Mapping the T cell repertoire to a complex gut bacterial community.

Certain bacterial strains from the microbiome induce a potent, antigen-specific T cell response1-5. However, the specificity of microbiome-induced T cells has not been explored at the strain level across the gut community. Here, we colonize germ-free mice with complex defined communities (roughly 100 bacterial strains) and profile T cell responses to each strain. The pattern of responses suggests that many T cells in the gut repertoire recognize several bacterial strains from the community. We constructed T cell hybridomas from 92 T cell receptor (TCR) clonotypes; by screening every strain in the community against each hybridoma, we find that nearly all the bacteria-specific TCRs show a one-to-many TCR-to-strain relationship, including 13 abundant TCR clonotypes that each recognize 18 Firmicutes. By screening three pooled bacterial genomic libraries, we discover that these 13 clonotypes share a single target: a conserved substrate-binding protein from an ATP-binding cassette transport system. Peripheral regulatory T cells and T helper 17 cells specific for an epitope from this protein are abundant in community-colonized and specific pathogen-free mice. Our work reveals that T cell recognition of commensals is focused on widely conserved, highly expressed cell-surface antigens, opening the door to new therapeutic strategies in which colonist-specific immune responses are rationally altered or redirected.

Altered Skin and Gut Microbiome in Hidradenitis Suppurativa.

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease characterized by the formation of nodules, abscesses, and fistulae at intertriginous sites. The skin-gut axis is an area of emerging research in inflammatory skin disease and is a potential contributory factor to the pathogenesis of HS. A total of 59 patients with HS provided fecal samples and nasal and skin swabs of affected sites for analysis. A total of 30 healthy controls provided fecal samples, and 20 healthy controls provided nasal and skin swabs. We performed bacterial 16S ribosomal RNA gene amplicon sequencing on total DNA derived from the samples. Microbiome alpha diversity was significantly lower in the fecal, skin, and nasal samples of individuals with HS, which may be secondary to disease biology or related to antibiotic usage. Ruminococcus gnavus was more abundant in the fecal microbiome of individuals with HS, which is also reported in Crohn's disease, suggesting comorbidity due to shared gut microbiota alterations. Finegoldia magna was overabundant in HS skin samples relative to that in the healthy controls. It is possible that local inflammation is driven by F. magna by promoting the formation of neutrophil extracellular traps. These alterations in both the gut and skin microbiome in HS warrant further exploration, and therapeutic strategies, including fecal microbiota transplant or bacteriotherapy, could be of benefit.

Intestinal microbial communities and Holdemanella isolated from HIV+/- men who have sex with men increase frequencies of lamina propria CCR5+ CD4+ T cells.

Men who have sex with men (MSM), regardless of HIV infection status, have an intestinal microbiome that is compositionally distinct from men who have sex with women (MSW) and women. We recently showed HIV-negative MSM have elevated levels of intestinal CD4+ T cells expressing CCR5, a critical co-receptor for HIV. Whether elevated expression of CCR5 is driven by the altered gut microbiome composition in MSM has not been explored. Here we used in vitro stimulation of gut Lamina Propria Mononuclear Cells (LPMCs) with whole intact microbial cells isolated from stool to demonstrate that fecal bacterial communities (FBCs) from HIV-positive/negative MSM induced higher frequencies of CCR5+ CD4+ T cells compared to FBCs from HIV-negative MSW and women. To identify potential microbial drivers, we related the frequency of CCR5+ CD4+ T cells to the abundance of individual microbial taxa in rectal biopsy of HIV-positive/negative MSM and controls, and Holdemanella biformis was strongly associated with increased frequency of CCR5+ CD4+ T cells. We used in vitro stimulation of gut LPMCs with the type strain of H. biformis, a second strain of H.biformis and an isolate of the closely related Holdemanella porci , cultured from either a HIV-positive or a HIV-negative MSM stool. H. porci elevated the frequency of both CCR5+ CD4+ T cells and the ratio of TNF-α/IL-10 Genomic comparisons of the 3 Holdemanella isolates revealed unique cell wall and capsular components, which may be responsible for their differences in immunogenicity. These findings describe a novel mechanism potentially linking intestinal dysbiosis in MSM to HIV transmission and mucosal pathogenesis.

Altered Actinobacteria and Firmicutes Phylum Associated Epitopes in Patients With Parkinson's Disease.

