Altering the intestinal microbiota during a critical developmental window has lasting metabolic consequences.

Acquisition of the intestinal microbiota begins at birth, and a stable microbial community develops from a succession of key organisms. Disruption of the microbiota during maturation by low-dose antibiotic exposure can alter host metabolism and adiposity. We now show that low-dose penicillin (LDP), delivered from birth, induces metabolic alterations and affects ileal expression of genes involved in immunity. LDP that is limited to early life transiently perturbs the microbiota, which is sufficient to induce sustained effects on body composition, indicating that microbiota interactions in infancy may be critical determinants of long-term host metabolic effects. In addition, LDP enhances the effect of high-fat diet induced obesity. The growth promotion phenotype is transferrable to germ-free hosts by LDP-selected microbiota, showing that the altered microbiota, not antibiotics per se, play a causal role. These studies characterize important variables in early-life microbe-host metabolic interaction and identify several taxa consistently linked with metabolic alterations. PAPERCLIP:

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.

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.

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.

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.

A probiotic modulates the microbiome and immunity in multiple sclerosis.

OBJECTIVE: Effect of a probiotic on the gut microbiome and peripheral immune function in healthy controls and relapsing-remitting multiple sclerosis (MS) patients. METHODS: MS patients (N = 9) and controls (N = 13) were orally administered a probiotic containing Lactobacillus, Bifidobacterium, and Streptococcus twice-daily for two months. Blood and stool specimens were collected at baseline, after completion of the 2-month treatment, and 3 months after discontinuation of therapy. Frozen peripheral blood mononuclear cells (PBMCs) were used for immune cell profiling. Stool samples were used for 16S rRNA profiling and metabolomics. RESULTS: Probiotic administration increased the abundance of several taxa known to be depleted in MS such as Lactobacillus. We found that probiotic use decreased the abundance of taxa previously associated with dysbiosis in MS, including Akkermansia and Blautia. Predictive metagenomic analysis revealed a decrease in the abundance of several KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways associated with altered gut microbiota function in MS patients, such as methane metabolism, following probiotic supplementation. At the immune level, probiotic administration induced an anti-inflammatory peripheral immune response characterized by decreased frequency of inflammatory monocytes, decreased mean fluorescence intensity (MFI) of CD80 on classical monocytes, as well as decreased human leukocyte antigen (HLA) D related MFI on dendritic cells. Probiotic administration was also associated with decreased expression of MS risk allele HLA-DQA1 in controls. Probiotic-induced increase in abundance of Lactobacillus and Bifidobacterium was associated with decreased expression of MS risk allele HLA.DPB1 in controls. INTERPRETATION: Our results suggest that probiotics could have a synergistic effect with current MS therapies. Ann Neurol 2018.

Introduction to the special issue for The anaerobe society of the America's 14th Biennial congress in Las Vegas.

In June 2018, the Anaerobe Society of the America's (ASA) held their 14th Biennial Congress in Las Vegas, Nevada. The Congress was attended by over 200 individuals from many different countries. The focus of the meeting was the fast-growing area of anaerobes in human and animal infectious disease, computational tools to understand basic biology and therapeutic development, the role of anaerobes in the microbiome, and clinical trials of novel bacterial-based therapies. To strengthen the community of researchers working on anaerobes, the congress held two training workshops on clinical bacteriology and anaerobes in the microbiome, several networking events, as well as a dinner which honored the lifetime achievement award given to Ellen Jo Baron. The meeting was also attended by the grandfather of anaerobic bacteriology and the founder of (ASA), Sydney Finegold, at the age of 97. In all, there was a broad diversity of research presented that showed new ways that anaerobes play a important role in health and disease.

A multivariate distance-based analytic framework for microbial interdependence association test in longitudinal study.

