Explore the vast range of titles available.

Diet influences health as a source of nutrients and toxins, and by shaping the composition of resident microbial populations. Previous studies have begun to map out associations between diet and the bacteria and viruses of the human gut microbiome. Here we investigate associations of diet with fungal and archaeal populations, taking advantage of samples from 98 well-characterized individuals. Diet was quantified using inventories scoring both long-term and recent diet, and archaea and fungi were characterized by deep sequencing of marker genes in DNA purified from stool. For fungi, we found 66 genera, with generally mutually exclusive presence of either the phyla Ascomycota or Basiodiomycota. For archaea, Methanobrevibacter was the most prevalent genus, present in 30% of samples. Several other archaeal genera were detected in lower abundance and frequency. Myriad associations were detected for fungi and archaea with diet, with each other, and with bacterial lineages. Methanobrevibacter and Candida were positively associated with diets high in carbohydrates, but negatively with diets high in amino acids, protein, and fatty acids. A previous study emphasized that bacterial population structure was associated primarily with long-term diet, but high Candida abundance was most strongly associated with the recent consumption of carbohydrates. Methobrevibacter abundance was associated with both long term and recent consumption of carbohydrates. These results confirm earlier targeted studies and provide a host of new associations to consider in modeling the effects of diet on the gut microbiome and human health.

The mammalian intestinal tract is colonized by trillions of beneficial commensal bacteria that are anatomically restricted to specific niches. However, the mechanisms that regulate anatomical containment remain unclear. Here, we show that interleukin-22 (IL-22)-producing innate lymphoid cells (ILCs) are present in intestinal tissues of healthy mammals. Depletion of ILCs resulted in peripheral dissemination of commensal bacteria and systemic inflammation, which was prevented by administration of IL-22. Disseminating bacteria were identified as Alcaligenes species originating from host lymphoid tissues. Alcaligenes was sufficient to promote systemic inflammation after ILC depletion in mice, and Alcaligenes-specific systemic immune responses were associated with Crohn's disease and progressive hepatitis C virus infection in patients. Collectively, these data indicate that ILCs regulate selective containment of lymphoid-resident bacteria to prevent systemic inflammation associated with chronic diseases.

Antibiotic use in humans has been associated with outgrowth of fungi. Here we used a murine model to investigate the gut microbiome over 76 days of treatment with vancomycin, ampicillin, neomycin, and metronidazole and subsequent recovery. Mouse stool was studied as a surrogate for the microbiota of the lower gastrointestinal tract. The abundance of fungi and bacteria was measured using quantitative PCR, and the proportional composition of the communities quantified using 454/Roche pyrosequencing of rRNA gene tags. Prior to treatment, bacteria outnumbered fungi by >3 orders of magnitude. Upon antibiotic treatment, bacteria dropped in abundance >3 orders of magnitude, so that the predominant 16S sequences detected became transients derived from food. Upon cessation of treatment, bacterial communities mostly returned to their previous numbers and types after 8 weeks, though communities remained detectably different from untreated controls. Fungal communities varied substantially over time, even in the untreated controls. Separate cages within the same treatment group showed radical differences, but mice within a cage generally behaved similarly. Fungi increased ∼40-fold in abundance upon antibiotic treatment but declined back to their original abundance after cessation of treatment. At the last time point, Candida remained more abundant than prior to treatment. These data show that 1) gut fungal populations change radically during normal mouse husbandry, 2) fungi grow out in the gut upon suppression of bacterial communities with antibiotics, and 3) perturbations due to antibiotics persist long term in both the fungal and bacterial microbiota.

Genetic variation is critical in microbial immune evasion and drug resistance, but variation has rarely been studied in complex heterogeneous communities such as the human microbiome. To begin to study natural variation, we analyzed DNA viruses present in the lower gastrointestinal tract of 12 human volunteers by determining 48 billion bases of viral DNA sequence. Viral genomes mostly showed low variation, but 51 loci of ∼100 bp showed extremely high variation, so that up to 96% of the viral genomes encoded unique amino acid sequences. Some hotspots of hypervariation were in genes homologous to the bacteriophage BPP-1 viral tail-fiber gene, which is known to be hypermutagenized by a unique reverse-transcriptase (RT)-based mechanism. Unexpectedly, other hypervariable loci in our data were in previously undescribed gene types, including genes encoding predicted Ig-superfamily proteins. Most of the hypervariable loci were linked to genes encoding RTs of a single clade, which we find is the most abundant clade among gut viruses but only a minor component of bacterial RT populations. Hypervariation was targeted to 5'-AAY-3' asparagine codons, which allows maximal chemical diversification of the encoded amino acids while avoiding formation of stop codons. These findings document widespread targeted hypervariation in the human gut virome, identify previously undescribed types of genes targeted for hypervariation, clarify association with RT gene clades, and motivate studies of hypervariation in the full human microbiome.

