Role of disease-associated tolerance in infectious superspreaders.

Natural populations show striking heterogeneity in their ability to transmit disease. For example, a minority of infected individuals known as superspreaders carries out the majority of pathogen transmission events. In a mouse model of Salmonella infection, a subset of infected hosts becomes superspreaders, shedding high levels of bacteria (>10(8) cfu per g of feces) but remain asymptomatic with a dampened systemic immune state. Here we show that superspreader hosts remain asymptomatic when they are treated with oral antibiotics. In contrast, nonsuperspreader Salmonella-infected hosts that are treated with oral antibiotics rapidly shed superspreader levels of the pathogen but display signs of morbidity. This morbidity is linked to an increase in inflammatory myeloid cells in the spleen followed by increased production of acute-phase proteins and proinflammatory cytokines. The degree of colonic inflammation is similar in antibiotic-treated superspreader and nonsuperspreader hosts, indicating that the superspreader hosts are tolerant of antibiotic-mediated perturbations in the intestinal tract. Importantly, neutralization of acute-phase proinflammatory cytokines in antibiotic-induced superspreaders suppresses the expansion of inflammatory myeloid cells and reduces morbidity. We describe a unique disease-associated tolerance to oral antibiotics in superspreaders that facilitates continued transmission of the pathogen.

Host-centric proteomics of stool: a novel strategy focused on intestinal responses to the gut microbiota.

The diverse community of microbes that inhabits the human bowel is vitally important to human health. Host-expressed proteins are essential for maintaining this mutualistic relationship and serve as reporters on the status of host-microbiota interaction. Therefore, unbiased and sensitive methods focused on host proteome characterization are needed. Herein we describe a novel method for applying shotgun proteomics to the analysis of feces, focusing on the secreted host proteome. We have conducted the most complete analysis of the extracellular mouse gut proteome to date by employing a gnotobiotic mouse model. Using mice colonized with defined microbial communities of increasing complexity or a complete human microbiota ('humanized'), we show that the complexity of the host stool proteome mirrors the complexity of microbiota composition. We further show that host responses exhibit signatures specific to the different colonization states. We demonstrate feasibility of this approach in human stool samples and provide evidence for a "core" stool proteome as well as personalized host response features. Our method provides a new avenue for noninvasive monitoring of host-microbiota interaction dynamics via host-produced proteins in stool.

A mutation rate model at the basepair resolution identifies the mutagenic effect of polymerase III transcription.

De novo mutations occur at substantially different rates depending on genomic location, sequence context and DNA strand. The success of methods to estimate selection intensity, infer demographic history and map rare disease genes, depends strongly on assumptions about the local mutation rate. Here we present Roulette, a genome-wide mutation rate model at basepair resolution that incorporates known determinants of local mutation rate. Roulette is shown to be more accurate than existing models. We use Roulette to refine the estimates of population growth within Europe by incorporating the full range of human mutation rates. The analysis of significant deviations from the model predictions revealed a tenfold increase in mutation rate in nearly all genes transcribed by polymerase III (Pol III), suggesting a new mutagenic mechanism. We also detected an elevated mutation rate within transcription factor binding sites restricted to sites actively used in testis and residing in promoters.

The Fibronectin-ILT3 Interaction Functions as a Stromal Checkpoint that Suppresses Myeloid Cells.

Suppressive myeloid cells inhibit antitumor immunity by preventing T-cell responses. Immunoglobulin-like transcript 3 (ILT3; also known as LILRB4) is highly expressed on tumor-associated myeloid cells and promotes their suppressive phenotype. However, the ligand that engages ILT3 within the tumor microenvironment and renders tumor-associated myeloid cells suppressive is unknown. Using a screening approach, we identified fibronectin as a functional ligand for ILT3. The interaction of fibronectin with ILT3 polarized myeloid cells toward a suppressive state, and these effects were reversed with an ILT3-specific antibody that blocked the interaction of ILT3 with fibronectin. Furthermore, ex vivo treatment of human tumor explants with anti-ILT3 reprogrammed tumor-associated myeloid cells toward a stimulatory phenotype. Thus, the ILT3-fibronectin interaction represents a "stromal checkpoint" through which the extracellular matrix actively suppresses myeloid cells. By blocking this interaction, tumor-associated myeloid cells may acquire a stimulatory phenotype, potentially resulting in increased antitumor T-cell responses.

LEAP2 Is an Endogenous Antagonist of the Ghrelin Receptor.

Ghrelin, an appetite-stimulatory hormone secreted by the stomach, was discovered as a ligand for the growth hormone secretagogue receptor (GHSR). Through GHSR, ghrelin stimulates growth hormone (GH) secretion, a function that evolved to protect against starvation-induced hypoglycemia. Though the biology mediated by ghrelin has been described in great detail, regulation of ghrelin action is poorly understood. Here, we report the discovery of liver-expressed antimicrobial peptide 2 (LEAP2) as an endogenous antagonist of GHSR. LEAP2 is produced in the liver and small intestine, and its secretion is suppressed by fasting. LEAP2 fully inhibits GHSR activation by ghrelin and blocks the major effects of ghrelin in vivo, including food intake, GH release, and maintenance of viable glucose levels during chronic caloric restriction. In contrast, neutralizing antibodies that block endogenous LEAP2 function enhance ghrelin action in vivo. Our findings reveal a mechanism for fine-tuning ghrelin action in response to changing environmental conditions.