Human microbiome is the collection of microbes living in and on the various parts of our body. The microbes living on our body in nature do not live alone. They act as integrated microbial community with massive competing and cooperating and contribute to our human health in a very important way. Most current analyses focus on examining microbial differences at a single time point, which do not adequately capture the dynamic nature of the microbiome data. With the advent of high-throughput sequencing and analytical tools, we are able to probe the interdependent relationship among microbial species through longitudinal study. Here, we propose a multivariate distance-based test to evaluate the association between key phenotypic variables and microbial interdependence utilizing the repeatedly measured microbiome data. Extensive simulations were performed to evaluate the validity and efficiency of the proposed method. We also demonstrate the utility of the proposed test using a well-designed longitudinal murine experiment and a longitudinal human study. The proposed methodology has been implemented in the freely distributed open-source R package and Python code.

Investigation of probiotics in multiple sclerosis.

None of the disease-modifying therapies (DMTs) currently being used for the management of multiple sclerosis (MS) are 100% effective. In addition, side effects associated with the use of these DMTs have limited the practice of combination therapy. Hence, there is a need for safe immunomodulatory agents to fine-tune the management of MS. The gut microbiome plays an important role in autoimmunity, and several studies have reported alterations in the gut microbiome of MS patients. Studies in animal model of MS have identified members of the gut commensal microflora that exacerbate or ameliorate neuroinflammation. Probiotics represent an oral, non-toxic immunomodulatory agent that could be used in combination with current MS therapy. We designed a pilot study to investigate the effect of VSL3 on the gut microbiome and peripheral immune system function in healthy controls and MS patients. VSL3 administration was associated with increased abundance of many taxa with enriched taxa predominated by Lactobacillus, Streptococcus, and Bifidobacterium species. At the immune level, VSL3 administration induced an anti-inflammatory peripheral immune response characterized by decreased frequency of intermediate monocytes (CD14highCD16low), decreased mean fluorescence intensity (MFI) of CD80 on classical monocytes as well as decreased human leukocyte antigen-antigen D related (HLA-DR) MFI on dendritic cells.

Prevalence of Fusobacterium necrophorum in Children Presenting with Pharyngitis.

Fusobacterium necrophorum, an obligate anaerobic bacterium, was recently reported to be an important cause of bacterial pharyngitis with a prevalence as high as that of group A Streptococcus (GAS) in adolescents and young adults. Importantly, F. necrophorum is the primary causative agent of the life-threatening Lemierre's syndrome, and screening of pharyngeal samples may be warranted for its early detection and prevention. The aim of this study was to determine the prevalences of F. necrophorum and groups A and C/G streptococci as agents of bacterial pharyngitis in children. Pharyngeal samples (n = 300) were collected from pediatric patients presenting to the emergency department with signs and symptoms of pharyngitis. Overall, 10 (3.3%), 79 (26.3%), and 4 (1.3%) patients were PCR positive for F. necrophorum, GAS, and group C/G streptococci, respectively. The prevalence of F. necrophorum was significantly higher in patients between the ages of 14 and 20 years at 13.5% than in patients aged 14 years and younger (1.9%, P < 0.001). All positive patients presented with signs and symptoms similar to GAS pharyngitis. Our data demonstrated a potential role for F. necrophorum as a pathogen of pharyngitis among young adults, but suggests that the prevalence of F. necrophorum is low in preadolescent patients.

Description of two novel members of the family Erysipelotrichaceae: Ileibacterium valens gen. nov., sp. nov. and Dubosiella newyorkensis, gen. nov., sp. nov., from the murine intestine, and emendation to the description of Faecalibaculum rodentium.