Recent evidence suggests that inflammation was participated in the pathogenesis of PD, thus, to understand the potential mechanism of gut microbiota in the pathogenesis of Parkinson's disease (PD), we performed a metagenomic analysis of fecal samples from PD patient and controls. Using a two-stage metagenome-wide association strategy, fecal DNA samples from 69 PD patients and 244 controls in three groups (comprising 66 spouses, 97 age-matched, and 81 normal samples, respectively) were analyzed, and differences between candidate gut microbiota and microbiota-associated epitopes (MEs) were compared. In the study, 27 candidate bacterial biomarkers and twenty-eight candidate epitope peptides were significantly different between the PD patients and control groups. Further, enriched 4 and 13 MEs in PD were positively associated with abnormal inflammatory indicators [neutrophil percentage (NEUT.1), monocyte count/percentage (MONO/MONO.1), white blood cell count (WBC)] and five candidate bacterial biomarkers (c_Actinobacteria, f_Bifidobacteriaceae, g_Bifidobacterium, o_Bifidobacteriales, p_Actinobacteria) from Actinobacteria phylum, and they were also positively associated with histidine degradation and proline biosynthesis pathways, respectively. Additionally, enriched 2 MEs and 1 ME in PD were positively associated with above inflammatory indicators and two bacteria (f_Lactobacillaceae, g_Lactobacillus) from Firmicutes phylum, and they were also positively associated with pyruvate fermentation to propanoate I and negatively associated with isopropanol biosynthesis, respectively. Of these MEs, two MEs from GROEL2, RPSC were derived from Mycobacterium tuberculosis, triggered the T cell immune response, as previously reported. Additionally, other candidate epitope peptides derived from Mycobacterium tuberculosis and Mycobacterium leprae may also have potential immune effects in PD. In all, the altered MEs in PD may relate to abnormalities in immunity and glutamate and propionate metabolism, which furthers our understanding of the pathogenesis of PD.

Reshaping of the gastrointestinal microbiome alters atherosclerotic plaque inflammation resolution in mice.

Since alterations in the intestinal microbiota may induce systemic inflammation and polarization of macrophages to the M1 state, the microbiome role in atherosclerosis, an M1-driven disease, requires evaluation. We aimed to determine if antibiotic (Abx) induced alterations to the intestinal microbiota interferes with atherosclerotic plaque inflammation resolution after lipid-lowering in mice. Hyperlipidemic Apoe-/- mice were fed a western diet to develop aortic atherosclerosis with aortas then transplanted into normolipidemic wild-type (WT) mice to model clinically aggressive lipid management and promote atherosclerosis inflammation resolution. Gut microbial composition pre and post-transplant was altered via an enteral antibiotic or not. Post aortic transplant, after Abx treatment, while plaque size did not differ, compared to Apoe-/- mice, Abx- WT recipient mice had a 32% reduction in CD68-expressing cells (p = 0.02) vs. a non-significant 12% reduction in Abx+ WT mice. A trend toward an M1 plaque CD68-expresing cell phenotype was noted in Abx+ mice. By 16S rRNA sequence analysis, the Abx+ mice had reduced alpha diversity and increased Firmicutes/Bacteroidetes relative abundance ratio with a correlation between gut Firmicutes abundance and plaque CD68-expressing cell content (p < 0.05). These results indicate that in a murine atherosclerotic plaque inflammation resolution model, antibiotic-induced microbiome perturbation may blunt the effectiveness of lipid-lowering to reduce the content of plaque inflammatory CD68-expressing cells.

Pectin in diet: Interactions with the human microbiome, role in gut homeostasis, and nutrient-drug interactions.

Pectins are a part of daily diet as well as food additives that are indigestible polysaccharides by human enzymes, however, they can be easily degraded by gut bacteria with the production of short chain fatty acids (SCFAs). Knowledge of pectin gut homeostasis and further how pectin affect gut bacterial communities is insufficient and limited. This review focuses on providing the whole story of how pectin functions as prebiotics in the gut. Understanding the interplay between functional and immunological responses inside animal or human gut as influenced by pectin in diets is provided. The interaction between pectin and gut microbiota is presented from both sides, in terms of how pectin affects gut microbiome and or the fermentation products produced in response by gut bacteria. This knowledge can be used to define preferred dietary pectins, targeting beneficial bacteria, and favoring balanced microbiota communities in the gut to maximize pectins' health benefits.

