Nathaniel Hubert

Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy.

Gut microbes affect immunotherapy The unleashing of antitumor T cell responses has ushered in a new era of cancer treatment. Although these therapies can cause dramatic tumor regressions in some patients, many patients inexplicably see no benefit. Mice have been used in two studies to investigate what might be happening. Specific members of the gut microbiota influence the efficacy of this type of immunotherapy (see the Perspective by Snyder et al.). Vétizou et al. found that optimal responses to anticytotoxic T lymphocyte antigen blockade required specific Bacteroides spp. Similarly, Sivan et al. discovered that Bifidobacterium spp. enhanced the efficacy of antiprogrammed cell death ligand 1 therapy. Science, this issue, p. 1079 and p. 1084; see also p. 1031 Gut microbes modulate the effectiveness of cancer immunotherapies in mice. [Also see Perspective by Snyder et al.] T cell infiltration of solid tumors is associated with favorable patient outcomes, yet the mechanisms underlying variable immune responses between individuals are not well understood. One possible modulator could be the intestinal microbiota. We compared melanoma growth in mice harboring distinct commensal microbiota and observed differences in spontaneous antitumor immunity, which were eliminated upon cohousing or after fecal transfer. Sequencing of the 16S ribosomal RNA identified Bifidobacterium as associated with the antitumor effects. Oral administration of Bifidobacterium alone improved tumor control to the same degree as programmed cell death protein 1 ligand 1 (PD-L1)–specific antibody therapy (checkpoint blockade), and combination treatment nearly abolished tumor outgrowth. Augmented dendritic cell function leading to enhanced CD8+ T cell priming and accumulation in the tumor microenvironment mediated the effect. Our data suggest that manipulating the microbiota may modulate cancer immunotherapy.

Effects of diurnal variation of gut microbes and high-fat feeding on host circadian clock function and metabolism.

Circadian clocks and metabolism are inextricably intertwined, where central and hepatic circadian clocks coordinate metabolic events in response to light-dark and sleep-wake cycles. We reveal an additional key element involved in maintaining host circadian rhythms, the gut microbiome. Despite persistence of light-dark signals, germ-free mice fed low or high-fat diets exhibit markedly impaired central and hepatic circadian clock gene expression and do not gain weight compared to conventionally raised counterparts. Examination of gut microbiota in conventionally raised mice showed differential diurnal variation in microbial structure and function dependent upon dietary composition. Additionally, specific microbial metabolites induced under low- or high-fat feeding, particularly short-chain fatty acids, but not hydrogen sulfide, directly modulate circadian clock gene expression within hepatocytes. These results underscore the ability of microbially derived metabolites to regulate or modify central and hepatic circadian rhythm and host metabolic function, the latter following intake of a Westernized diet.

Gene-targeted microfluidic cultivation validated by isolation of a gut bacterium listed in Human Microbiome Project's Most Wanted taxa

Significance Obtaining cultures of microbes is essential for developing knowledge of bacterial genetics and physiology, but many microbes with potential biomedical significance identified from metagenomic studies have not yet been cultured due to the difficulty of identifying growth conditions, isolation, and characterization. We developed a microfluidics-based, genetically targeted approach to address these challenges. This approach corrects sampling bias from differential bacterial growth kinetics, enables the use of growth stimulants available only in small quantities, and allows targeted isolation and cultivation of a previously uncultured microbe from the human cecum that belongs to the high-priority group of the Human Microbiome Project’s “Most Wanted” list. This workflow could be leveraged to isolate novel microbes and focus cultivation efforts on biomedically important targets. This paper describes a microfluidics-based workflow for genetically targeted isolation and cultivation of microorganisms from complex clinical samples. Data sets from high-throughput sequencing suggest the existence of previously unidentified bacterial taxa and functional genes with high biomedical importance. Obtaining isolates of these targets, preferably in pure cultures, is crucial for advancing understanding of microbial genetics and physiology and enabling physical access to microbes for further applications. However, the majority of microbes have not been cultured, due in part to the difficulties of both identifying proper growth conditions and characterizing and isolating each species. We describe a method that enables genetically targeted cultivation of microorganisms through a combination of microfluidics and on- and off-chip assays. This method involves (i) identification of cultivation conditions for microbes using growth substrates available only in small quantities as well as the correction of sampling bias using a “chip wash” technique; and (ii) performing on-chip genetic assays while also preserving live bacterial cells for subsequent scale-up cultivation of desired microbes, by applying recently developed technology to create arrays of individually addressable replica microbial cultures. We validated this targeted approach by cultivating a bacterium, here referred to as isolate microfluidicus 1, from a human cecal biopsy. Isolate microfluidicus 1 is, to our knowledge, the first successful example of targeted cultivation of a microorganism from the high-priority group of the Human Microbiome Project’s “Most Wanted” list, and, to our knowledge, the first cultured representative of a previously unidentified genus of the Ruminococcaceae family.

