks and then maintained on a high-salt diet or a normal diet for an additional 12 weeks. Experimental endpoints were haemodynamics, behaviour, microglial gene expression (bulk RNA sequencing), brain inflammation (brain tissue quantitative PCR) and microbiome diversity (16S RNA sequencing). High-salt diet did not affect systemic blood pressure or heart rate in sham or injured mice. High-salt diet increased anxiety-like behaviour in injured mice compared to sham mice fed with high-salt diet and injured mice fed with normal diet. Increased anxiety in injured mice that received a high-salt diet was associated with microgliosis and a proinflammatory microglial transcriptomic signature, including upregulation in interferon-gamma, interferon-beta and oxidative stress-related pathways. Accordingly, we found upregulation of tumour necrosis factor-alpha and interferon-gamma mRNA in the brain tissue of high salt diet-fed injured mice. High-salt diet had a larger effect on the gut microbiome composition than repetitive closed head injury. Increases in gut microbes in the families Lachnospiraceae, Erysipelotrichaceae and Clostridiaceae were positively correlated with anxiety-like behaviours. In contrast, Muribaculaceae, Acholeplasmataceae and Lactobacillaceae were negatively correlated with anxiety in injured mice that received a high-salt diet, a time-dependent effect. The findings suggest that high-salt diet, administered after a recovery period, may affect neurologic outcomes following mild repetitive head injury, including the development of anxiety. This effect was linked to microbiome dysregulation and an exacerbation of microglial inflammation, which may be physiological targets to prevent behavioural sequelae in the chronic period after mild repetitive head injury. The data suggest an important contribution of diet in determining long-term outcomes after mild repetitive head injury.

Dietary protein modulates intestinal dendritic cells to establish mucosal homeostasis.

Dietary proteins are taken up by intestinal dendritic cells (DCs), cleaved into peptides, loaded to major histocompatibility complexes, and presented to T cells to generate an immune response. Amino acid (AA)-diets do not have the same effects because AAs cannot bind to major histocompatibility complex to activate T cells. Here, we show that impairment in regulatory T cell generation and loss of tolerance in mice fed a diet lacking whole protein is associated with major transcriptional changes in intestinal DCs including downregulation of genes related to DC maturation, activation and decreased gene expression of immune checkpoint molecules. Moreover, the AA-diet had a profound effect on microbiome composition, including an increase in Akkermansia muciniphilia and Oscillibacter and a decrease in Lactococcus lactis and Bifidobacterium. Although microbiome transfer experiments showed that AA-driven microbiome modulates intestinal DC gene expression, most of the unique transcriptional change in DC was linked to the absence of whole protein in the diet. Our findings highlight the importance of dietary proteins for intestinal DC function and mucosal tolerance.

Bacteroidota inhibit microglia clearance of amyloid-beta and promote plaque deposition in Alzheimer's disease mouse models.

The gut microbiota and microglia play critical roles in Alzheimer's disease (AD), and elevated Bacteroides is correlated with cerebrospinal fluid amyloid-ß (Aß) and tau levels in AD. We hypothesize that Bacteroides contributes to AD by modulating microglia. Here we show that administering Bacteroides fragilis to APP/PS1-21 mice increases Aß plaques in females, modulates cortical amyloid processing gene expression, and down regulates phagocytosis and protein degradation microglial gene expression. We further show that administering Bacteroides fragilis to aged wild-type male and female mice suppresses microglial uptake of Aß1-42 injected into the hippocampus. Depleting murine Bacteroidota with metronidazole decreases amyloid load in aged 5xFAD mice, and activates microglial pathways related to phagocytosis, cytokine signaling, and lysosomal degradation. Taken together, our study demonstrates that members of the Bacteroidota phylum contribute to AD pathogenesis by suppressing microglia phagocytic function, which leads to impaired Aß clearance and accumulation of amyloid plaques.

Gamma-delta T cells suppress microbial metabolites that activate striatal neurons and induce repetitive/compulsive behavior in mice.

