Opposing diet, microbiome, and metabolite mechanisms regulate inflammatory bowel disease in a genetically susceptible host.

Inflammatory bowel diseases (IBDs) are chronic conditions characterized by periods of spontaneous intestinal inflammation and are increasing in industrialized populations. Combined with host genetics, diet and gut bacteria are thought to contribute prominently to IBDs, but mechanisms are still emerging. In mice lacking the IBD-associated cytokine, interleukin-10, we show that a fiber-deprived gut microbiota promotes the deterioration of colonic mucus, leading to lethal colitis. Inflammation starts with the expansion of natural killer cells and altered immunoglobulin-A coating of some bacteria. Lethal colitis is then driven by Th1 immune responses to increased activities of mucin-degrading bacteria that cause inflammation first in regions with thinner mucus. A fiber-free exclusive enteral nutrition diet also induces mucus erosion but inhibits inflammation by simultaneously increasing an anti-inflammatory bacterial metabolite, isobutyrate. Our findings underscore the importance of focusing on microbial functions-not taxa-contributing to IBDs and that some diet-mediated functions can oppose those that promote disease.

Salivary microbiome changes distinguish response to chemoradiotherapy in patients with oral cancer.

BACKGROUND: Oral squamous cell carcinoma (SCC) is associated with oral microbial dysbiosis. In this unique study, we compared pre- to post-treatment salivary microbiome in patients with SCC by 16S rRNA gene sequencing and examined how microbiome changes correlated with the expression of an anti-microbial protein. RESULTS: Treatment of SCC was associated with a reduction in overall bacterial richness and diversity. There were significant changes in the microbial community structure, including a decrease in the abundance of Porphyromonaceae and Prevotellaceae and an increase in Lactobacillaceae. There were also significant changes in the microbial community structure before and after treatment with chemoradiotherapy, but not with surgery alone. In patients treated with chemoradiotherapy alone, several bacterial populations were differentially abundant between responders and non-responders before and after therapy. Microbiome changes were associated with a change in the expression of DMBT1, an anti-microbial protein in human saliva. Additionally, we found that salivary DMBT1, which increases after treatment, could serve as a post-treatment salivary biomarker that links to microbial changes. Specifically, post-treatment increases in human salivary DMBT1 correlated with increased abundance of Gemella spp., Pasteurellaceae spp., Lactobacillus spp., and Oribacterium spp. This is the first longitudinal study to investigate treatment-associated changes (chemoradiotherapy and surgery) in the oral microbiome in patients with SCC along with changes in expression of an anti-microbial protein in saliva. CONCLUSIONS: The composition of the oral microbiota may predict treatment responses; salivary DMBT1 may have a role in modulating the oral microbiome in patients with SCC. After completion of treatment, 6 months after diagnosis, patients had a less diverse and less rich oral microbiome. Leptotrichia was a highly prevalent bacteria genus associated with disease. Expression of DMBT1 was higher after treatment and associated with microbiome changes, the most prominent genus being Gemella Video Abstract.

Unravelling specific diet and gut microbial contributions to inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic condition characterized by periods of spontaneous intestinal inflammation and is increasing in industrialized populations. Combined with host genetic predisposition, diet and gut bacteria are thought to be prominent features contributing to IBD, but little is known about the precise mechanisms involved. Here, we show that low dietary fiber promotes bacterial erosion of protective colonic mucus, leading to lethal colitis in mice lacking the IBD-associated cytokine, interleukin-10. Diet-induced inflammation is driven by mucin-degrading bacteria-mediated Th1 immune responses and is preceded by expansion of natural killer T cells and reduced immunoglobulin A coating of some bacteria. Surprisingly, an exclusive enteral nutrition diet, also lacking dietary fiber, reduced disease by increasing bacterial production of isobutyrate, which is dependent on the presence of a specific bacterial species, Eubacterium rectale. Our results illuminate a mechanistic framework using gnotobiotic mice to unravel the complex web of diet, host and microbial factors that influence IBD.

A steamed broccoli sprout diet preparation that reduces colitis via the gut microbiota.

Sulforaphane is a bioactive metabolite with anti-inflammatory activity and is derived from the glucosinolate glucoraphanin, which is highly abundant in broccoli sprouts. However, due to its inherent instability its use as a therapeutic against inflammatory diseases has been limited. There are few studies to investigate a whole food approach to increase sulforaphane levels with therapeutic effect and reduce inflammation. In the current study, using a mouse model of inflammatory bowel disease, we investigated the ability of steamed broccoli sprouts to ameliorate colitis and the role of the gut microbiota in mediating any effects. We observed that despite inactivation of the plant myrosinase enzyme responsible for the generation of sulforaphane via steaming, measurable levels of sulforaphane were detectable in the colon tissue and feces of mice after ingestion of steamed broccoli sprouts. In addition, this preparation of broccoli sprouts was also capable of reducing chemically-induced colitis. This protective effect was dependent on the presence of an intact microbiota, highlighting an important role for the gut microbiota in the metabolism of cruciferous vegetables to generate bioactive metabolites and promote their anti-inflammatory effects.