The Association of Targeted Gut Microbiota with Body Composition in Type 2 Diabetes Mellitus.

The association between body composition and gut microbiota in type 2 diabetes mellitus (DM) remains unknown. To elucidate the correlation of body composition and gut microbiota, we conducted a clinical study to enroll 179 patients with type 2 DM. Body composition of lean tissue index (LTI) and fat tissue index was measured by Body Composition Monitor. Eight pairs of 16S rRNA gene primers specific to Firmicutes, Bacteroidetes, the Clostridium leptum group, Bacteroides, Bifidobacterium, Akkermansia muciniphila, Escherichia coli, and Faecalibacterium prausnitzii were used to measure their abundance by quantitative polymerase chain reaction. The results showed that type 2 DM with higher abundance of phylum Firmicutes and a higher ratio of phyla Firmicutes to Bacteroidetes (phyla F/B ratio) had higher LTI. This significant correlation between phyla F/B ratio and LTI was especially evident in type 2 DM with high body mass index, and independent of glycemic control or dipeptidyl peptidase-4 inhibitor usage. In conclusion, our study demonstrated the positive association of LTI with the abundance of phylum Firmicutes and the phyla F/B ratio in type 2 DM.

CD8 T cells drive anorexia, dysbiosis, and blooms of a commensal with immunosuppressive potential after viral infection.

Infections elicit immune adaptations to enable pathogen resistance and/or tolerance and are associated with compositional shifts of the intestinal microbiome. However, a comprehensive understanding of how infections with pathogens that exhibit distinct capability to spread and/or persist differentially change the microbiome, the underlying mechanisms, and the relative contribution of individual commensal species to immune cell adaptations is still lacking. Here, we discovered that mouse infection with a fast-spreading and persistent (but not a slow-spreading acute) isolate of lymphocytic choriomeningitis virus induced large-scale microbiome shifts characterized by increased Verrucomicrobia and reduced Firmicute/Bacteroidetes ratio. Remarkably, the most profound microbiome changes occurred transiently after infection with the fast-spreading persistent isolate, were uncoupled from sustained viral loads, and were instead largely caused by CD8 T cell responses and/or CD8 T cell-induced anorexia. Among the taxa enriched by infection with the fast-spreading virus, Akkermansia muciniphila, broadly regarded as a beneficial commensal, bloomed upon starvation and in a CD8 T cell-dependent manner. Strikingly, oral administration of A. muciniphila suppressed selected effector features of CD8 T cells in the context of both infections. Our findings define unique microbiome differences after chronic versus acute viral infections and identify CD8 T cell responses and downstream anorexia as driver mechanisms of microbial dysbiosis after infection with a fast-spreading virus. Our data also highlight potential context-dependent effects of probiotics and suggest a model in which changes in host behavior and downstream microbiome dysbiosis may constitute a previously unrecognized negative feedback loop that contributes to CD8 T cell adaptations after infections with fast-spreading and/or persistent pathogens.

Melatonin controls microbiota in colitis by goblet cell differentiation and antimicrobial peptide production through Toll-like receptor 4 signalling.

Microbial dysbiosis has long been postulated to be associated with the pathogenesis of inflammatory bowel disease (IBD). Although evidence supporting the anti-colitic effects of melatonin have been accumulating, it is not clear how melatonin affects the microbiota. Herein, we investigated the effects of melatonin on the microbiome in colitis and identified involvement of Toll-like receptor (TLR) 4 signalling in the effects. Melatonin improved dextran sulfate sodium (DSS)-induced colitis and reverted microbial dysbiosis in wild-type (WT) mice but not in TLR4 knockout (KO) mice. Induction of goblet cells was observed with melatonin administration, which was accompanied by suppression of Il1b and Il17a and induction of melatonin receptor and Reg3ß, an antimicrobial peptide (AMP) against Gram-negative bacteria. In vitro, melatonin treatment of HT-29 intestinal epithelial cells promotes mucin and wound healing and inhibits growth of Escherichia coli. Herein, we showed that melatonin significantly increases goblet cells, Reg3ß, and the ratio of Firmicutes to Bacteriodetes by suppressing Gram-negative bacteria through TLR4 signalling. Our study suggests that sensing of bacteria through TLR4 and regulation of bacteria through altered goblet cells and AMPs is involved in the anti-colitic effects of melatonin. Melatonin may have use in therapeutics for IBD.