Comparison of brush and biopsy sampling methods of the ileal pouch for assessment of mucosa-associated microbiota of human subjects

BackgroundMucosal biopsy is the most common sampling technique used to assess microbial communities associated with the intestinal mucosa. Biopsies disrupt the epithelium and can be associated with complications such as bleeding. Biopsies sample a limited area of the mucosa, which can lead to potential sampling bias. In contrast to the mucosal biopsy, the mucosal brush technique is less invasive and provides greater mucosal coverage, and if it can provide equivalent microbial community data, it would be preferable to mucosal biopsies.ResultsWe compared microbial samples collected from the intestinal mucosa using either a cytology brush or mucosal biopsy forceps. We collected paired samples from patients with ulcerative colitis (UC) who had previously undergone colectomy and ileal pouch anal anastomosis (IPAA), and profiled the microbial communities of the samples by sequencing V4-V6 or V4-V5 16S rRNA-encoding gene amplicons. Comparisons of 177 taxa in 16 brush-biopsy sample pairs had a mean R2 of 0.94. We found no taxa that varied significantly between the brush and biopsy samples after adjusting for multiple comparisons (false discovery rate ≤0.05). We also tested the reproducibility of DNA amplification and sequencing in 25 replicate pairs and found negligible variation (mean R2 = 0.99). A qPCR analysis of the two methods showed that the relative yields of bacterial DNA to human DNA were several-fold higher in the brush samples than in the biopsies.ConclusionsMucosal brushing is preferred to mucosal biopsy for sampling the epithelial-associated microbiota. Although both techniques provide similar assessments of the microbial community composition, the brush sampling method has relatively more bacterial to host DNA, covers a larger surface area, and is less traumatic to the epithelium than the mucosal biopsy.

Chronic sleep disruption alters gut microbiota, induces systemic and adipose tissue inflammation and insulin resistance in mice.

Chronic sleep fragmentation (SF) commonly occurs in human populations, and although it does not involve circadian shifts or sleep deprivation, it markedly alters feeding behaviors ultimately promoting obesity and insulin resistance. These symptoms are known to be related to the host gut microbiota. Mice were exposed to SF for 4 weeks and then allowed to recover for 2 weeks. Taxonomic profiles of fecal microbiota were obtained prospectively, and conventionalization experiments were performed in germ-free mice. Adipose tissue insulin sensitivity and inflammation, as well as circulating measures of inflammation, were assayed. Effect of fecal water on colonic epithelial permeability was also examined. Chronic SF-induced increased food intake and reversible gut microbiota changes characterized by the preferential growth of highly fermentative members of Lachnospiraceae and Ruminococcaceae and a decrease of Lactobacillaceae families. These lead to systemic and visceral white adipose tissue inflammation in addition to altered insulin sensitivity in mice, most likely via enhanced colonic epithelium barrier disruption. Conventionalization of germ-free mice with SF-derived microbiota confirmed these findings. Thus, SF-induced metabolic alterations may be mediated, in part, by concurrent changes in gut microbiota, thereby opening the way for gut microbiome-targeted therapeutics aimed at reducing the major end-organ morbidities of chronic SF.

Multiphasic analysis of the temporal development of the distal gut microbiota in patients following ileal pouch anal anastomosis.