Testing Practices, Interpretation, and Diagnostic Evaluation of Iron Deficiency Anemia by US Primary Care Physicians.

Importance: Recognition of iron deficiency anemia (IDA) is important to initiate timely evaluation for gastrointestinal tract cancer. Retrospective studies have reported delays in diagnostic evaluation of IDA as a common factor associated with delayed diagnosis of colorectal cancer. Objective: To assess how US primary care physicians (PCPs) approach testing for anemia, interpret iron laboratory studies, and refer patients with IDA for gastrointestinal endoscopy. Design, Setting, and Participants: This survey study, conducted in August 2019, included members of the American College of Physicians Internal Medicine Insiders Panel, a nationally representative group of American College of Physicians membership, who self-identified as PCPs. Participants completed a vignette-based survey to assess practices related to screening for anemia, interpretation of laboratory-based iron studies, and appropriate diagnostic evaluation of IDA. Main Outcomes and Measures: Descriptive statistics based on survey responses were evaluated for frequency of anemia screening, correct interpretation of iron laboratory studies, and proportion of patients with new-onset IDA referred for gastrointestinal tract evaluation. Results: Of 631 PCPs who received an invitation to participate in the survey, 356 (56.4%) responded and 31 (4.9%) were excluded, for an adjusted eligible sample size of 600, yielding 325 completed surveys (response rate, 54.2%). Of the 325 participants who completed surveys, 180 (55.4%) were men; age of participants was not assessed. The mean (SD) duration of clinical experience was 19.8 (11.2) years (range, 1.0-45.0 years). A total of 250 participants (76.9%) screened at least some patients for anemia. Interpretation of iron studies was least accurate in a scenario of a borderline low ferritin level (40 ng/mL) with low transferrin saturation (2%); 86 participants (26.5%) incorrectly responded that this scenario did not indicate IDA, and 239 (73.5%) correctly identified this scenario as IDA. Of 312 participants, 170 (54.5%) recommended bidirectional endoscopy (upper endoscopy and colonoscopy) for new IDA for women aged 65 years; of 305 respondents, 168 (55.1%) recommended bidirectional endoscopy for men aged 65 years. Conclusions and Relevance: In this survey study, US PCPs' self-reported testing practices for anemia suggest overuse of screening laboratory tests, misinterpretation of iron studies, and underuse of bidirectional endoscopy for evaluation of new-onset IDA. Both misinterpretation of iron studies and underuse of bidirectional endoscopy can lead to delayed diagnosis of gastrointestinal tract cancers and warrant additional interventions.

Generation of systemic antitumour immunity via the in situ modulation of the gut microbiome by an orally administered inulin gel.

The performance of immune-checkpoint inhibitors, which benefit only a subset of patients and can cause serious immune-related adverse events, underscores the need for strategies that induce T-cell immunity with minimal toxicity. The gut microbiota has been implicated in the outcomes of patients following cancer immunotherapy, yet manipulating the gut microbiome to achieve systemic antitumour immunity is challenging. Here we show in multiple murine tumour models that inulin-a widely consumed dietary fibre-formulated as a 'colon-retentive' orally administered gel can effectively modulate the gut microbiome in situ, induce systemic memory-T-cell responses and amplify the antitumour activity of the checkpoint inhibitor anti-programmed cell death protein-1 (α-PD-1). Orally delivered inulin-gel treatments increased the relative abundances of key commensal microorganisms and their short-chain-fatty-acid metabolites, and led to enhanced recall responses for interferon-γ+CD8+ T cells as well as to the establishment of stem-like T-cell factor-1+PD-1+CD8+ T cells within the tumour microenvironment. Gels for the in situ modulation of the gut microbiome may be applicable more broadly to treat pathologies associated with a dysregulated gut microbiome.

Gut microbiota and systemic immunity in health and disease.

The mammalian intestine is colonized by trillions of microorganisms that have co-evolved with the host in a symbiotic relationship. Although the influence of the gut microbiota on intestinal physiology and immunity is well known, mounting evidence suggests a key role for intestinal symbionts in controlling immune cell responses and development outside the gut. Although the underlying mechanisms by which the gut symbionts influence systemic immune responses remain poorly understood, there is evidence for both direct and indirect effects. In addition, the gut microbiota can contribute to immune responses associated with diseases outside the intestine. Understanding the complex interactions between the gut microbiota and the host is thus of fundamental importance to understand both immunity and human health.

High fat stems tumor immune surveillance.

