Effect of Mutant and Engineered High-Acetate-Producing Saccharomyces cerevisiae var. boulardii Strains in Dextran Sodium Sulphate-Induced Colitis.

Acetate-producing Saccharomyces cerevisiae var. boulardii strains could exert improved effects on ulcerative colitis, which here, was preclinically evaluated in an acute dextran sodium sulphate induced model of colitis. Nine-week-old female mice were divided into 12 groups, receiving either drinking water or 2.75% dextran sodium sulphate for 7 days, combined with a daily gavage of various treatments with different levels of acetate accumulation: sham control (phosphate buffered saline, no acetate), non-probiotic control (Baker's yeast, no acetate), probiotic control (Enterol®, transient acetate), and additionally several Saccharomyces cerevisiae var. boulardii strains with respectively no, high, and extra-high acetate accumulation. Disease activity was monitored daily, and feces samples were collected at different timepoints. On day 14, the mice were sacrificed, upon which blood and colonic tissue were collected for analysis. Disease activity in inflamed mice was lower when treated with the high-acetate-producing strain compared to sham and non-probiotic controls. The non-acetate-producing strain showed higher disease activity compared to the acetate-producing strains. Accordingly, higher histologic inflammation was observed in non- or transient-acetate-producing strains compared to the sham control, whereas this increase was not observed for high- and extra-high-acetate-producing strains upon induction of inflammation. These anti-inflammatory findings were confirmed by transcriptomic analysis of differentially expressed genes. Moreover, only the strain with the highest acetate production was superior in maintaining a stable gut microbial alpha-diversity upon inflammation. These findings support new possibilities for acetate-mediated management of inflammation in inflammatory bowel disease by administrating high-acetate-producing Saccharomyces cerevisae var. boulardii strains.

Rectal organoid morphology analysis (ROMA) as a novel physiological assay for diagnostic classification in cystic fibrosis.

BACKGROUND: Diagnosing cystic fibrosis (CF) is not always straightforward, in particular when sweat chloride concentration (SCC) is intermediate and <2 CF-causing CFTR variants are identified. The physiological CFTR assays proposed in the guidelines, nasal potential difference and intestinal current measurement, are not readily available nor feasible at all ages. Rectal organoid morphology analysis (ROMA) was previously shown to discriminate between organoids from subjects with and without CF based on a distinct phenotypical difference: compared with non-CF organoids, CF organoids have an irregular shape and lack a visible lumen. The current study serves to further explore the role of ROMA when a CF diagnosis is inconclusive. METHODS: Organoid morphology was analysed using the previously established ROMA protocol. Two indices were calculated: the circularity index to quantify the roundness of organoids and the intensity ratio as a measure of the presence of a central lumen. RESULTS: Rectal organoids from 116 subjects were cultured and analysed together with the 189 subjects from the previous study. ROMA almost completely discriminated between CF and non-CF. ROMA indices correlated with SCC, pancreatic status and genetics, demonstrating convergent validity. For cases with an inconclusive diagnosis according to current guidelines, ROMA provided additional diagnostic information, with a diagnostic ROMA classification for 18 of 24 (75%). DISCUSSION: ROMA provides additional information to support a CF diagnosis when SCC and genetics are insufficient for diagnostic classification. ROMA is standardised and can be centralised, allowing future inclusion in the diagnostic work-up as first-choice physiological assay in case of an unclear diagnosis.

Rigorous Donor Selection for Fecal Microbiota Transplantation in Active Ulcerative Colitis: Key Lessons From a Randomized Controlled Trial Halted for Futility.