Bacteroidetes and Firmicutes levels in gut microbiota and effects of hosts TLR2/TLR4 gene expression levels in adult type 1 diabetes patients in Istanbul, Turkey.

AIM: The aim of this study was to determine and compare the levels of both Bacteroidetes and Firmicutes in the gut microbiota and TLR2/TLR4 gene expression in the blood of patients with type 1 diabetes mellitus (T1DM) and healthy individuals. These results may serve as a preliminary assessment to guide future research. METHOD: Between January and October 2014, stool and blood samples were collected from 53 adult T1DM patients and 53 age- and gender-matched healthy individuals. Bacteroidetes and Firmicutes levels were assessed from stool sample DNA and TLR2 and TLR4 expression levels were analyzed via qPCR using RNA from EDTA blood samples from both patients and healthy controls. RESULTS: The amounts of Bacteroidetes and Firmicutes were statistically significantly higher and lower, respectively, in the T1DM group than in the healthy control group (p < 0.001 and p < 0.001, respectively). In addition, the Firmicutes/Bacteroidetes ratios in patients with T1DM were significantly lower than in healthy controls. The TLR4 and TLR2 gene expression levels in T1DM patients were significantly upregulated and downregulated, respectively, compared to those in the control group. CONCLUSION: Our data are the first to show a relationship between T1DM and gut microbiota in our country. In addition, our results provide information about the connections between T1DM, gut microbiota, and TLR2 and TLR4 expression. We believe that Bacteroidetes and Firmicutes in the gut microbiota may play a role in the autoimmune process of T1DM and that these findings should be further investigated in the future.

Depletion of Foxp3+ regulatory T cells is accompanied by an increase in the relative abundance of Firmicutes in the murine gut microbiome.

A reciprocal interaction exists between the gut microbiota and the immune system. Regulatory T (Treg) cells are important for controlling immune responses and for maintaining the intestinal homeostasis but their precise influence on the gut microbiota is unclear. We studied the effects of Treg cell depletion on inflammation of the intestinal mucosa and analysed the gut microbiota before and after depletion of Treg cells using the DEpletion of REGulatory T cells (DEREG) mouse model. DNA was extracted from stool samples of DEREG mice and wild-type littermates at different time-points before and after diphtheria toxin application to deplete Treg cells in DEREG mice. The V3/V4 region of the 16S rRNA gene was used for studying the gut microbiota with Illumina MiSeq paired ends sequencing. Multidimensional scaling separated the majority of gut microbiota samples from late time-points after Treg cell depletion in DEREG mice from samples of early time-points before Treg cell depletion in these mice and from gut microbiota samples of wild-type mice. Treg cell depletion in DEREG mice was accompanied by an increase in the relative abundance of the phylum Firmicutes and by intestinal inflammation in DEREG mice 20 days after Treg cell depletion, indicating that Treg cells influence the gut microbiota composition. In addition, the variables cage, breeding and experiment number were associated with differences in the gut microbiota composition and these variables should be respected in murine studies.

Amish (Rural) vs. non-Amish (Urban) Infant Fecal Microbiotas Are Highly Diverse and Their Transplantation Lead to Differences in Mucosal Immune Maturation in a Humanized Germfree Piglet Model.

The gut microbiome plays an important role in the immune system development, maintenance of normal health status, and in disease progression. In this study, we comparatively examined the fecal microbiomes of Amish (rural) and non-Amish (urban) infants and investigated how they could affect the mucosal immune maturation in germ-free piglets that were inoculated with the two types of infant fecal microbiota (IFM). Differences in microbiome diversity and structure were noted between the two types of fecal microbiotas. The fecal microbiota of the non-Amish (urban) infants had a greater relative abundance of Actinobacteria and Bacteroidetes phyla, while that of the Amish (rural) counterparts was dominated by Firmicutes. Amish infants had greater species richness compared with the non-Amish infants' microbiota. The fecal microbiotas of the Amish and the non-Amish infants were successfully transplanted into germ-free piglets, and the diversity and structure of the microbiota in the transplanted piglets remained similar at phylum level but not at the genus level. Principal coordinates analysis (PCoA) based on Weighted-UniFrac distance revealed distinct microbiota structure in the intestines of the transplanted piglets. Shotgun metagenomic analysis also revealed clear differences in functional diversity of fecal microbiome between Amish and non-Amish donors as well as microbiota transplanted piglets. Specific functional features were enriched in either of the microbiota transplanted piglet groups directly corresponding to the predominance of certain bacterial populations in their gut environment. Some of the colonized bacterial genera were correlated with the frequency of important lymphoid and myeloid immune cells in the ileal submucosa and mesenteric lymph nodes (MLN), both important for mucosal immune maturation. Overall, this study demonstrated that transplantation of diverse IFM into germ-free piglets largely recapitulates the differences in gut microbiota structure between rural (Amish) and urban (non-Amish) infants. Thus, fecal microbiota transplantation to germ-free piglets could be a useful large animal model system for elucidating the impact of gut microbiota on the mucosal immune system development. Future studies can focus on determining the additional advantages of the pig model over the rodent model.

