Exploring the Potential of Breast Microbiota as Biomarker for Breast Cancer and Therapeutic Response.

Breast cancer tissue contains its own unique microbiota. Emerging preclinical data indicates that breast microbiota dysbiosis contributes to breast cancer initiation and progression. Furthermore, the breast microbiota may be a promising biomarker for treatment selection and prognosis. Differences in breast microbiota composition have been found between breast cancer subtypes and disease severities that may contribute to immunosuppression, enabling tumor cells to evade immune destruction. Interactions between breast microbiota, gut microbiota, and immune system are proposed, all forming potential targets to increase therapeutic efficacy. In addition, because the gut microbiota affects the host immune system and systemic availability of estrogen and bile acids known to influence tumor biology, gut microbiota modulation could be used to manipulate breast microbiota composition. Identifying breast and gut microbial compositions that respond positively to certain anticancer therapeutics could significantly reduce cancer burden. Additional research is needed to unravel the complexity of breast microbiota functioning and its interactions with the gut and the immune system. In this review, developments in the understanding of breast microbiota and its interaction with the immune system and the gut microbiota are discussed. Furthermore, the biomarker potential of breast microbiota is evaluated in conjunction with possible strategies to target microbiota in order to improve breast cancer treatment.

Serology of Streptococcus gallolyticus subspecies gallolyticus and its association with colorectal cancer and precursors.

Streptococcus gallolyticus subspecies gallolyticus (SGG) is potentially associated with colorectal cancer (CRC) and its precursors. A previous case-control study measured antibody responses to SGG pilus proteins Gallo2178 and Gallo2179 and identified significant associations with a small fraction of CRC cases. We aimed at replicating and expanding these findings in an independent study including additional SGG antigens and explored the association with precancerous lesions. We applied multiplex serology to measure antibodies to eleven SGG proteins in serum samples of a screening colonoscopy trial (BliTz study) including participants diagnosed with either non-advanced adenoma (NAA, n = 30), advanced adenoma (AA, n = 100), CRC (n = 50) or controls (n = 228). In addition, we analyzed CRC samples (n = 318) from patients recruited in a clinical setting (DACHSplus study). The association of antibody responses to SGG pilus proteins Gallo2178 and Gallo2179 with CRC was replicated with 4% positive DACHSplus cases compared to 0% positive BliTz controls. Positivity to two or more proteins of a newly defined panel of six SGG markers was significantly associated with CRC in the DACHSplus study (OR: 1.81, 95% CI: 1.07-3.06). Odds for CRC, AA and NAA in the BliTz study were also increased with antibody responses to SGG, and the association was significant for NAA (OR: 2.98, 95% CI: 1.18-7.57). Antibody responses to SGG are associated with CRC and its precursors. The newly identified SGG six-marker panel and associations found with precancerous lesions should be further explored.

Association of Streptococcus gallolyticus subspecies gallolyticus with colorectal cancer: Serological evidence.

The colonic opportunist Streptococcus gallolyticus subspecies gallolyticus (SGG) is potentially associated with colorectal cancer (CRC). Large-scale seroepidemiological data for SGG antibodies and their possible association with CRC is currently missing. Associations between CRC and antibody responses to SGG were examined in 576 CRC cases and 576 controls matched by sex, age and province from a population-based multicase-control project (MCC-Spain). MCC-Spain was conducted between 2008 and 2013 in 12 Spanish provinces. Antibody responses to recombinant affinity-purified SGG pilus proteins Gallo1569, 2039, 2178 and 2179 were analysed by multiplex serology. Polyomavirus (PyV) JC VP1 and PyV 6 VP1 proteins served as disease-specificity controls. In the control population, antibody responses to pilus proteins were mostly weak. Antibody responses to individual pilus proteins Gallo2039 (OR: 1.58, 95% CI: 1.09-2.28), Gallo2178 (OR: 1.58, 95% CI: 1.09-2.30) and Gallo2179 (OR: 1.45, 95% CI: 1.00-2.11) were significantly associated with CRC risk. The association was stronger for positivity to two or more pilus proteins of Gallo1569, Gallo2178 and Gallo2179 (OR:1.93, 95% CI: 1.04-3.56) and for double-positivity to Gallo2178 and Gallo2179 (OR: 3.54, 95% CI: 1.49-8.44). The association between SGG infection and CRC risk was stronger among individuals younger than 65 years. For the first time we demonstrated a statistically significant association of exposure to SGG antigens and CRC in a large seroepidemiological study. These results should stimulate further studies on the role of SGG in CRC pathogenesis.