Intestinal γδ T cells play an important role in shaping the gut microbiota, which is critical not only for maintaining intestinal homeostasis but also for controlling brain function and behavior. Here, we found that mice deficient for γδ T cells (γδ-/-) developed an abnormal pattern of repetitive/compulsive (R/C) behavior, which was dependent on the gut microbiota. Colonization of WT mice with γδ-/- microbiota induced R/C behavior whereas colonization of γδ-/- mice with WT microbiota abolished the R/C behavior. Moreover, γδ-/- mice had elevated levels of the microbial metabolite 3-phenylpropanoic acid in their cecum, which is a precursor to hippurate (HIP), a metabolite we found to be elevated in the CSF. HIP reaches the striatum and activates dopamine type 1 (D1R)-expressing neurons, leading to R/C behavior. Altogether, these data suggest that intestinal γδ T cells shape the gut microbiota and their metabolites and prevent dysfunctions of the striatum associated with behavior modulation.

Vγ1 and Vγ4 gamma-delta T cells play opposing roles in the immunopathology of traumatic brain injury in males.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality. The innate and adaptive immune responses play an important role in the pathogenesis of TBI. Gamma-delta (γδ) T cells have been shown to affect brain immunopathology in multiple different conditions, however, their role in acute and chronic TBI is largely unknown. Here, we show that γδ T cells affect the pathophysiology of TBI as early as one day and up to one year following injury in a mouse model. TCRδ-/- mice are characterized by reduced inflammation in acute TBI and improved neurocognitive functions in chronic TBI. We find that the Vγ1 and Vγ4 γδ T cell subsets play opposing roles in TBI. Vγ4 γδ T cells infiltrate the brain and secrete IFN-γ and IL-17 that activate microglia and induce neuroinflammation. Vγ1 γδ T cells, however, secrete TGF-ß that maintains microglial homeostasis and dampens TBI upon infiltrating the brain. These findings provide new insights on the role of different γδ T cell subsets after brain injury and lay down the principles for the development of targeted γδ T-cell-based therapy for TBI.

Gamma-delta T cells modulate the microbiota and fecal micro-RNAs to maintain mucosal tolerance.

BACKGROUND: Gamma-delta (γδ) T cells are a major cell population in the intestinal mucosa and are key mediators of mucosal tolerance and microbiota composition. Little is known about the mechanisms by which intestinal γδ T cells interact with the gut microbiota to maintain tolerance. RESULTS: We found that antibiotic treatment impaired oral tolerance and depleted intestinal γδ T cells, suggesting that the gut microbiota is necessary to maintain γδ T cells. We also found that mice deficient for γδ T cells (γδ-/-) had an altered microbiota composition that led to small intestine (SI) immune dysregulation and impaired tolerance. Accordingly, colonizing WT mice with γδ-/- microbiota resulted in SI immune dysregulation and loss of tolerance whereas colonizing γδ-/- mice with WT microbiota normalized mucosal immune responses and restored mucosal tolerance. Moreover, we found that SI γδ T cells shaped the gut microbiota and regulated intestinal homeostasis by secreting the fecal micro-RNA let-7f. Importantly, oral administration of let-7f to γδ-/- mice rescued mucosal tolerance by promoting the growth of the γδ-/--microbiota-depleted microbe Ruminococcus gnavus. CONCLUSIONS: Taken together, we demonstrate that γδ T cell-selected microbiota is necessary and sufficient to promote mucosal tolerance, is mediated in part by γδ T cell secretion of fecal micro-RNAs, and is mechanistically linked to restoration of mucosal immune responses. Video Abstract.

The emerging role of the microbiome in Alzheimer's disease.

Alzheimer's disease (AD) is the most prevalent form of dementia and can be influenced by genetic and environmental factors. Recent studies suggest that the intestinal microbiota is altered in AD patients when compared to healthy individuals and may play a role in disease onset and progression. Aging is the greatest risk factor for AD, and age-related changes in the microbiota can affect processes that contribute to cognitive decline. The microbiota may affect AD by modulating peripheral and central immunity or by secreting factors that influence neurogenesis or neuronal cell death. Finally, probiotic and dietary interventions that target the microbiome may have therapeutic potential to prevent or treat AD.

Identification of environmental factors that promote intestinal inflammation.

Genome-wide association studies have identified risk loci linked to inflammatory bowel disease (IBD)1-a complex chronic inflammatory disorder of the gastrointestinal tract. The increasing prevalence of IBD in industrialized countries and the augmented disease risk observed in migrants who move into areas of higher disease prevalence suggest that environmental factors are also important determinants of IBD susceptibility and severity2. However, the identification of environmental factors relevant to IBD and the mechanisms by which they influence disease has been hampered by the lack of platforms for their systematic investigation. Here we describe an integrated systems approach, combining publicly available databases, zebrafish chemical screens, machine learning and mouse preclinical models to identify environmental factors that control intestinal inflammation. This approach established that the herbicide propyzamide increases inflammation in the small and large intestine. Moreover, we show that an AHR-NF-κB-C/EBPß signalling axis operates in T cells and dendritic cells to promote intestinal inflammation, and is targeted by propyzamide. In conclusion, we developed a pipeline for the identification of environmental factors and mechanisms of pathogenesis in IBD and, potentially, other inflammatory diseases.