Commensal Microbes Affect Host Humoral Immunity to Bordetella pertussis Infection.

As important players in the host defense system, commensal microbes and the microbiota influence multiple aspects of host physiology. Bordetella pertussis infection is highly contagious among humans. However, the roles of the microbiota in B. pertussis pathogenesis are poorly understood. Here, we show that antibiotic-mediated depletion of the microbiota results in increased susceptibility to B. pertussis infection during the early stage. The increased susceptibility was associated with a marked impairment of the systemic IgG, IgG2a, and IgG1 antibody responses to B. pertussis infection after antibiotic treatment. Furthermore, the microbiota impacted the short-lived plasma cell responses as well as the recall responses of memory B cells to B. pertussis infection. Finally, we found that the dysbiosis caused by antibiotic treatment affects CD4+ T cell generation and PD-1 expression on CD4+ T cells and thereby perturbs plasma cell differentiation. Our results have revealed the importance of commensal microbes in modulating host immune responses to B. pertussis infection and support the possibility of controlling the severity of B. pertussis infection in humans by manipulating the microbiota.

Lung inflammation and disease: A perspective on microbial homeostasis and metabolism.

It is now well appreciated that the human microbiome plays a significant role in a number of processes in the body, significantly affecting its metabolic, inflammatory, and immune homeostasis. Recent research has revealed that almost every mucosal surface in the human body is associated with a resident commensal microbiome of its own. While the gut microbiome and its role in regulation of host metabolism along with its alteration in a disease state has been well studied, there is a lacuna in understanding the resident microbiota of other mucosal surfaces. Among these, the scientific information on the role of lung microbiota in pulmonary diseases is currently severely limited. Historically, lungs have been considered to be sterile and lung diseases have only been studied in the context of bacterial pathogenesis. Recently however, studies have revealed a resilient microbiome in the upper and lower respiratory tracts and there is increased evidence on its central role in respiratory diseases. Knowledge of lung microbiome and its metabolic fallout (local and systemic) is still in its nascent stages and attracting immense interest in recent times. In this review, we will provide a perspective on lung-associated metabolic disorders defined for lung diseases (e.g., chronic obstructive pulmonary disease, asthma, and respiratory depression due to infection) and correlate it with lung microbial perturbation. Such perturbations may be due to altered biochemical or metabolic stress as well. Finally, we will draw evidence from microbiome and classical microbiology literature to demonstrate how specific lung morbidities associate with specific metabolic characteristics of the disease, and with the role of microbiome in this context. © 2018 IUBMB Life, 71(1):152-165, 2019.

Correlations between intestinal innate immune genes and cecal microbiota highlight potential for probiotic development for immune modulation in poultry.

Immune function is influenced by the diversity and stability of the intestinal microbiota. A likely trade-off of immune function for growth has been demonstrated in heavier breeds of poultry that have been genetically selected for growth and feed efficiency traits. We investigated the expression of selected innate immune genes and genes encoding products involved in intestinal barrier function to determine whether function changes could be consistently linked to the phenotypic expression of feed conversion ratio (FCR), a common measure of performance within poultry broiler flocks. In addition, we compared individual cecal microbial composition with innate immune gene expression. Samples were utilised from two replicate trials termed P1E1 and P1E2. High (n = 12) and low (n = 12) performing birds were selected based on their individual FCR data from each replicate and combined for microbiota phylogenetic composition and immune gene expression analysis. Toll-like receptor 1 (TLR1La) and zonula occludens 1 (ZO1) were differentially expressed between high- and low-performing broilers. Several taxa were correlated with FCR; of these, unclassified YS2 and ZO1 were also positively correlated with each other. Interactions between taxa and differentially expressed innate immune genes between P1E1 and P1E2 were much greater compared to relationships between high- and low-performing birds. At the level of phylum, reciprocal correlations between tight junction proteins and Toll-like receptors with Bacteroidetes and Firmicutes were evident, as were correlations at the genus level.