To better characterize murine intestinal microbiota, a large number (187) of Gram-positive-staining, rod- and coccoid-shaped, and facultatively or strictly anaerobic bacteria were isolated from small and large intestinal contents from mice. Based on 16S rRNA gene sequencing, a total 115 isolates formed three phylogenetically distinct clusters located within the family Erysipelotrichaceae. Group 1, as represented by strain NYU-BL-A3T, was most closely related to Allobaculum stercoricanis, with 16S rRNA gene sequence similarity values of 87.7 %. A second group, represented by NYU-BL-A4T, was most closely related to Faecalibaculum rodentium, with 86.6 % 16S rRNA gene sequence similarity. A third group had a nearly identical 16S rRNA gene sequence (99.9 %) compared with the recently described Faecalibaculum rodentium, also recovered from a laboratory mouse; however, this strain had a few differences in biochemical characteristics, which are detailed in an emended description. The predominant (>10 %) cellular fatty acids of strain NYU-BL-A3T were C16 : 0 and C18 : 0, and those of strain NYU-BL-A4T were C10 : 0, C16 : 0, C18 : 0 and C18 : 1ω9c. The two groups could also be distinguished by multiple biochemical reactions, with the group represented by NYU-BL-A4T being considerably more active. Based on phylogenetic, biochemical and chemotaxonomic criteria, two novel genera are proposed, Ileibacterium valens gen. nov., sp. nov. with NYU-BL-A3T (=ATCC TSD-63T=DSM 103668T) as the type strain and Dubosiella newyorkensis gen. nov., sp. nov. with NYU-BL-A4T (=ATCC TSD-64T=DSM 103457T) as the type strain.

The nonfermentable dietary fiber hydroxypropyl methylcellulose modulates intestinal microbiota.

Diet influences host metabolism and intestinal microbiota; however, detailed understanding of this tripartite interaction is limited. To determine whether the nonfermentable fiber hydroxypropyl methylcellulose (HPMC) could alter the intestinal microbiota and whether such changes correlated with metabolic improvements, C57B/L6 mice were normalized to a high-fat diet (HFD), then either maintained on HFD (control), or switched to HFD supplemented with 10% HPMC, or a low-fat diet (LFD). Compared to control treatment, both LFD and HPMC reduced weight gain (11.8 and 5.7 g, respectively), plasma cholesterol (23.1 and 19.6%), and liver triglycerides (73.1 and 44.6%), and, as revealed by 454-pyrosequencing of the microbial 16S rRNA gene, decreased microbial α-diversity and differentially altered intestinal microbiota. Both LFD and HPMC increased intestinal Erysipelotrichaceae (7.3- and 12.4-fold) and decreased Lachnospiraceae (2.0- and 2.7-fold), while only HPMC increased Peptostreptococcaceae (3.4-fold) and decreased Ruminococcaceae (2.7-fold). Specific microorganisms were directly linked with weight change and metabolic parameters in HPMC and HFD mice, but not in LFD mice, indicating that the intestinal microbiota may play differing roles during the two dietary modulations. This work indicates that HPMC is a potential prebiotic fiber that influences intestinal microbiota and improves host metabolism.

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.

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.

Impaired fitness of Mycobacterium africanum despite secretion of ESAT-6.

BACKGROUND: When compared with Mycobacterium tuberculosis, individuals that live in the same household as an active case of smear-positive pulmonary tuberculosis exposed to M. africanum progress less frequently to active disease within 2 years. A putative ESX-1 secretion apparatus member, Rv3879c, is mutated in M. africanum, and individuals infected with M. africanum less frequently demonstrate T-cell responses to the ESX-1-secreted virulence factor ESAT-6 than those infected with M. tuberculosis. We hypothesized that less frequent progression is caused by impaired secretion of ESAT-6. METHODS: We analyzed in vivo growth and in vitro secretion of ESAT-6 and CFP-10, comparing M. tuberculosis to M. africanum and a strain of M. africanum complemented with M. tuberculosis Rv3879c. RESULTS: ESAT-6 and CFP-10 secretion were similar for all strains, although these were enriched in M. africanum cell lysates, suggesting a modest ESX-1 secretion defect unrelated to the Rv3879c mutation. In mice, M. africanum demonstrated smaller bacterial population sizes than M. tuberculosis but similar numbers and frequencies of ESAT-6-responsive T cells in the lungs. CONCLUSIONS: These results confirm impaired fitness of M. africanum in vivo and indicate that Rv3879c is not required for secretion of ESAT-6 or for its presentation as an antigen to T cells in vivo.

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.

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.