RAS testing practices and RAS mutation prevalence among patients with metastatic colorectal cancer: results from a Europe-wide survey of pathology centres.

BACKGROUND: Treatment options for patients with metastatic colorectal cancer (mCRC) include anti-epithelial growth factor therapies, which, in Europe, are indicated in patients with RAS wild-type tumours only and require prior mutation testing of "hot-spot" codons in exons 2, 3 and 4 of KRAS and NRAS. The aim of this study was to evaluate the implementation of RAS testing methods and estimate the RAS mutation prevalence in mCRC patients. METHODS: Overall, 194 pathology laboratories were invited to complete an online survey. Participating laboratories were asked to provide information on their testing practices and aggregated RAS mutation data from 20 to 30 recently tested patients with mCRC. RESULTS: A total of 96 (49.5 %) laboratories across 24 European countries completed the survey. All participants tested KRAS exon 2, codons 12 and 13. Seventy (72.9 %) laboratories reported complete testing of all RAS hot-spot codons, and three (3.1 %) reported only testing KRAS exon 2. Sixty-nine (71.9 %) laboratories reported testing >80 patients yearly for RAS mutation status. Testing was typically performed within the reporting institution (93.8 %, n = 90), at the request of a treating oncologist (89.5 %, n = 85); testing methodology varied by laboratory and by individual codon tested. For laboratory RAS testing, turnaround times were ≤10 working days for the majority of institutions (90.6 %, n = 87). The overall crude RAS mutation prevalence was 48.5 % (95 % confidence interval: 46.4-50.6) for laboratories testing all RAS hot-spot codons. Prevalence estimates varied significantly by primary tumour location, approximate number of patients tested yearly and indication given for RAS testing. CONCLUSION: Our findings indicate a rapid uptake of RAS testing in the majority of European pathology laboratories.

The Bacteroides fragilis toxin gene is prevalent in the colon mucosa of colorectal cancer patients.

BACKGROUND: Enterotoxigenic Bacteroides fragilis (ETBF) produces the Bacteroides fragilis toxin, which has been associated with acute diarrheal disease, inflammatory bowel disease, and colorectal cancer (CRC). ETBF induces colon carcinogenesis in experimental models. Previous human studies have demonstrated frequent asymptomatic fecal colonization with ETBF, but no study has investigated mucosal colonization that is expected to impact colon carcinogenesis. METHODS: We compared the presence of the bft gene in mucosal samples from colorectal neoplasia patients (cases, n = 49) to a control group undergoing outpatient colonoscopy for CRC screening or diagnostic workup (controls, n = 49). Single bacterial colonies isolated anaerobically from mucosal colon tissue were tested for the bft gene with touch-down polymerase chain reaction. RESULTS: The mucosa of cases was significantly more often bft-positive on left (85.7%) and right (91.7%) tumor and/or paired normal tissues compared with left and right control biopsies (53.1%; P = .033 and 55.5%; P = .04, respectively). Detection of bft was concordant in most paired mucosal samples from individual cases or controls (75% cases; 67% controls). There was a trend toward increased bft positivity in mucosa from late- vs early-stage CRC patients (100% vs 72.7%, respectively; P = .093). In contrast to ETBF diarrheal disease where bft-1 detection dominates, bft-2 was the most frequent toxin isotype identified in both cases and controls, whereas multiple bft isotypes were detected more frequently in cases (P ≤ .02). CONCLUSIONS: The bft gene is associated with colorectal neoplasia, especially in late-stage CRC. Our results suggest that mucosal bft exposure is common and may be a risk factor for developing CRC.