BackgroundThe indigenous gut microbiota are thought to play a crucial role in the development and maintenance of the abnormal inflammatory responses that are the hallmark of inflammatory bowel disease. Direct tests of the role of the gut microbiome in these disorders are typically limited by the fact that sampling of the microbiota generally occurs once disease has become manifest. This limitation could potentially be circumvented by studying patients who undergo total proctocolectomy with ileal pouch anal anastomosis (IPAA) for the definitive treatment of ulcerative colitis. A subset of patients who undergo IPAA develops an inflammatory condition known as pouchitis, which is thought to mirror the pathogenesis of ulcerative colitis. Following the development of the microbiome of the pouch would allow characterization of the microbial community that predates the development of overt disease.ResultsWe monitored the development of the pouch microbiota in four patients who underwent IPAA. Mucosal and luminal samples were obtained prior to takedown of the diverting ileostomy and compared to samples obtained 2, 4 and 8 weeks after intestinal continuity had been restored. Through the combined analysis of 16S rRNA-encoding gene amplicons, targeted 16S amplification and microbial cultivation, we observed major changes in structure and function of the pouch microbiota following ileostomy. There is a relative increase in anaerobic microorganisms with the capacity for fermentation of complex carbohydrates, which corresponds to the physical stasis of intestinal contents in the ileal pouch. Compared to the microbiome structure encountered in the colonic mucosa of healthy individuals, the pouch microbial community in three of the four individuals was quite distinct. In the fourth patient, a community that was much like that seen in a healthy colon was established, and this patient also had the most benign clinical course of the four patients, without the development of pouchitis 2 years after IPAA.ConclusionsThe microbiota that inhabit the ileal-anal pouch of patients who undergo IPAA for treatment of ulcerative colitis demonstrate significant structural and functional changes related to the restoration of fecal flow. Our preliminary results suggest once the pouch has assumed the physiologic role previously played by the intact colon, the precise structure and function of the pouch microbiome, relative to a normal colonic microbiota, will determine if there is establishment of a stable, healthy mucosal environment or the reinitiation of the pathogenic cascade that results in intestinal inflammation.

Mutual reinforcement of pathophysiological host-microbe interactions in intestinal stasis models

Chronic diseases arise when there is mutual reinforcement of pathophysiological processes that cause an aberrant steady state. Such a sequence of events may underlie chronic constipation, which has been associated with dysbiosis of the gut. In this study we hypothesized that assemblage of microbial communities, directed by slow gastrointestinal transit, affects host function in a way that reinforces constipation and further maintains selection on microbial communities. In our study, we used two models – an opioid‐induced constipation model in mice, and a humanized mouse model where germ‐free mice were colonized with stool from a patient with constipation‐predominant irritable bowel syndrome (IBS‐C) in humans. We examined the impact of pharmacologically (loperamide)‐induced constipation (PIC) and IBS‐C on the structural and functional profile of the gut microbiota. Germ‐free (GF) mice were colonized with microbiota from PIC donor mice and IBS‐C patients to determine how the microbiota affects the host. PIC and IBS‐C promoted changes in the gut microbiota, characterized by increased relative abundance of Bacteroides ovatus and Parabacteroides distasonis in both models. PIC mice exhibited decreased luminal concentrations of butyrate in the cecum and altered metabolic profiles of the gut microbiota. Colonization of GF mice with PIC‐associated mice cecal or human IBS‐C fecal microbiota significantly increased GI transit time when compared to control microbiota recipients. IBS‐C‐associated gut microbiota also impacted colonic contractile properties. Our findings support the concept that constipation is characterized by disease‐associated steady states caused by reinforcement of pathophysiological factors in host‐microbe interactions.

Early Transcriptomic Changes in the Ileal Pouch Provide Insight into the Molecular Pathogenesis of Pouchitis and Ulcerative Colitis