Intestinal Inflammation in Chilean Infants Fed With Bovine Formula vs. Breast Milk and Its Association With Their Gut Microbiota.

Introduction: Compared to bovine formula (BF), breast milk (BM) has unique properties. In the newborn intestine, there is a homeostatic balance between the counterparts of the immune system, which allows a physiological inflammation, modulated by the gut microbiota. Many studies have attempted to understand the effect of BF vs. BM, and the changes in the gut microbiota, but few also focus on intestinal inflammation. Methods: We conducted a cohort study of newborn infants during their first 3 months. In stool samples taken at 1 and 3 months (timepoints T1 and T3), we quantified calprotectin, IL-8 and α1-antitrypsin by ELISA and we evaluated the expression of IL8 and IL1ß genes by RT-qPCR. To determine the microbiota composition, the 16S rRNA gene was amplified and sequenced using 454 pyrosequencing. Sequences were clustered into operational taxonomic units (OTUs). Results: In total 15 BM and 10 BF infants were enrolled. In the BM group, we found calprotectin and α1-antitrypsin levels were significantly elevated at T3 compared to T1; no differences were found between T1 and T3 in the BF group. A comparison between the BM and BF groups showed that calprotectin levels at T1 were lower in the BM than the BF group; this difference was not observed at T3. For IL-8 levels, we found no differences between groups. A gene expression analysis of the IL8 and IL1ß genes showed that infants from the BF group at T1 have a significantly increased expression of these markers compared to the BM group. Gut microbiota analyses revealed that the phylum Bacteroidetes was higher in BM than BF, whereas Firmicutes were higher in BF. A redundancy analysis and ANOVA showed BM has a community structure statistically different to BF at T1 but not at T3. Compared to BF, BM at T1 showed a higher representation of Enterococcus, Streptococcus, Enterobacter, Lactococcus, and Propionibacterium. Conclusions: We found a basal state of inflammation in the infants' intestine based on inflammation markers. One month after birth, infants receiving BF exhibited higher levels of inflammation compared to BM.

TRIF deficiency protects non-obese diabetic mice from type 1 diabetes by modulating the gut microbiota and dendritic cells.

The incidence of type 1 diabetes (T1D) is determined by both genetic and environmental factors. In recent years, the gut microbiota have been identified to be an important environmental factor that could modify diabetes susceptibility. We have previously shown that Myeloid differentiation primary response gene 88 (MyD88), a major adaptor protein downstream of most innate immune Toll-like receptor (TLR) signaling, is important for mediating diabetes susceptibility in the non-obese diabetic (NOD) mouse model of human T1D. Here we report the role of TIR-domain-containing adapter-inducing interferon-ß (TRIF) in T1D development, as TRIF is an important adaptor protein downstream of TLR3 and TLR4 signaling. We found that TRIF-deficient (TRIF-/-) NOD mice were protected from development of diabetes, but only when housed with TRIF-deficient (TRIF-/-) NOD mice. When housed with TRIF-sufficient wild type (WT, i.e., TRIF+/+) NOD mice, the mice developed diabetes. We further investigated the gut microbiota as a potential cause for the altered diabetes development. Interestingly, TRIF-/-NOD mice had a different microbiota composition compared to WT NOD mice, only if they were housed with TRIF-/-NOD mice. However, the composition of gut microbiota in the TRIF-/-NOD mice was indistinguishable from WT NOD mice, if they were housed with WT NOD mice. The difference in the gut microbiota in TRIF-/-NOD mice, due to cohousing, accorded with the diabetes development in TRIF-/-NOD mice. Comparing the gut microbiota in TRIF-/- and WT NOD mice, we identified changes in percentage of Sutterella, Rikenella and Turicibacter species. Moreover, bacteria from WT NOD mice induced significantly stronger inflammatory immune responses in vitro compared to those from TRIF-/-NOD mice. Further immunological analysis revealed impaired function of dendritic cells and reduced T cell activation and proliferation in TRIF-/-NOD mice. Our data show that TRIF-deficiency protects NOD mice from diabetes development through alteration of the gut microbiota and reduced immune cell activation; however, that protection is over-ridden upon exposure to WT NOD bacteria. Therefore exposure to different microbiota can modify disease susceptibility determined by genetic factors related to innate immunity.