BACKGROUND & AIMS: Rigorous donor preselection on microbiota level, strict anaerobic processing, and repeated fecal microbiota transplantation (FMT) administration were hypothesized to improve FMT induction of remission in ulcerative colitis (UC). METHODS: The RESTORE-UC trial was a multi-centric, double-blind, sham-controlled, randomized trial. Patients with moderate to severe UC (defined by total Mayo 4-10) were randomly allocated to receive 4 anaerobic-prepared allogenic or autologous donor FMTs. Allogenic donor material was selected after a rigorous screening based on microbial cell count, enterotype, and the abundance of specific genera. The primary endpoint was steroid-free clinical remission (total Mayo ≤2, no sub-score >1) at week 8. A pre-planned futility analysis was performed after 66% (n = 72) of intended inclusions (n = 108). Quantitative microbiome profiling (n = 44) was performed at weeks 0 and 8. RESULTS: In total, 72 patients were included, of which 66 received at least 1 FMT (allogenic FMT, n = 30 and autologous FMT, n = 36). At week 8, respectively, 3 and 5 patients reached the primary endpoint of steroid-free clinical remission (P = .72), indicating no treatment difference of at least 5% in favor of allogenic FMT. Hence, the study was stopped due to futility. Microbiome analysis showed numerically more enterotype transitions upon allogenic FMT compared with autologous FMT, and more transitions were observed when patients were treated with a different enterotype than their own at baseline (P = .01). Primary response was associated with lower total Mayo scores, lower bacterial cell counts, and higher Bacteroides 2 prevalence at baseline. CONCLUSION: The RESTORE-UC trial did not meet its primary endpoint of increased steroid-free clinical remission at week 8. Further research should additionally consider patient selection, sterilized sham-control, increased frequency, density, and viability of FMT prior to administration. CLINICALTRIALS: gov, Number: NCT03110289.

Directed biomechanical compressive forces enhance fusion efficiency in model placental trophoblast cultures.

The syncytiotrophoblast is a multinucleated structure that arises from fusion of mononucleated cytotrophoblasts, to sheath the placental villi and regulate transport across the maternal-fetal interface. Here, we ask whether the dynamic mechanical forces that must arise during villous development might influence fusion, and explore this question using in vitro choriocarcinoma trophoblast models. We demonstrate that mechanical stress patterns arise around sites of localized fusion in cell monolayers, in patterns that match computational predictions of villous morphogenesis. We then externally apply these mechanical stress patterns to cell monolayers and demonstrate that equibiaxial compressive stresses (but not uniaxial or equibiaxial tensile stresses) enhance expression of the syndecan-1 and loss of E-cadherin as markers of fusion. These findings suggest that the mechanical stresses that contribute towards sculpting the placental villi may also impact fusion in the developing tissue. We then extend this concept towards 3D cultures and demonstrate that fusion can be enhanced by applying low isometric compressive stresses to spheroid models, even in the absence of an inducing agent. These results indicate that mechanical stimulation is a potent activator of cellular fusion, suggesting novel avenues to improve experimental reproductive modelling, placental tissue engineering, and understanding disorders of pregnancy development.

SCFAs switch stem cell fate through HDAC inhibition to improve barrier integrity in 3D intestinal organoids from patients with obesity.

Stem cells are a keystone of intestinal homeostasis, but their function could be shifted during energy imbalance or by crosstalk with microbial metabolites in the stem cell niche. This study reports the effect of obesity and microbiota-derived short-chain fatty acids (SCFAs) on intestinal stem cell (ISC) fate in human crypt-derived intestinal organoids (enteroids). ISC fate decision was impaired in obesity, resulting in smaller enteroids with less outward protruding crypts. Our key finding is that SCFAs switch ISC commitment to the absorptive enterocytes, resulting in reduced intestinal permeability in obese enteroids. Mechanistically, SCFAs act as HDAC inhibitors in stem cells to enhance Notch signaling, resulting in transcriptional activation of the Notch target gene HES1 to promote enterocyte differentiation. In summary, targeted reprogramming of ISC fate, using HDAC inhibitors, may represent a potential, robust therapeutic strategy to improve gut integrity in obesity.

High Acetate Concentration Protects Intestinal Barrier and Exerts Anti-Inflammatory Effects in Organoid-Derived Epithelial Monolayer Cultures from Patients with Ulcerative Colitis.