Background: Ulcerative colitis (UC) only involves the colonic mucosa. Yet, nearly 50% of patients with UC who undergo total proctocolectomy with ileal pouch anal anastomosis develop UC-like inflammation of the ileal pouch (pouchitis). By contrast, patients with familial adenomatous polyposis (FAP) with ileal pouch anal anastomosis develop pouchitis far less frequently. We hypothesized that pathogenic events associated with the development of UC are recapitulated by colonic-metaplastic transcriptomic reprogramming of the UC pouch. Methods: We prospectively sampled pouch and prepouch ileum mucosal biopsies in patients with UC with ileal pouch anal anastomosis 4, 8, and 12 months after their pouch was in continuity. Mucosal samples were also obtained from patients with FAP. Transcriptional profiles of the UC and FAP pouch and prepouch ileum were investigated via RNA sequencing and compared with data from a previously published microarray study. Results: Unlike patients with FAP, subjects with UC exhibited a large set of differentially expressed genes between the pouch and prepouch ileum as early as 4 months after pouch functionalization. Functional pathway analysis of differentially expressed genes in the UC pouch revealed an enhanced state of immune/inflammatory response and extracellular matrix remodeling. Moreover, >70% of differentially expressed genes mapped to published inflammatory bowel diseases microarray data sets displayed directional changes consistent with active UC but not with Crohns disease. Conclusions: The UC pouch, well before histologic inflammation, already displays a systems-level gain of colon-associated genes and loss of ileum-associated genes. Patients with UC exhibit a unique transcriptomic response to ileal pouch creation that can be observed well before disease and may in part explain their susceptibility to the development of pouchitis.BACKGROUND Ulcerative colitis (UC) only involves the colonic mucosa. Yet, nearly 50% of patients with UC who undergo total proctocolectomy with ileal pouch anal anastomosis develop UC-like inflammation of the ileal pouch (pouchitis). By contrast, patients with familial adenomatous polyposis (FAP) with ileal pouch anal anastomosis develop pouchitis far less frequently. We hypothesized that pathogenic events associated with the development of UC are recapitulated by colonic-metaplastic transcriptomic reprogramming of the UC pouch. METHODS We prospectively sampled pouch and prepouch ileum mucosal biopsies in patients with UC with ileal pouch anal anastomosis 4, 8, and 12 months after their pouch was in continuity. Mucosal samples were also obtained from patients with FAP. Transcriptional profiles of the UC and FAP pouch and prepouch ileum were investigated via RNA sequencing and compared with data from a previously published microarray study. RESULTS Unlike patients with FAP, subjects with UC exhibited a large set of differentially expressed genes between the pouch and prepouch ileum as early as 4 months after pouch functionalization. Functional pathway analysis of differentially expressed genes in the UC pouch revealed an enhanced state of immune/inflammatory response and extracellular matrix remodeling. Moreover, >70% of differentially expressed genes mapped to published inflammatory bowel diseases microarray data sets displayed directional changes consistent with active UC but not with Crohns disease. CONCLUSIONS The UC pouch, well before histologic inflammation, already displays a systems-level gain of colon-associated genes and loss of ileum-associated genes. Patients with UC exhibit a unique transcriptomic response to ileal pouch creation that can be observed well before disease and may in part explain their susceptibility to the development of pouchitis.

Data on changes to mucosal inflammation and the intestinal microbiota following dietary micronutrients in genetically susceptible hosts

These data support the findings that dietary micronutrients influence the inflammatory responses and intestinal microbial community structure and function in a model of pouchitis-like small bowel inflammation reported in “Dietary Antioxidant Micronutrients Alter Mucosal Inflammatory Risk in a Murine Model of Genetic and Microbial Susceptibility” (Pierre et al., 2018) [1]. Briefly, wild-type and IL-10 deficient mice underwent surgical placement of small intestinal self-filling loops (SFL) and were subsequently fed purified control diet (CONT) or control diet supplemented with 4 micronutrients (AOX), retinoic acid, Vitamin C, Vitamin E, and selenium, for 14 days. These data include changes in host markers, such as body weight, mucosal levels of myeloperoxidase and syndecan-1, and luminal IgA and IgG levels. These data also include changes in the microbial compartment, including 16S community structure in the self-filling loop, conventionalized germ-free mice, and microbial substrate preference performed through anaerobic bacterial culturing of SLF CONT and AOX microbiota.

High-resolution tracking of microbial colonization in Fecal Microbiota Transplantation experiments via metagenome-assembled genomes

Fecal microbiota transplantation (FMT) is an effective treatment for recurrent Clostridium difficile infection and shows promise for treating other medical conditions associated with intestinal dysbioses. However, we lack a sufficient understanding of which microbial populations successfully colonize the recipient gut, and the widely used approaches to study the microbial ecology of FMT experiments fail to provide enough resolution to identify populations that are likely responsible for FMT-derived benefits. Here we used shotgun metagenomics to reconstruct 97 metagenome-assembled genomes (MAGs) from fecal samples of a single donor and followed their distribution in two FMT recipients to identify microbial populations with different colonization properties. Our analysis of the occurrence and distribution patterns post-FMT revealed that 22% of the MAGs transferred from the donor to both recipients and remained abundant in their guts for at least eight weeks. Most MAGs that successfully colonized the recipient gut belonged to the order Bacteroidales. The vast majority of those that lacked evidence of colonization belonged to the order Clostridiales and colonization success was negatively correlated with the number of genes related to sporulation. Although our dataset showed a link between taxonomy and the ability of a MAG to colonize the recipient gut, we also identified MAGs with different colonization properties that belong to the same taxon, highlighting the importance of genome-resolved approaches to explore the functional basis of colonization and to identify targets for cultivation, hypothesis generation, and testing in model systems for mechanistic insights.