Microbial dysbiosis associated with impaired intestinal Na+/H+ exchange accelerates and exacerbates colitis in ex-germ free mice.

Intestinal epithelial Na+/H+ exchange facilitated by the apical NHE3 (Slc9a3) is a highly regulated process inhibited by intestinal pathogens and in inflammatory bowel diseases. NHE3-/- mice develop spontaneous, bacterially mediated colitis, and IBD-like dysbiosis. Disruption of epithelial Na+/H+ exchange in IBD may thus represent a host response contributing to the altered gut microbial ecology, and may play a pivotal role in modulating the severity of inflammation in a microbiome-dependent manner. To test whether microbiome fostered in an NHE3-deficient environment is able to drive mucosal immune responses affecting the onset or severity of colitis, we performed a series of cohousing experiments and fecal microbiome transplants into germ-free Rag-deficient or IL-10-/- mice. We determined that in the settings where the microbiome of NHE3-deficient mice was stably engrafted in the recipient host, it was able accelerate the onset and amplify severity of experimental colitis. NHE3-deficiency was characterized by the reduction in pH-sensitive butyrate-producing Firmicutes families Lachnospiraceae and Ruminococcaceae (Clostridia clusters IV and XIVa), with an expansion of inflammation-associated Bacteroidaceae. We conclude that the microbiome fostered by impaired epithelial Na+/H+ exchange enhances the onset and severity of colitis through disruption of the gut microbial ecology.

Restructuring of the Gut Microbiome by Intermittent Fasting Prevents Retinopathy and Prolongs Survival in db/db Mice.

Intermittent fasting (IF) protects against the development of metabolic diseases and cancer, but whether it can prevent diabetic microvascular complications is not known. In db/db mice, we examined the impact of long-term IF on diabetic retinopathy (DR). Despite no change in glycated hemoglobin, db/db mice on the IF regimen displayed significantly longer survival and a reduction in DR end points, including acellular capillaries and leukocyte infiltration. We hypothesized that IF-mediated changes in the gut microbiota would produce beneficial metabolites and prevent the development of DR. Microbiome analysis revealed increased levels of Firmicutes and decreased Bacteroidetes and Verrucomicrobia. Compared with db/db mice on ad libitum feeding, changes in the microbiome of the db/db mice on IF were associated with increases in gut mucin, goblet cell number, villi length, and reductions in plasma peptidoglycan. Consistent with the known modulatory effects of Firmicutes on bile acid (BA) metabolism, measurement of BAs demonstrated a significant increase of tauroursodeoxycholate (TUDCA), a neuroprotective BA, in db/db on IF but not in db/db on AL feeding. TGR5, the TUDCA receptor, was found in the retinal primary ganglion cells. Expression of TGR5 did not change with IF or diabetes. However, IF reduced retinal TNF-α mRNA, which is a downstream target of TGR5 activation. Pharmacological activation of TGR5 using INT-767 prevented DR in a second diabetic mouse model. These findings support the concept that IF prevents DR by restructuring the microbiota toward species producing TUDCA and subsequent retinal protection by TGR5 activation.

Computer-guided design of optimal microbial consortia for immune system modulation.

Manipulation of the gut microbiota holds great promise for the treatment of diseases. However, a major challenge is the identification of therapeutically potent microbial consortia that colonize the host effectively while maximizing immunologic outcome. Here, we propose a novel workflow to select optimal immune-inducing consortia from microbiome compositicon and immune effectors measurements. Using published and newly generated microbial and regulatory T-cell (Treg) data from germ-free mice, we estimate the contributions of twelve Clostridia strains with known immune-modulating effect to Treg induction. Combining this with a longitudinal data-constrained ecological model, we predict the ability of every attainable and ecologically stable subconsortium in promoting Treg activation and rank them by the Treg Induction Score (TrIS). Experimental validation of selected consortia indicates a strong and statistically significant correlation between predicted TrIS and measured Treg. We argue that computational indexes, such as the TrIS, are valuable tools for the systematic selection of immune-modulating bacteriotherapeutics.