Bacterial responses to a simulated colon tumor microenvironment.

One of the few bacteria that have been consistently linked to colorectal cancer (CRC) is the opportunistic pathogen Streptococcus gallolyticus. Infections with this bacterium are generally regarded as an indicator for colonic malignancy, while the carriage rate of this bacterium in the healthy large intestine is relatively low. We speculated that the physiological changes accompanying the development of CRC might favor the colonization of this bacterium. To investigate whether colon tumor cells can support the survival of S. gallolyticus, this bacterium was grown in spent medium of malignant colonocytes to simulate the altered metabolic conditions in the CRC microenvironment. These in vitro simulations indicated that S. gallolyticus had a significant growth advantage in these spent media, which was not observed for other intestinal bacteria. Under these conditions, bacterial responses were profiled by proteome analysis and metabolic shifts were analyzed by (1)H-NMR-spectroscopy. In silico pathway analysis of the differentially expressed proteins and metabolite analysis indicated that this advantage resulted from the increased utilization of glucose, glucose derivates, and alanine. Together, these data suggest that tumor cell metabolites facilitate the survival of S. gallolyticus, favoring its local outgrowth and providing a possible explanation for the specific association of S. gallolyticus with colonic malignancy.

The influence of Helicobacter pylori, proton pump inhibitor, and obesity on the gastric microbiome in relation to gastric cancer development.

Helicobacter pylori infection is still the main risk factor for the development of gastric cancer (GC). We explore the scientific evidence for the role of the gastric microbiome beyond Helicobacter pylori (H. pylori) in gastric carcinogenesis. The composition of the gastric microbiome in healthy individuals, in presence and absence of H. pylori infection, in proton pump inhibitor (PPI)-users, obese individuals, and GC patients was investigated. Possible mechanisms for microbial involvement, limitations of available research and options for future studies are provided. A common finding amongst studies was increased levels of Streptococcus, Prevotella, Neisseria, and Actinomyces in healthy individuals or those with H. pylori-negative gastritis. In PPI-users the risk for GC increases with the treatment duration, and the gastric microbiome shifts, with the most consistent increase in the genus Streptococcus. Similarly, in obese individuals, Streptococcus was the most abundant genus, with an increased risk for cardia GC. The genera Streptococcus, Lactobacillus and Prevotella were found to be more prominent in GC patients in multiple studies. Potential mechanisms of non-H. pylori microbiota contributing to GC are linked to lipopolysaccharide production, contribution to inflammatory pathways, and the formation of N-nitroso compounds and reactive oxygen species. In conclusion, the knowledge of the gastric microbiome in GC is mainly descriptive and based on sequencing of gastric mucosal samples. For a better mechanistic understanding of microbes in GC development, longitudinal cohorts including precancerous lesions, different regions in the stomach, and subtypes of GC, and gastric organoid models for diffuse and intestinal type GC should be employed.

Partial associations of dietary iron, smoking and intestinal bacteria with colorectal cancer risk.

Smoking and high red meat intake have been associated with colorectal cancer (CRC) risk. Increased iron exposure may be a common factor, favoring the colonization of certain bacterial pathogens that preferentially grow in an iron-rich luminal environment. We analyzed the data from a population-based case-control study of CRC and measured antibody levels against flagelin of Salmonella (FliC), one of the irontrophic bacteria, in 2 independent blood collections. The risk of CRC synergistically increased by combined exposures to heme iron intake and pack-yr (PY) of cigarette smoking (P value for the interaction = 0.039 on the continuous scale). There was a marginally significant interaction between heme iron intake and PY in increasing FliC antibody in the U.S. control subjects (P = 0.055), although no iron or smoking data were available for Dutch samples. Furthermore, FliC antibody levels were significantly higher in patients with colorectal polyps and cancer than in controls in both Dutch (3.93 vs. 2.23) (P = 0.014) and U.S. samples (6.65 vs. 4.37) (P < 0.001). Potential roles of iron from cigarette smoking and dietary heme in CRC through altering irontrophic luminal bacterial population may warrant further investigation.