Short-chain fatty acids as well as their bacterial producers are of increasing interest in inflammatory bowel diseases. Although less studied compared to butyrate, acetate might also be of interest as it may be less toxic to epithelial cells, stimulate butyrate-producing bacteria by cross-feeding, and have anti-inflammatory and barrier-protective properties. Moreover, one of the causative factors of the probiotic potency of Saccharomyces cerevisae var. boulardii is thought to be its high acetate production. Therefore, the objective was to preclinically assess the effects of high acetate concentrations on inflammation and barrier integrity in organoid-based monolayer cultures from ulcerative colitis patients. Confluent organoid-derived colonic epithelial monolayers (n = 10) were exposed to basolateral inflammatory stimulation or control medium. After 24 h, high acetate or control medium was administered apically for an additional 48 h. Changes in TEER were measured after 48 h. Expression levels of barrier genes and inflammatory markers were determined by qPCR. Pro-inflammatory proteins in the supernatant were quantified using the MSD platform. Increased epithelial resistance was observed with high acetate administration in both inflamed and non-inflamed conditions, together with decreased expression levels of IL8 and TNFα and CLDN1. Upon high acetate administration to inflamed monolayers, upregulation of HIF1α, MUC2, and MKI67, and a decrease of the majority of pro-inflammatory cytokines was observed. In our patient-derived human epithelial cell culture model, a protective effect of high acetate administration on epithelial resistance, barrier gene expression, and inflammatory protein production was observed. These findings open up new possibilities for acetate-mediated management of barrier defects and inflammation in IBD.

Microbiota, not host origin drives ex vivo intestinal epithelial responses.

Microbial dysbiosis is an established finding in patients with inflammatory bowel disease (IBD), but host-microbial interactions are poorly understood. We aimed to unravel the effect of microbiota exposure on intestinal epithelial cells. Confluent Transwell® organoid monolayers of eight UC patients and eight non-IBD controls were co-cultured for six hours with microbiota (3x108 cells) of UC patients or a healthy volunteer (HV), in the presence or absence of an inflammatory cytokine mix. Transepithelial electrical resistance (TEER), fluorescein isothiocyanate (FITC) dextran measurements, and RNA sequencing were performed on epithelial cells, and 16S rRNA sequencing on microbiota samples before and after co-culture. Transcriptomic response following microbiota exposure was not different between epithelial cells from UC patients or non-IBD controls. Following UC microbiota exposure, but not HV microbiota, a strong decrease in epithelial barrier integrity was observed in both UC and HV epithelial cells by TEER and FITC dextran measurements. Exposure of inflamed epithelium to UC microbiota induced transcriptomic stress pathways including activation of EGR1, MAPK and JAK/STAT signaling, as well as AP-1 family and FOSL transcripts. Stress responses after HV microbiota stimulation were milder. We conclude that not the epithelial cell origin (UC versus non-IBD) but the microbial donor drives transcriptomic responses, as exposure to UC microbiota was sufficient to induce stress responses in all epithelial cells. Further research on therapies to restore the microbial balance, to remove the constant trigger of dysbiosis, is required.

Severity of the S1251N allele in cystic fibrosis is affected by the presence of the F508C variant in cis.

BACKGROUND: In cystic fibrosis (CF), genotype-phenotype correlation is complicated by the large number of CFTR variants, the influence of modifier genes, environmental effects, and the existence of complex alleles. We document the importance of complex alleles, in particular the F508C variant present in cis with the S1251N disease-causing variant, by detailed analysis of a patient with CF, with the [S1251N;F508]/G542X genotype and a very mild phenotype, contrasting it to that of four subjects with the [S1251N;F508C]/F508del genotype and classical CF presentation. METHODS: Genetic differences were identified by Sanger sequencing and CFTR function was quantified using rectal organoids in rectal organoid morphology analysis (ROMA) and forskolin-induced swelling (FIS) assays. CFTR variants were further characterised in CF bronchial epithelial (CFBE) cell lines. The impact of involved amino acid changes in the CFTR 3D protein structure was evaluated. RESULTS: Organoids of the patient [S1251N;F508] with mild CF phenotype confirmed the CF diagnosis but showed higher residual CFTR function compared to the four others [S1251N;F508C]. CFBE cell lines showed a decrease in [S1251N;F508C]-CFTR function but not in processing when compared to [S1251N;F508]-CFTR. Analysis of the 3D CFTR structure suggested an additive deleterious effect of the combined presence of S1251N and F508C with respect to NBD1-2 dimerisation. CONCLUSIONS: In vitro and in silico data show that the presence of F508C in cis with S1251N decreases CFTR function without affecting processing. Complex CFTR alleles play a role in clinical phenotype and their identification is relevant in the context of personalised medicine for each patient with CF.

