Stephanie Grunberg

Innate Lymphoid Cells Promote Anatomical Containment of Lymphoid-Resident Commensal Bacteria

Protecting Against a Barrier Breach In order to coexist peacefully, a “firewall” exists that keeps the commensal bacteria that reside in our intestines and associated lymphoid tissue contained. Several diseases and infections, however, lead to a breach in this barrier, which leads to chronic inflammation and pathology. Sonnenberg et al. (p. 1321) found that in mice, innate lymphoid cells (ILCs) are critically important for the anatomical containment of commensal bacteria in an interleukin-22 (IL-22)–dependent manner. ILC depletion or blockade of IL-22 led to loss of bacterial containment and systemic inflammation. Lymphocytes prevent bacteria from spreading beyond gut-associated lymphoid tissues and causing systemic inflammation. 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.

Innate lymphoid cells regulate CD4 + T-cell responses to intestinal 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. Alterations in ILC responses are associated with multiple chronic human diseases, including inflammatory bowel disease, implicating a role for ILCs in disease pathogenesis. Owing to an inability to target ILCs selectively, experimental studies assessing ILC function have predominantly used mice lacking adaptive immune cells. 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.

Archaea and Fungi of the Human Gut Microbiome: Correlations with Diet and Bacterial Residents

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.

Hypervariable loci in the human gut virome

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.

Histone deacetylase 3 coordinates commensal-bacteria-dependent intestinal homeostasis

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. 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 (HDAC3ΔIEC mice) exhibited extensive dysregulation of IEC-intrinsic gene expression, including decreased basal expression of genes associated with antimicrobial defence. Critically, conventionally housed HDAC3ΔIEC mice demonstrated loss of Paneth cells, impaired IEC function and alterations in the composition of intestinal commensal bacteria. In addition, HDAC3ΔIEC 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 HDAC3ΔIEC 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.

Fungi of the murine gut: episodic variation and proliferation during antibiotic treatment.

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.

A tool kit for quantifying eukaryotic rRNA gene sequences from human microbiome samples

Eukaryotic microorganisms are important but understudied components of the human microbiome. Here we present a pipeline for analysis of deep sequencing data on single cell eukaryotes. We designed a new 18S rRNA gene-specific PCR primer set and compared a published rRNA gene internal transcribed spacer (ITS) gene primer set. Amplicons were tested against 24 specimens from defined eukaryotes and eight well-characterized human stool samples. A software pipeline https://sourceforge.net/projects/brocc/ was developed for taxonomic attribution, validated against simulated data, and tested on pyrosequence data. This study provides a well-characterized tool kit for sequence-based enumeration of eukaryotic organisms in human microbiome samples.

Fungal Signature in the Gut Microbiota of Pediatric Patients With Inflammatory Bowel Disease.

Background: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. Methods:A 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. Results:Patients with IBD (Crohns 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. Conclusions:Pediatric 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.

Alterations of the Subgingival Microbiota in Pediatric Crohn's Disease Studied Longitudinally in Discovery and Validation Cohorts.

Background:Oral manifestations are common in Crohns 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. Methods:Pediatric patients with CD were recruited from The Childrens 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). Results:Most 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. Conclusions:Subgingival 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.

An Oxygen Equilibrium at the Host-Microbial Interface Determined by Phosphorescent Nanoprobe Technology: P-257 YI

BACKGROUND: The intestines of all mammals are colonized with a diverse microbiota that provide metabolic benefits to their hosts. However, this symbiotic relationship can break down when resident bacteria opportunistically invade the intestinal barrier, leading to pathologies such as inflammatory bowel disease (IBD) and bacteremia. As a result, epithelial cell innate immune responses play an essential role in preventing bacterial invasion of host tissues and maintaining a symbiotic host-bacterial relationship. Autophagy is emerging as an important component of innate immunity. Mounting evidence suggests that dysregulation of autophagy can lead to inflammatory bowel disease. Additionally, increased bacterial invasion of epithelial cells is a hallmark of IBD. Little is known, however, about the role of autophagy in controlling interactions between intestinal bacteria and the intestinal epithelium in vivo. METHODS: The present study was conducted to evaluate the role of autophagy in the epithelial cells of the small intestine. We utilized a known intestinal pathogen, Salmonella typhimurium, and monitored enterocyte autophagy activation in gnotobiotic (germ-free) and conventionally (CV) raised wild-type mice following intragastric bacterial challenge. Autophagy activation was analyzed via immunofluorescence, Western blot analysis and electron microscopy. We next investigated the role of Toll-like receptors (TLRs) on autophagy activation by utilizing mice deficient in myeloid differentiation protein (MyD88), an adaptor molecule essential for TLR signaling. Finally, we demonstrated a critical role for epithelial cell autophagy by generating mice lacking Atg5, a critical autophagy protein, specifically in the epithelial cells of the small intestine, and monitoring the ability of Salmonella to translocate to extraintestinal sites including the liver and spleen. RESULTS: In this study, we demonstrate that small intestinal epithelial cell autophagy is essential for protection against tissue invasion by intestinal pathogens and opportunistically invasive commensals. We show that small intestinal autophagy is an early innate immune response that functions in an epithelial cell-intrinsic MyD88 dependent, NOD2-independent manner. Utilizing mice deficient in small intestinal epithelial cell autophagy (Atg5, we have determined that epithelial cell autophagy is required to limit pathogen dissemination to extraintestinal sites, including the liver and spleen. CONCLUSION(S): This study represents the first comprehensive and mechanistic in vivo dissection of the microbiota capable of activating epithelial cell autophagy, the pattern recognition receptors (PRRs) required for this activation, as well as the critical antibacterial role of epithelial cell autophagy in the intestine. A deeper understanding of how small intestine epithelial cell autophagy functions will be crucial in understanding how autophagy mutations predispose for IBD development and thus improve treatment modalities for both Crohn’s Disease and ulcerative colitis