Oral manifestations are common in Crohn's disease (CD). Here we characterized the subgingival microbiota in pediatric patients with CD initiating therapy and after 8 weeks to identify microbial community features associated with CD and therapy.MethodsPediatric patients with CD were recruited from The Children's Hospital of Pennsylvania. Healthy control subjects were recruited from primary care or orthopedics clinic. Subgingival plaque samples were collected at initiation of therapy and after 8 weeks. Treatment exposures included 5-ASAs, immunomodulators, steroids, and infliximab. The microbiota was characterized by 16S rRNA gene sequencing. The study was repeated in separate discovery (35 CD, 43 healthy) and validation cohorts (43 CD, 31 healthy).ResultsMost subjects in both cohorts demonstrated clinical response after 8 weeks of therapy (discovery cohort 88%, validation cohort 79%). At week 0, both antibiotic exposure and disease state were associated with differences in bacterial community composition. Seventeen genera were identified in the discovery cohort as candidate biomarkers, of which 11 were confirmed in the validation cohort. Capnocytophaga, Rothia, and TM7 were more abundant in CD relative to healthy controls. Other bacteria were reduced in abundance with antibiotic exposure among CD subjects. CD-associated genera were not enriched compared with healthy controls after 8 weeks of therapy.ConclusionsSubgingival microbial community structure differed with CD and antibiotic use. Results in the discovery cohort were replicated in a separate validation cohort. Several potentially pathogenic bacterial lineages were associated with CD but were not diminished in abundance by antibiotic treatment, suggesting targets for additional surveillance.

Inflammatory bowel disease (IBD) involves dysregulation of mucosal immunity in response to environmental factors such as the gut microbiota. The bacterial microbiota is often altered in IBD, but the connection to disease is not fully clarified and gut fungi have recently been suggested to play a role as well. In this study, we compared microbes from all 3 domains of life—bacteria, archaea, and eukaryota—in pediatric patients with IBD and healthy controls.MethodsA stool sample was collected from patients with IBD (n = 32) or healthy control subjects (n = 90), and bacterial, archaeal, and fungal communities were characterized by deep sequencing of rRNA gene segments specific to each domain.ResultsPatients with IBD (Crohn's disease or ulcerative colitis) had lower bacterial diversity and distinctive fungal communities. Two lineages annotating as Candida were significantly more abundant in patients with IBD (P = 0.0034 and P = 0.00038, respectively), whereas a lineage annotating as Cladosporium was more abundant in healthy subjects (P = 0.0025). There were no statistically significant differences in archaea, which were rare in pediatric samples compared with those from adults.ConclusionsPediatric IBD is associated with reduced diversity in both fungal and bacterial gut microbiota. Specific Candida taxa were found to be increased in abundance in the IBD samples. These data emphasize the potential importance of fungal microbiota signatures as biomarkers of pediatric IBD, supporting their possible role in disease pathogenesis.