Mining the microbiota to identify gut commensals modulating neuroinflammation in a mouse model of multiple sclerosis.

BACKGROUND: The gut microbiome plays an important role in autoimmunity including multiple sclerosis and its mouse model called experimental autoimmune encephalomyelitis (EAE). Prior studies have demonstrated that the multiple sclerosis gut microbiota can contribute to disease, hence making it a potential therapeutic target. In addition, antibiotic treatment has been shown to ameliorate disease in the EAE mouse model of multiple sclerosis. Yet, to this date, the mechanisms mediating these antibiotic effects are not understood. Furthermore, there is no consensus on the gut-derived bacterial strains that drive neuroinflammation in multiple sclerosis. RESULTS: Here, we characterized the gut microbiome of untreated and vancomycin-treated EAE mice over time to identify bacteria with neuroimmunomodulatory potential. We observed alterations in the gut microbiota composition following EAE induction. We found that vancomycin treatment ameliorates EAE, and that this protective effect is mediated via the microbiota. Notably, we observed increased abundance of bacteria known to be strong inducers of regulatory T cells, including members of Clostridium clusters XIVa and XVIII in vancomycin-treated mice during the presymptomatic phase of EAE, as well as at disease peak. We identified 50 bacterial taxa that correlate with EAE severity. Interestingly, several of these taxa exist in the human gut, and some of them have been implicated in multiple sclerosis including Anaerotruncus colihominis, a butyrate producer, which had a positive correlation with disease severity. We found that Anaerotruncus colihominis ameliorates EAE, and this is associated with induction of RORγt+ regulatory T cells in the mesenteric lymph nodes. CONCLUSIONS: We identified vancomycin as a potent modulator of the gut-brain axis by promoting the proliferation of bacterial species that induce regulatory T cells. In addition, our findings reveal 50 gut commensals as regulator of the gut-brain axis that can be used to further characterize pathogenic and beneficial host-microbiota interactions in multiple sclerosis patients. Our findings suggest that elevated Anaerotruncus colihominis in multiple sclerosis patients may represent a protective mechanism associated with recovery from the disease. Video Abstract.

Clinical Trial-Ready Patient Cohorts for Multiple System Atrophy: Coupling Biospecimen and iPSC Banking to Longitudinal Deep-Phenotyping.

Multiple system atrophy (MSA) is a fatal neurodegenerative disease of unknown etiology characterized by widespread aggregation of the protein alpha-synuclein in neurons and glia. Its orphan status, biological relationship to Parkinson's disease (PD), and rapid progression have sparked interest in drug development. One significant obstacle to therapeutics is disease heterogeneity. Here, we share our process of developing a clinical trial-ready cohort of MSA patients (69 patients in 2 years) within an outpatient clinical setting, and recruiting 20 of these patients into a longitudinal "n-of-few" clinical trial paradigm. First, we deeply phenotype our patients with clinical scales (UMSARS, BARS, MoCA, NMSS, and UPSIT) and tests designed to establish early differential diagnosis (including volumetric MRI, FDG-PET, MIBG scan, polysomnography, genetic testing, autonomic function tests, skin biopsy) or disease activity (PBR06-TSPO). Second, we longitudinally collect biospecimens (blood, CSF, stool) and clinical, biometric, and imaging data to generate antecedent disease-progression scores. Third, in our Mass General Brigham SCiN study (stem cells in neurodegeneration), we generate induced pluripotent stem cell (iPSC) models from our patients, matched to biospecimens, including postmortem brain. We present 38 iPSC lines derived from MSA patients and relevant disease controls (spinocerebellar ataxia and PD, including alpha-synuclein triplication cases), 22 matched to whole-genome sequenced postmortem brain. iPSC models may facilitate matching patients to appropriate therapies, particularly in heterogeneous diseases for which patient-specific biology may elude animal models. We anticipate that deeply phenotyped and genotyped patient cohorts matched to cellular models will increase the likelihood of success in clinical trials for MSA.