In a recent issue of Cell Stem Cell, Beyaz et al. show that high-fat diets promote tumorigenesis by reducing major histocompatibility complex (MHC) class II expression in intestinal stem cells. Dietary modulation of epithelial MHC II expression is regulated by the gut microbiota.

Understanding Covalent versus Spin-Orbit Coupling Contributions to Temperature-Dependent Electron Spin Relaxation in Cupric and Vanadyl Phthalocyanines.

Recent interest in transition-metal complexes as potential quantum bits (qubits) has reinvigorated the investigation of fundamental contributions to electron spin relaxation in various ligand scaffolds. From quantum computers to chemical and biological sensors, interest in leveraging the quantum properties of these molecules has opened a discussion of the requirements to maintain coherence over a large temperature range, including near room temperature. Here we compare temperature-, magnetic field position-, and concentration-dependent electron spin relaxation in copper(II) phthalocyanine (CuPc) and vanadyl phthalocyanine (VOPc) doped into diamagnetic hosts. While VOPc demonstrates coherence up to room temperature, CuPc coherence times become rapidly T1-limited with increasing temperature, despite featuring a more covalent ground-state wave function than VOPc. As rationalized by a ligand field model, this difference is ascribed to different spin-orbit coupling (SOC) constants for Cu(II) versus V(IV). The manifestation of SOC contributions to spin-phonon coupling and electron spin relaxation in different ligand fields is discussed, allowing for a further understanding of the competing roles of SOC and covalency in electron spin relaxation.

NOD1 and NOD2 in inflammatory and infectious diseases.

It has been long recognized that NOD1 and NOD2 are critical players in the host immune response, primarily by their sensing bacterial peptidoglycan-conserved motifs. Significant advances have been made from efforts that characterize their upstream activators, assembly of signaling complexes, and activation of downstream signaling pathways. Disruption in NOD1 and NOD2 signaling has also been associated with impaired host defense and resistance to the development of inflammatory diseases. In this review, we will describe how NOD1 and NOD2 sense microbes and cellular stress to regulate host responses that can affect disease pathogenesis and outcomes.

Gut Microbiota Modulate CD8 T Cell Responses to Influence Colitis-Associated Tumorigenesis.

There is increasing evidence that gut microbiome perturbations, also known as dysbiosis, can influence colorectal cancer development. To understand the mechanisms by which the gut microbiome modulates cancer susceptibility, we examine two wild-type mouse colonies with distinct gut microbial communities that develop significantly different tumor numbers using a mouse model of inflammation-associated tumorigenesis. We demonstrate that adaptive immune cells contribute to the different tumor susceptibilities associated with the two microbial communities. Mice that develop more tumors have increased colon lamina propria CD8+ IFNγ+ T cells before tumorigenesis but reduced CD8+ IFNγ+ T cells in tumors and adjacent tissues compared with mice that develop fewer tumors. Notably, intratumoral T cells in mice that develop more tumors exhibit increased exhaustion. Thus, these studies suggest that microbial dysbiosis can contribute to colon tumor susceptibility by hyperstimulating CD8 T cells to promote chronic inflammation and early T cell exhaustion, which can reduce anti-tumor immunity.

A Method for Isolation and Proteomic Analysis of Outer Membrane Vesicles from Fecal Samples by LC-MS/MS.

Outer membrane vesicles (OMVs) are nanosized spheres secreted by bacteria that are similar to the vesicles known as exosomes, which are secreted by most mammalian cell types. In contrast to many studies focusing on optimizing methods for enriching exosomes from biological fluid, few studies have been conducted to investigate outer membrane vesicles from fecal samples. Herein, we have developed a pipeline comprised of membrane filtration and multiple cycles of ultracentrifugation (UC) to isolate OMVs from fecal samples for proteomics analysis, where multiple cycles of UC are required for removal of contaminants. By iTRAQ labeling quantitative proteomics analysis, different filter sizes (0.22 µm and 0.45 µm) were compared in terms of their performance in enriching OMVs and eliminating background fecal material. Using the 0.45 µm filter, a slightly higher protein yield was obtained but no additional contaminating proteins from bacteria were identified compared to those from the 0.22 µm filter. The 0.45 µm filter together with the multiple cycles of UC were thus used to isolate OMVs for proteomics analysis. To our knowledge, this is the first study profiling a large number of OMV proteins from fecal samples. Such capabilities may help provide valuable information in understanding the communication between the host and microbiota, which is critical in preventing cancer and disease development.

Flavonoids and Colorectal Cancer Prevention.

Colorectal cancer (CRC) is the third most common cancer, but despite advances in treatment, it remains the second most common cause of cancer-related mortality. Prevention may, therefore, be a key strategy in reducing colorectal cancer deaths. Given reports of an inverse association between fruit and vegetable consumption with colorectal cancer risk, there has been significant interest in understanding the metabolism and bioactivity of flavonoids, which are highly abundant in fruits and vegetables and account for their pigmentation. In this review, we discuss host and microbiota-mediated metabolism of flavonoids and the potential mechanisms by which flavonoids can exert protective effects against colon tumorigenesis, including regulation of signaling pathways involved in apoptosis, cellular proliferation, and inflammation and modulation of the gut microbiome.