Group 3 innate lymphoid cells are shown to process and present antigen and to control CD4 + T-cell responses to intestinal commensal bacteria through an MHC-class-II-dependent mechanism. Immune response to intestinal bacteria The recently characterized innate lymphoid cells (ILCs) can be classified functionally into three groups. Group 1 ILCs produce interferon-γ, group 2 cells express interleukin (IL)-5, IL-13 and amphiregulin, and group 3 ILCs produce IL-17A and IL-22. The function of ILCs in the presence of adaptive immunity and their potential to influence adaptive immune cell responses are largely unknown. A study in mice now shows that group 3 ILCs process and present antigen and control CD4 + T-cell responses to intestinal commensal bacteria through an MHC-class-II-dependent mechanism. This finding may be relevant to the pathogenesis of chronic human diseases associated with inflammatory host immune responses to commensal bacteria. Innate lymphoid cells (ILCs) are a recently characterized family of immune cells that have critical roles in cytokine-mediated regulation of intestinal epithelial cell barrier integrity 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 . Alterations in ILC responses are associated with multiple chronic human diseases, including inflammatory bowel disease, implicating a role for ILCs in disease pathogenesis 3 , 8 , 11 , 12 , 13 . Owing to an inability to target ILCs selectively, experimental studies assessing ILC function have predominantly used mice lacking adaptive immune cells 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 . However, in lymphocyte-sufficient hosts ILCs are vastly outnumbered by CD4 + T cells, which express similar profiles of effector cytokines. Therefore, the function of ILCs in the presence of adaptive immunity and their potential to influence adaptive immune cell responses remain unknown. To test this, we used genetic or antibody-mediated depletion strategies to target murine ILCs in the presence of an adaptive immune system. We show that loss of retinoic-acid-receptor-related orphan receptor-γt-positive (RORγt + ) ILCs was associated with dysregulated adaptive immune cell responses against commensal bacteria and low-grade systemic inflammation. Remarkably, ILC-mediated regulation of adaptive immune cells occurred independently of interleukin (IL)-17A, IL-22 or IL-23. Genome-wide transcriptional profiling and functional analyses revealed that RORγt + ILCs express major histocompatibility complex class II (MHCII) and can process and present antigen. However, rather than inducing T-cell proliferation, ILCs acted to limit commensal bacteria-specific CD4 + T-cell responses. Consistent with this, selective deletion of MHCII in murine RORγt + ILCs resulted in dysregulated commensal bacteria-dependent CD4 + T-cell responses that promoted spontaneous intestinal inflammation. These data identify that ILCs maintain intestinal homeostasis through MHCII-dependent interactions with CD4 + T cells that limit pathological adaptive immune cell responses to commensal bacteria.

This work identifies a role for intestinal epithelial cell (IEC)-intrinsic expression of histone deacetylase 3 in regulating commensal-bacteria-dependent gene expression and intestinal homeostasis; IEC-specific HDAC3 deficiency gives rise to Paneth cell abnormalities, impaired intestinal barrier function, and increased DSS-induced intestinal inflammation in commensal-bacteria-containing, but not germ-free, mice. HDAC3 role in intestinal homeostasis Epigenetic mechanisms alter the transcriptional response to environmental cues and thus represent a mechanism that can link host genetic predisposition and environmental triggers in the pathogenesis of inflammatory bowel disease (IBD). This study identifies a previously unrecognized role for intestinal epithelial cell (IEC)-intrinsic expression of the epigenome-modifying enzyme histone deacetylase 3 (HDAC3) in regulating intestinal barrier function and susceptibility to commensal-driven inflammation. IEC-specific HDAC3 deficiency in mice is shown to give rise to Paneth-cell abnormalities, rectal prolapse, increased susceptibility to dextran sodium sulphate-induced colitis and increased barrier permeability in commensal-containing but not germ-free animals. The development and severity of inflammatory bowel diseases and other chronic inflammatory conditions can be influenced by host genetic and environmental factors, including signals derived from commensal bacteria 1 , 2 , 3 , 4 , 5 , 6 . However, the mechanisms that integrate these diverse cues remain undefined. Here we demonstrate that mice with an intestinal epithelial cell (IEC)-specific deletion of the epigenome-modifying enzyme histone deacetylase 3 (HDAC3 ΔIEC mice) exhibited extensive dysregulation of IEC-intrinsic gene expression, including decreased basal expression of genes associated with antimicrobial defence. Critically, conventionally housed HDAC3 ΔIEC mice demonstrated loss of Paneth cells, impaired IEC function and alterations in the composition of intestinal commensal bacteria. In addition, HDAC3 ΔIEC mice showed significantly increased susceptibility to intestinal damage and inflammation, indicating that epithelial expression of HDAC3 has a central role in maintaining intestinal homeostasis. Re-derivation of HDAC3 ΔIEC mice into germ-free conditions revealed that dysregulated IEC gene expression, Paneth cell homeostasis and intestinal barrier function were largely restored in the absence of commensal bacteria. Although the specific mechanisms through which IEC-intrinsic HDAC3 expression regulates these complex phenotypes remain to be determined, these data indicate that HDAC3 is a critical factor that integrates commensal-bacteria-derived signals to calibrate epithelial cell responses required to establish normal host–commensal relationships and maintain intestinal homeostasis.