Bacterial Oncotraits Rather than Spatial Organization Are Associated with Dysplasia in Ulcerative Colitis.

BACKGROUND AND AIMS: Colonic bacterial biofilms are frequently present in ulcerative colitis [UC] and may increase dysplasia risk through pathogens expressing oncotraits. This prospective cohort study aimed to determine [1] the association of oncotraits and longitudinal biofilm presence with dysplasia risk in UC, and [2] the relation of bacterial composition with biofilms and dysplasia risk. METHODS: Faeces and left- and right-sided colonic biopsies were collected from 80 UC patients and 35 controls. Oncotraits [FadA of Fusobacterium, BFT of Bacteroides fragilis, colibactin [ClbB] and Intimin [Eae] of Escherichia coli] were assessed in faecal DNA with multiplex quantitative polymerase chain reaction [qPCR]. Biopsies were screened for biofilms [n = 873] with 16S rRNA fluorescent in situ hybridiation. Shotgun metagenomic sequencing [n = 265], and ki67-immunohistochemistry were performed. Associations were determined with a mixed-effects regression model. RESULTS: Biofilms were highly prevalent in UC patients [90.8%] with a median persistence of 3 years (interquartile range [IQR] 2-5 years). Biofilm-positive biopsies showed increased epithelial hypertrophy [p = 0.025] and a reduced Shannon diversity independent of disease status [p = 0.015], but were not significantly associated with dysplasia in UC: adjusted odds ratio [aOR] 1.45, 95% confidence interval [CI] 0.63-3.40. In contrast, ClbB independently associated with dysplasia [aOR 7.16, 95% CI 1.75-29.28], and FadA and Fusobacteriales were associated with a decreased dysplasia risk in UC [aOR 0.23, 95% CI 0.06-0.83, p <0.01]. CONCLUSIONS: Biofilms are a hallmark of UC; however, because of their high prevalence are a poor biomarker for dysplasia. In contrast, colibactin presence and FadA absence independently associate with dysplasia in UC and might therefore be valuable biomarkers for future risk stratification and intervention strategies.

Novel clues on the specific association of Streptococcus gallolyticus subsp gallolyticus with colorectal cancer.

BACKGROUND: The prevalence of Streptococcus gallolyticus subsp gallolyticus ( Streptococcus bovis biotype I) endocarditis is in general low but very often linked to colorectal cancer. Therefore, this study aimed to reveal the virulence characteristics that distinguish this opportunistic pathogen from a panel of (closely related) intestinal bacteria. METHODS: The route of infection was reconstructed in vitro with adhesion, invasion, and translocation assays on differentiated Caco-2 cells. Furthermore, cellular immune responses upon infection and bacterial biofilm formation were analyzed in a comparative manner. RESULTS: S. gallolyticus subsp gallolyticus strains were demonstrated to have a relative low adhesiveness and could not internalize epithelial cells. However, these bacteria were uniquely able to paracellularly cross a differentiated epithelium without inducing epithelial interleukin 8 or 1ß responses. Importantly, they had an outstanding ability to form biofilms on collagen-rich surfaces, which in vivo are found at damaged heart valves and (pre)cancerous sites with a displaced epithelium. CONCLUSIONS: Together, these data show that S. gallolyticus subsp gallolyticus has a unique repertoire of virulence factors that facilitate infection through (pre)malignant colonic lesions and subsequently can provide this bacterium with a competitive advantage in (1) evading the innate immune system and (2) forming resistant vegetations at collagen-rich sites in susceptible patients with colorectal cancer.

Nutrition or nature: using elementary flux modes to disentangle the complex forces shaping prokaryote pan-genomes.