The NLRP6 Inflammasome Recognizes Lipoteichoic Acid and Regulates Gram-Positive Pathogen Infection.

The activator and composition of the NLRP6 inflammasome remain poorly understood. We find that lipoteichoic acid (LTA), a molecule produced by Gram-positive bacteria, binds and activates NLRP6. In response to cytosolic LTA or infection with Listeria monocytogenes, NLRP6 recruited caspase-11 and caspase-1 via the adaptor ASC. NLRP6 activation by LTA induced processing of caspase-11, which promoted caspase-1 activation and interleukin-1ß (IL-1ß)/IL-18 maturation in macrophages. Nlrp6-/- and Casp11-/- mice were less susceptible to L. monocytogenes infection, which was associated with reduced pathogen loads and impaired IL-18 production. Administration of IL-18 to Nlrp6-/- or Casp11-/- mice restored the susceptibility of mutant mice to L. monocytogenes infection. These results reveal a previously unrecognized innate immunity pathway triggered by cytosolic LTA that is sensed by NLRP6 and exacerbates systemic Gram-positive pathogen infection via the production of IL-18.

Myc-Associated Zinc Finger Protein Regulates the Proinflammatory Response in Colitis and Colon Cancer via STAT3 Signaling.

Myc-associated zinc finger (MAZ) is a transcription factor highly upregulated in chronic inflammatory disease and several human cancers. In the present study, we found that MAZ protein is highly expressed in human ulcerative colitis and colon cancer. However, the precise role for MAZ in the progression of colitis and colon cancer is not well defined. To determine the function of MAZ, a novel mouse model of intestinal epithelial cell-specific MAZ overexpression was generated. Expression of MAZ in intestinal epithelial cells was sufficient to enhance inflammatory injury in two complementary models of colitis. Moreover, MAZ expression increased tumorigenesis in an in vivo model of inflammation-induced colon cancer and was important for growth of human colon cancer cell lines in vitro and in vivo Mechanistically, MAZ is critical in the regulation of oncogenic STAT3 signaling. MAZ-expressing mice have enhanced STAT3 activation in the acute response to colitis. Moreover, MAZ was essential for cytokine- and bacterium-induced STAT3 signaling in colon cancer cells. Furthermore, we show that STAT3 is essential for MAZ-induced colon tumorigenesis using a chemical inhibitor. These data indicate an important functional role for MAZ in the inflammatory progression of colon cancer through regulation of STAT3 signaling and suggest that MAZ is a potential therapeutic target to dampen STAT3 signaling in colon cancer.

The Role of the Gut Microbiome in Colorectal Cancer.

There is increasing evidence that the gut microbiome, which consists of trillions of microbes representing over 1,000 species of bacteria with over 3 million genes, significantly impacts intestinal health and disease. The gut microbiota not only is capable of promoting intestinal homeostasis and antitumor responses but can also contribute to chronic dysregulated inflammation as well as have genotoxic effects that lead to carcinogenesis. Whether the gut microbiota maintains health or promotes colon cancer may ultimately depend on the composition of the gut microbiome and the balance within the microbial community of protective and detrimental bacterial populations. Disturbances in the normal balanced state of a healthful microbiome, known as dysbiosis, have been observed in patients with colorectal cancer (CRC); however, whether these alterations precede and cause CRC remains to be determined. Nonetheless, studies in mice strongly suggest that the gut microbiota can modulate susceptibility to CRC, and therefore may serve as both biomarkers and therapeutic targets.

NLRP6 Protects Il10-/- Mice from Colitis by Limiting Colonization of Akkermansia muciniphila.

Dysfunction in host immune responses and pathologic alterations in the gut microbiota, referred to as dysbiosis, can both contribute to the development of inflammatory bowel disease (IBD). However, it remains unclear how specific changes in host immunity or the microbiota cause disease. We previously demonstrated that the loss of the innate immune receptor NLRP6 in mice resulted in impaired production of interleukin-18 (IL-18) and increased susceptibility to epithelial-induced injury. Here, we show that NLRP6 is important for suppressing the development of spontaneous colitis in the Il10-/- mice model of IBD and that NLRP6 deficiency results in the enrichment of Akkermansia muciniphila. A. muciniphila was sufficient for promoting intestinal inflammation in both specific-pathogen-free and germ-free Il10-/- mice. Our results demonstrate that A. muciniphila can act as a pathobiont to promote colitis in a genetically susceptible host and that NLRP6 is a key regulator of its abundance.