Inflammatory bowel disease (IBD) is associated with a dysbiotic intestinal microbiota. As oral manifestations are commonly seen in Crohn's disease (CD), we hypothesized that the oral microbiota may also be dysbiotic in patients with IBD. Our aim was to characterize the subgingival oral microbiota in a longitudinal cohort of pediatric patients with CD and healthy control (HC) subjects to determine whether there is an overall association with disease as well as with specific CD phenotypes.MethodsCD cohort included new diagnoses whose treatment exposures included 5-ASAs, immunomodulators and steroids, and patients initiated on infliximab (IFX) therapy. Subgingival plaque samples were obtained longitudinally from both patients with CD and healthy controls at 2 time points, week 0 and week 8. In the IFX group, samples were obtained from patients with CD prior to IFX and at week 8 of therapy. Antibiotic exposure was evaluated in a subgroup analysis. Samples were analyzed by 16S rRNA gene sequencing. Disease activity was quantified using the Pediatric Crohn's Disease Activity Index (PCDAI) and by measuring fecal calprotectin (FC) levels.ResultsSubgingival samples were obtained from 35 patients with CD (28 on IFX and 7 new diagnoses) and 46 HC subjects. At time point 2, 80% and 87% of patients with CD demonstrated decrease in PCDAI and FC respectively, reflecting improvement in disease status. No subject had clinical evidence of gingivitis. The subgingival microbiota was different between CD and HC at the first timepoint (unweighted UniFrac, P < 0.03), where there was an increase in the relative abundance of Campylobacter genus in CD (weighted UniFrac, P < 0.001). A comparison of the microbiota between the 2 time points in patients with CD did not show any association with disease activity. Antibiotic exposure was associated with alterations in the subgingival microbiota (weighted and unweighted UniFrac, P < 0.001) with taxanomic alterations reminiscent of those previously described in patients treated for periodontitis. Finally, patients with CD who had extra-intestinal manifestations of joint pain were more similar to each other (beta dispersion, P < 0.02) and exhibited smaller alterations in their subgingival microbiota between the 2 time points (unweighted and weighted Unifrac, P < 0.03 and P < 0.004, respectively) compared to patients with CD without joint pain.ConclusionsIn the absence of gingivitis, the structure of the subgingival microbiota in patients with CD is intrinsically different from that in HC subjects where the proportional abundance of Campylobacter is associated with disease. Within patients with CD, systemic influences associated with the development of a specific extra-intestinal manifestation may also impact upon the subgingival microbiota. Going forward, these data provide hypotheses for testing in future validation data sets. Surprisingly, there was no effect of disease activity, emphasizing the stability of these disease intrinsic associations. Nevertheless, extrinsic environmental factors, such as the use of antibiotics, result in predictable effects on the subgingival microbiota in CD.

IBD is associated with a dysbiotic gut flora. As oral manifestations are commonly found in Crohn disease (CD), we hypothesized that the oral microbiome may also be dysbiotic in patients with IBD. We characterized the subgingival oral microbiota in a longitudinal cohort of pediatric patients with CD and healthy controls to determine association with disease activity.MethodsSubgingival plaque samples were obtained longitudinally from patients with CD and healthy controls at 2 time points. Samples were obtained from patients with CD prior to infliximab and at week 8 of therapy. Samples were analyzed by 16S rDNA 454 sequencing. Clinical records and diet inventories were obtained. Disease activity was measured by Pediatric Crohn Disease Activity Index (PCDAI) and fecal calprotectin (FCP).Results13 patients with CD and 13 controls were included. 85% of patients with CD demonstrated a decrease in PCDAI from severe disease, mean 37.5, to mild or quiescent disease at week 8, mean 13. There was a 77% decrease in the FCP from a mean 819, to a mean 401 at week 8. We used unweighted UniFrac to monitor changes in community membership, and found that the CD community showed greater dispersion than controls (P < 0.0001), suggestive of dysbiosis in the oral bacterial community. These alterations were observed in the absence of clinical gingivitis.Conclusion(s)This is the first study to demonstrate significant alterations in the subgingival oral microbiome in the absence of clinical gingivitis. Increased dispersion of the cohorts with CD suggests loss of regulation of the normal community. Additional analyses are underway to identify the taxa responsible for this finding. Newly diagnosed patients are being recruited to determine disease effect on the oral and gut microbiome.