BACKGROUND: Microbial pan-genomes are shaped by a complex combination of stochastic and deterministic forces. Even closely related genomes exhibit extensive variation in their gene content. Understanding what drives this variation requires exploring the interactions of gene products with each other and with the organism's external environment. However, to date, conceptual models of pan-genome dynamics often represent genes as independent units and provide limited information about their mechanistic interactions. RESULTS: We simulated the stochastic process of gene-loss using the pooled genome-scale metabolic reaction networks of 46 taxonomically diverse bacterial and archaeal families as proxies for their pan-genomes. The frequency by which reactions are retained in functional networks when stochastic gene loss is simulated in diverse environments allowed us to disentangle the metabolic reactions whose presence depends on the metabolite composition of the external environment (constrained by "nutrition") from those that are independent of the environment (constrained by "nature"). By comparing the frequency of reactions from the first group with their observed frequencies in bacterial and archaeal families, we predicted the metabolic niches that shaped the genomic composition of these lineages. Moreover, we found that the lineages that were shaped by a more diverse metabolic niche also occur in more diverse biomes as assessed by global environmental sequencing datasets. CONCLUSION: We introduce a computational framework for analyzing and interpreting pan-reactomes that provides novel insights into the ecological and evolutionary drivers of pan-genome dynamics.

Correction: Protocol of the Healthy Brain Study: An accessible resource for understanding the human brain and how it dynamically and individually operates in its bio-social context.

[This corrects the article DOI: 10.1371/journal.pone.0260952.].

Surface-affinity profiling to identify host-pathogen interactions.

Proteolytic treatment of intact bacterial cells has proven to be a convenient approach for the identification of surface-exposed proteins. This class of proteins directly interacts with the outside world, for instance, during adherence to human epithelial cells. Here, we aimed to identify host receptor proteins by introducing a preincubation step in which bacterial cells were first allowed to capture human proteins from epithelial cell lysates. Using Streptococcus gallolyticus as a model bacterium, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of proteolytically released peptides yielded the identification of a selective number of human epithelial proteins that were retained by the bacterial surface. Of these potential receptors for bacterial interference, (cyto)keratin-8 (CK8) was verified as the most significant hit, and its surface localization was investigated by subcellular fractionation and confocal microscopy. Interestingly, bacterial enolase could be assigned as an interaction partner of CK8 by MS/MS analysis of cross-linked protein complexes and complementary immunoblotting experiments. As surface-exposed enolase has a proposed role in epithelial adherence of several Gram-positive pathogens, its interaction with CK8 seems to point toward a more general virulence mechanism. In conclusion, our study shows that surface-affinity profiling is a valuable tool to identify novel adhesin-receptor pairs, which advocates its application in other hybrid biological systems.

Clinical Importance of Streptococcus gallolyticus infection among colorectal cancer patients: systematic review and meta-analysis.

BACKGROUND: Streptococcus bovis has long been associated with colorectal cancer (CRC). However, not all genospecies are as closely related to CRC. With this systematic review, we aim to increase the awareness of the association between S. bovis biotype I (Streptococcus gallolyticus) and CRC and urge for uniform molecular microbiological classification. METHODS: In January 2011, the PubMed database was searched for all studies that investigated the association between S. bovis, infective endocarditis (IE), and CRC. A total of 191 studies were screened for eligibility and yielded 52 case reports and 31 case series, of which 11 were used for meta-analysis on the association between S. bovis biotype, IE, and adenomas/carcinomas (CRC). RESULTS: Among the S. bovis-infected patients who underwent colonic evaluation, the median percentage of patients who had concomitant adenomas/carcinomas was 60% (interquartile range, 22%), which largely exceeds the disease rate reported in the general asymptomatic population. Meta-analysis showed that patients with S. bovis biotype I infection had a strongly increased risk of having CRC (pooled odds ratio [OR], 7.26; 95% confidence interval [CI], 3.94-13.36) and IE (pooled OR, 16.61; 95% CI, 8.85-31.16), compared with S. bovis biotype II-infected patients. Notably, CRC occurred more often among patients with S. bovis IE than among patients with S. bovis infection at other sites (pooled OR, 3.72; 95% CI, 2.03-6.81). CONCLUSIONS: Our meta-analysis clearly indicates that S. bovis should no longer be regarded as a single species in clinical practice, because S. gallolyticus (S. bovis biotype I) infection, in particular, has an unambiguous association with CRC.