Seasonal cycling in the gut microbiome of the Hadza hunter-gatherers of Tanzania.

Although humans have cospeciated with their gut-resident microbes, it is difficult to infer features of our ancestral microbiome. Here, we examine the microbiome profile of 350 stool samples collected longitudinally for more than a year from the Hadza hunter-gatherers of Tanzania. The data reveal annual cyclic reconfiguration of the microbiome, in which some taxa become undetectable only to reappear in a subsequent season. Comparison of the Hadza data set with data collected from 18 populations in 16 countries with varying lifestyles reveals that gut community membership corresponds to modernization: Notably, the taxa within the Hadza that are the most seasonally volatile similarly differentiate industrialized and traditional populations. These data indicate that some dynamic lineages of microbes have decreased in prevalence and abundance in modernized populations.

Host-Microbiota Interactions in the Pathogenesis of Antibiotic-Associated Diseases.

Improved understanding of the interplay between host and microbes stands to illuminate new avenues for disease diagnosis, treatment, and prevention. Here, we provide a high-resolution view of the dynamics between host and gut microbiota during antibiotic-induced intestinal microbiota depletion, opportunistic Salmonella typhimurium and Clostridium difficile pathogenesis, and recovery from these perturbed states in a mouse model. Host-centric proteome and microbial community profiles provide a nuanced longitudinal view, revealing the interdependence between host and microbiota in evolving dysbioses. Time- and condition-specific molecular and microbial signatures are evident and clearly distinguished from pathogen-independent inflammatory fingerprints. Our data reveal that mice recovering from antibiotic treatment or C. difficile infection retain lingering signatures of inflammation, despite compositional normalization of the microbiota, and host responses could be rapidly and durably relieved through fecal transplant. These experiments demonstrate insights that emerge from the combination of these orthogonal, untargeted approaches to the gastrointestinal ecosystem.

The effect of microbial colonization on the host proteome varies by gastrointestinal location.

Endogenous intestinal microbiota have wide-ranging and largely uncharacterized effects on host physiology. Here, we used reverse-phase liquid chromatography-coupled tandem mass spectrometry to define the mouse intestinal proteome in the stomach, jejunum, ileum, cecum and proximal colon under three colonization states: germ-free (GF), monocolonized with Bacteroides thetaiotaomicron and conventionally raised (CR). Our analysis revealed distinct proteomic abundance profiles along the gastrointestinal (GI) tract. Unsupervised clustering showed that host protein abundance primarily depended on GI location rather than colonization state and specific proteins and functions that defined these locations were identified by random forest classifications. K-means clustering of protein abundance across locations revealed substantial differences in host protein production between CR mice relative to GF and monocolonized mice. Finally, comparison with fecal proteomic data sets suggested that the identities of stool proteins are not biased to any region of the GI tract, but are substantially impacted by the microbiota in the distal colon.

Monitoring host responses to the gut microbiota.

The gastrointestinal (GI) ecosystem is increasingly understood to be a fundamental component of health, and has been identified as a new focal point for diagnosing, correcting and preventing countless disorders. Shotgun DNA sequencing has emerged as the dominant technology for determining the genetic and microbial composition of the gut microbiota. This technology has linked microbiota dysbioses to numerous GI diseases including inflammatory bowel disease, obesity and allergy, and to non-GI diseases like autism and depression. The importance of establishing causality in the deterioration of the host-microbiota relationship is well appreciated; however, discovery of candidate molecules and pathways that underlie mechanisms remains a major challenge. Targeted approaches, transcriptional assays, cytokine panels and imaging analyses, applied to animals, have yielded important insight into host responses to the microbiota. However, non-invasive, hypothesis-independent means of measuring host responses in humans are necessary to keep pace with similarly unbiased sequencing efforts that monitor microbes. Mass spectrometry-based proteomics has served this purpose in many other fields, but stool proteins exist in such diversity and dynamic range as to overwhelm conventional proteomics technologies. Focused analysis of host protein secretion into the gut lumen and monitoring proteome-level dynamics in stool provides a tractable route toward non-invasively evaluating dietary, microbial, surgical or pharmacological intervention efficacies. This review is intended to guide GI biologists and clinicians through the methods currently used to elucidate host responses in the gut, with a specific focus on mass spectrometry-based shotgun proteomics applied to the study of host protein dynamics within the GI ecosystem.

Quantitative Imaging of Gut Microbiota Spatial Organization.

Genomic technologies have significantly advanced our understanding of the composition and diversity of host-associated microbial populations. However, their spatial organization and functional interactions relative to the host have been more challenging to study. Here we present a pipeline for the assessment of intestinal microbiota localization within immunofluorescence images of fixed gut cross-sections that includes a flexible software package, BacSpace, for high-throughput quantification of microbial organization. Applying this pipeline to gnotobiotic and human microbiota-colonized mice, we demonstrate that elimination of microbiota-accessible carbohydrates (MACs) from the diet results in thinner mucus in the distal colon, increased proximity of microbes to the epithelium, and heightened expression of the inflammatory marker REG3ß. Measurements of microbe-microbe proximity reveal that a MAC-deficient diet alters monophyletic spatial clustering. Furthermore, we quantify the invasion of Helicobacter pylori into the glands of the mouse stomach relative to host mitotic progenitor cells, illustrating the generalizability of this approach.