The microbiota restrains neurodegenerative microglia in a model of amyotrophic lateral sclerosis.

BACKGROUND: The gut microbiota can affect neurologic disease by shaping microglia, the primary immune cell in the central nervous system (CNS). While antibiotics improve models of Alzheimer's disease, Parkinson's disease, multiple sclerosis, and the C9orf72 model of amyotrophic lateral sclerosis (ALS), antibiotics worsen disease progression the in SOD1G93A model of ALS. In ALS, microglia transition from a homeostatic to a neurodegenerative (MGnD) phenotype and contribute to disease pathogenesis, but whether this switch can be affected by the microbiota has not been investigated. RESULTS: In this short report, we found that a low-dose antibiotic treatment worsened motor function and decreased survival in the SOD1 mice, which is consistent with studies using high-dose antibiotics. We also found that co-housing SOD1 mice with wildtype mice had no effect on disease progression. We investigated changes in the microbiome and found that antibiotics reduced Akkermansia and butyrate-producing bacteria, which may be beneficial in ALS, and cohousing had little effect on the microbiome. To investigate changes in CNS resident immune cells, we sorted spinal cord microglia and found that antibiotics downregulated homeostatic genes and increased neurodegenerative disease genes in SOD1 mice. Furthermore, antibiotic-induced changes in microglia preceded changes in motor function, suggesting that this may be contributing to disease progression. CONCLUSIONS: Our findings suggest that the microbiota play a protective role in the SOD1 model of ALS by restraining MGnD microglia, which is opposite to other neurologic disease models, and sheds new light on the importance of disease-specific interactions between microbiota and microglia. Video abstract.

PD-L1+ and XCR1+ dendritic cells are region-specific regulators of gut homeostasis.

The intestinal mucosa constitutes an environment of closely regulated immune cells. Dendritic cells (DC) interact with the gut microbiome and antigens and are important in maintaining gut homeostasis. Here, we investigate DC transcriptome, phenotype and function in five anatomical locations of the gut lamina propria (LP) which constitute different antigenic environments. We show that DC from distinct gut LP compartments induce distinct T cell differentiation and cytokine secretion. We also find that PD-L1+ DC in the duodenal LP and XCR1+ DC in the colonic LP comprise distinct tolerogenic DC subsets that are crucial for gut homeostasis. Mice lacking PD-L1+ and XCR1+ DC have a proinflammatory gut milieu associated with an increase in Th1/Th17 cells and a decrease in Treg cells and have exacerbated disease in the models of 5-FU-induced mucositis and DSS-induced colitis. Our findings identify PD-L1+ and XCR1+ DC as region-specific physiologic regulators of intestinal homeostasis.

Self-tunable engineered yeast probiotics for the treatment of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a complex chronic inflammatory disorder of the gastrointestinal tract. Extracellular adenosine triphosphate (eATP) produced by the commensal microbiota and host cells activates purinergic signaling, promoting intestinal inflammation and pathology. Based on the role of eATP in intestinal inflammation, we developed yeast-based engineered probiotics that express a human P2Y2 purinergic receptor with up to a 1,000-fold increase in eATP sensitivity. We linked the activation of this engineered P2Y2 receptor to the secretion of the ATP-degrading enzyme apyrase, thus creating engineered yeast probiotics capable of sensing a pro-inflammatory molecule and generating a proportional self-regulated response aimed at its neutralization. These self-tunable yeast probiotics suppressed intestinal inflammation in mouse models of IBD, reducing intestinal fibrosis and dysbiosis with an efficacy similar to or higher than that of standard-of-care therapies usually associated with notable adverse events. By combining directed evolution and synthetic gene circuits, we developed a unique self-modulatory platform for the treatment of IBD and potentially other inflammation-driven pathologies.

Regulation of splenic monocyte homeostasis and function by gut microbial products.

Splenic Ly6Chigh monocytes are innate immune cells involved in the regulation of central nervous system-related diseases. Recent studies have reported the shaping of peripheral immune responses by the gut microbiome via mostly unexplored pathways. In this study, we report that a 4-day antibiotic treatment eliminates certain families of the Bacteroidetes, Firmicutes, Tenericutes, and Actinobacteria phyla in the gut and reduces the levels of multiple pattern recognition receptor (PRR) ligands in the serum. Reduction of PRR ligands was associated with reduced numbers and perturbed function of splenic Ly6Chigh monocytes, which acquired an immature phenotype producing decreased levels of inflammatory cytokines and exhibiting increased phagocytic and anti-microbial abilities. Addition of PRR ligands in antibiotic-treated mice restored the number and functions of splenic Ly6Chigh monocytes. Our data identify circulating PRR ligands as critical regulators of the splenic Ly6Chigh monocyte behavior and suggest possible intervention pathways to manipulate this crucial immune cell subset.