Optimized bacterial DNA isolation method for microbiome analysis of human tissues.

Recent advances in microbiome sequencing have rendered new insights into the role of the microbiome in human health with potential clinical implications. Unfortunately, the presence of host DNA in tissue isolates has hampered the analysis of host-associated bacteria. Here, we present a DNA isolation protocol for tissue, optimized on biopsies from resected human colons (~2-5 mm in size), which includes reduction of human DNA without distortion of relative bacterial abundance at the phylum level. We evaluated which concentrations of Triton and saponin lyse human cells and leave bacterial cells intact, in combination with DNAse treatment to deplete released human DNA. Saponin at a concentration of 0.0125% in PBS lysed host cells, resulting in a 4.5-fold enrichment of bacterial DNA while preserving the relative abundance of Firmicutes, Bacteroidetes, γ-Proteobacteria, and Actinobacteria assessed by qPCR. Our optimized protocol was validated in the setting of two large clinical studies on 521 in vivo acquired colon biopsies of 226 patients using shotgun metagenomics. The resulting bacterial profiles exhibited alpha and beta diversities that are similar to the diversities found by 16S rRNA amplicon sequencing. A direct comparison between shotgun metagenomics and 16S rRNA amplicon sequencing of 15 forceps tissue biopsies showed similar bacterial profiles and a similar Shannon diversity index between the sequencing methods. Hereby, we present the first protocol for enriching bacterial DNA from tissue biopsies that allows efficient isolation of all bacteria. Our protocol facilitates analysis of a wide spectrum of bacteria of clinical tissue samples improving their applicability for microbiome research.

Streptococcus gallolyticus Increases Expression and Activity of Aryl Hydrocarbon Receptor-Dependent CYP1 Biotransformation Capacity in Colorectal Epithelial Cells.

Objective: The opportunistic pathogen Streptococcus gallolyticus is one of the few intestinal bacteria that has been consistently linked to colorectal cancer (CRC). This study aimed to identify novel S. gallolyticus-induced pathways in colon epithelial cells that could further explain how S. gallolyticus contributes to CRC development. Design and Results: Transcription profiling of in vitro cultured CRC cells that were exposed to S. gallolyticus revealed the specific induction of oxidoreductase pathways. Most prominently, CYP1A and ALDH1 genes that encode phase I biotransformation enzymes were responsible for the detoxification or bio-activation of toxic compounds. A common feature is that these enzymes are induced through the Aryl hydrocarbon receptor (AhR). Using the specific inhibitor CH223191, we showed that the induction of CYP1A was dependent on the AhR both in vitro using multiple CRC cell lines as in vivo using wild-type C57bl6 mice colonized with S. gallolyticus. Furthermore, we showed that CYP1 could also be induced by other intestinal bacteria and that a yet unidentified diffusible factor from the S. galloltyicus secretome (SGS) induces CYP1A enzyme activity in an AhR-dependent manner. Importantly, priming CRC cells with SGS increased the DNA damaging effect of the polycyclic aromatic hydrocarbon 3-methylcholanthrene. Conclusion: This study shows that gut bacteria have the potential to modulate the expression of biotransformation pathways in colonic epithelial cells in an AhR-dependent manner. This offers a novel theory on the contribution of intestinal bacteria to the etiology of CRC by modifying the capacity of intestinal epithelial or (pre-)cancerous cells to (de)toxify dietary components, which could alter intestinal susceptibility to DNA damaging events.

Production of inactivated gram-positive and gram-negative species with preserved cellular morphology and integrity.

There are many approaches available to produce inactive bacteria by termination of growth, each with a different efficacy, impact on cell integrity, and potential for application in standardized inactivation protocols. The aim of this study was to compare these approaches and develop a standardized protocol for generation of inactivated Gram-positive and Gram-negative bacteria, yielding cells that are metabolically dead with retained cellular integrity i.e., preserving the surface and limited leakage of intracellular proteins and DNA. These inactivated bacteria are required for various applications, for instance, when investigating receptor-triggered signaling or bacterial contact-dependent analysis of cell lines requiring long incubation times. We inactivated eight different bacterial strains of different species by treatment with beta-propiolactone, ethanol, formalin, sodium hydroxide, and pasteurization. Inactivation efficacy was determined by culturing, and cell wall integrity assessed by quantifying released DNA, bacterial membrane and intracellular DNA staining, and visualization by scanning electron microscopy. Based on these results, we discuss the bacterial inactivation methods, and their advantages and disadvantages to study host-microbe interactions with inactivated bacteria.

Higher Prevalence of Bacteroides fragilis in Crohn's Disease Exacerbations and Strain-Dependent Increase of Epithelial Resistance.

Bacteroides fragilis has previously been linked to Crohn's disease (CD) exacerbations, but results are inconsistent and underlying mechanisms unknown. This study investigates the epidemiology of B. fragilis and its virulence factors bft (enterotoxin) and ubiquitin among 181 CD patients and the impact on the intestinal epithelial barrier in vitro. The prevalence of B. fragilis was significantly higher in active (n = 69/88, 78.4%) as compared to remissive (n = 58/93, 62.4%, p = 0.018) CD patients. Moreover, B. fragilis was associated with intestinal strictures. Interestingly, the intestinal barrier function, as examined by transepithelial electrical resistance (TEER) measurements of Caco-2 monolayers, increased when exposed to secretomes of bft-positive (bft-1 and bft-2 isotype; increased TEER ∼160%, p < 0.001) but not when exposed to bft-negative strains. Whole metagenome sequencing and metabolomics, respectively, identified nine coding sequences and two metabolites that discriminated TEER-increasing from non-TEER-increasing strains. This study revealed a higher B. fragilis prevalence during exacerbation. Surprisingly, bft-positive secretomes increased epithelial resistance, but we excluded Bft as the likely causative factor.

G-protein coupled receptor 35 (GPR35) regulates the colonic epithelial cell response to enterotoxigenic Bacteroides fragilis.

G protein-coupled receptor (GPR)35 is highly expressed in the gastro-intestinal tract, predominantly in colon epithelial cells (CEC), and has been associated with inflammatory bowel diseases (IBD), suggesting a role in gastrointestinal inflammation. The enterotoxigenic Bacteroides fragilis (ETBF) toxin (BFT) is an important virulence factor causing gut inflammation in humans and animal models. We identified that BFT signals through GPR35. Blocking GPR35 function in CECs using the GPR35 antagonist ML145, in conjunction with shRNA knock-down and CRISPRcas-mediated knock-out, resulted in reduced CEC-response to BFT as measured by E-cadherin cleavage, beta-arrestin recruitment and IL-8 secretion. Importantly, GPR35 is required for the rapid onset of ETBF-induced colitis in mouse models. GPR35-deficient mice showed reduced death and disease severity compared to wild-type C57Bl6 mice. Our data support a role for GPR35 in the CEC and mucosal response to BFT and underscore the importance of this molecule for sensing ETBF in the colon.

Mechanisms of Immune Checkpoint Inhibitor-Mediated Colitis.

Immune checkpoint inhibitors (ICIs) have provided tremendous clinical benefit in several cancer types. However, systemic activation of the immune system also leads to several immune-related adverse events. Of these, ICI-mediated colitis (IMC) occurs frequently and is the one with the highest absolute fatality. To improve current treatment strategies, it is important to understand the cellular mechanisms that induce this form of colitis. In this review, we discuss important pathways that are altered in IMC in mouse models and in human colon biopsy samples. This reveals a complex interplay between several types of immune cells and the gut microbiome. In addition to a mechanistic understanding, patients at risk should be identifiable before ICI therapy. Here we propose to focus on T-cell subsets that interact with bacteria after inducing epithelial damage. Especially, intestinal resident immune cells are of interest. This may lead to a better understanding of IMC and provides opportunities for prevention and management.