Gut Microbiome in Progressive Multiple Sclerosis.

OBJECTIVE: This study was undertaken to investigate the gut microbiome in progressive multiple sclerosis (MS) and how it relates to clinical disease. METHODS: We sequenced the microbiota from healthy controls and relapsing-remitting MS (RRMS) and progressive MS patients and correlated the levels of bacteria with clinical features of disease, including Expanded Disability Status Scale (EDSS), quality of life, and brain magnetic resonance imaging lesions/atrophy. We colonized mice with MS-derived Akkermansia and induced experimental autoimmune encephalomyelitis (EAE). RESULTS: Microbiota ß-diversity differed between MS patients and controls but did not differ between RRMS and progressive MS or differ based on disease-modifying therapies. Disease status had the greatest effect on the microbiome ß-diversity, followed by body mass index, race, and sex. In both progressive MS and RRMS, we found increased Clostridium bolteae, Ruthenibacterium lactatiformans, and Akkermansia and decreased Blautia wexlerae, Dorea formicigenerans, and Erysipelotrichaceae CCMM. Unique to progressive MS, we found elevated Enterobacteriaceae and Clostridium g24 FCEY and decreased Blautia and Agathobaculum. Several Clostridium species were associated with higher EDSS and fatigue scores. Contrary to the view that elevated Akkermansia in MS has a detrimental role, we found that Akkermansia was linked to lower disability, suggesting a beneficial role. Consistent with this, we found that Akkermansia isolated from MS patients ameliorated EAE, which was linked to a reduction in RORγt+ and IL-17-producing γδ T cells. INTERPRETATION: Whereas some microbiota alterations are shared in relapsing and progressive MS, we identified unique bacteria associated with progressive MS and clinical measures of disease. Furthermore, elevated Akkermansia in MS may be a compensatory beneficial response in the MS microbiome. ANN NEUROL 2021;89:1195-1211.

Correction: Helminth Colonization Is Associated with Increased Diversity of the Gut Microbiota.

[This corrects the article DOI: 10.1371/journal.pntd.0002880.].

Clostridium bolteae is elevated in neuromyelitis optica spectrum disorder in India and shares sequence similarity with AQP4.

OBJECTIVE: To understand the role of gut microbiome in influencing the pathogenesis of neuromyelitis optica spectrum disorders (NMOSDs) among patients of south Indian origin. METHODS: In this case-control study, stool and blood samples were collected from 39 patients with NMOSD, including 17 with aquaporin 4 IgG antibodies (AQP4+) and 36 matched controls. 16S ribosomal RNA (rRNA) sequencing was used to investigate the gut microbiome. Peripheral CD4+ T cells were sorted in 12 healthy controls, and in 12 patients with AQP4+ NMOSD, RNA was extracted and immune gene expression was analyzed using the NanoString nCounter human immunology kit code set. RESULTS: Microbiota community structure (beta diversity) differed between patients with AQP4+ NMOSD and healthy controls (p < 0.001, pairwise PERMANOVA test). Linear discriminatory analysis effect size identified several members of the microbiota that were altered in patients with NMOSD, including an increase in Clostridium bolteae (effect size 4.23, p 0.00007). C bolteae was significantly more prevalent (p = 0.02) among patients with AQP4-IgG+ NMOSD (n = 8/17 subjects) compared with seronegative patients (n = 3/22) and was absent among healthy stool samples. C bolteae has a highly conserved glycerol uptake facilitator and related aquaporin protein (p59-71) that shares sequence homology with AQP4 peptide (p92-104), positioned within an immunodominant (AQP4 specific) T-cell epitope (p91-110). Presence of C bolteae correlated with expression of inflammatory genes associated with both innate and adaptive immunities and particularly involved in plasma cell differentiation, B cell chemotaxis, and Th17 activation. CONCLUSION: Our study described elevated levels of C bolteae associated with AQP4+ NMOSD among Indian patients. It is possible that this organism may be causally related to the immunopathogenesis of this disease in susceptible individuals.