Tailoring Multi-omics to Inflammatory Bowel Diseases: All for One and One for All.

Inflammatory bowel disease [IBD] has a multifactorial origin and originates from a complex interplay of environmental factors with the innate immune system at the intestinal epithelial interface in a genetically susceptible individual. All these factors make its aetiology intricate and largely unknown. Multi-omic datasets obtained from IBD patients are required to gain further insights into IBD biology. We here review the landscape of multi-omic data availability in IBD and identify barriers and gaps for future research. We also outline the various technical and non-technical factors that influence the utility and interpretability of multi-omic datasets and thereby the study design of any research project generating such datasets. Coordinated generation of multi-omic datasets and their systemic integration with clinical phenotypes and environmental exposures will not only enhance understanding of the fundamental mechanisms of IBD but also improve therapeutic strategies. Finally, we provide recommendations to enable and facilitate generation of multi-omic datasets.

Rectal organoid morphology analysis (ROMA) as a promising diagnostic tool in cystic fibrosis.

Diagnosing cystic fibrosis (CF) when sweat chloride is not in the CF range and less than 2 disease-causing CFTR mutations are found requires physiological CFTR assays, which are not always feasible or available. We developed a new physiological CFTR assay based on the morphological differences between rectal organoids from subjects with and without CF. In organoids from 167 subjects with and 22 without CF, two parameters derived from a semi-automated image analysis protocol (rectal organoid morphology analysis, ROMA) fully discriminated CF subjects with two disease-causing mutations from non-CF subjects (p<0.001). ROMA, feasible at all ages, can be centralised to improve standardisation.

Intestinal Receptor of SARS-CoV-2 in Inflamed IBD Tissue Seems Downregulated by HNF4A in Ileum and Upregulated by Interferon Regulating Factors in Colon.

BACKGROUND: Patients with inflammatory bowel disease [IBD] are considered immunosuppressed, but do not seem more vulnerable for COVID-19. Nevertheless, intestinal inflammation has shown to be an important risk factor for SARS-CoV-2 infection and prognosis. Therefore, we investigated the role of intestinal inflammation on the viral intestinal entry mechanisms, including ACE2, in IBD. METHODS: We collected inflamed and uninflamed mucosal biopsies from Crohn's disease [CD] [n = 193] and ulcerative colitis [UC] [n = 158] patients, and from 51 matched non-IBD controls for RNA sequencing, differential gene expression, and co-expression analysis. Organoids from UC patients were subjected to an inflammatory mix and processed for RNA sequencing. Transmural ileal biopsies were processed for single-cell [sc] sequencing. Publicly available colonic sc-RNA sequencing data, and microarrays from tissue pre/post anti-tumour necrosis factor [TNF] therapy, were analysed. RESULTS: In inflamed CD ileum, ACE2 was significantly decreased compared with control ileum [p = 4.6E-07], whereas colonic ACE2 was higher in inflamed colon of CD/UC compared with control [p = 8.3E-03; p = 1.9E-03]. Sc-RNA sequencing confirmed this ACE2 dysregulation and exclusive epithelial ACE2 expression. Network analyses highlighted HNF4A as key regulator of ileal ACE2, and pro-inflammatory cytokines and interferon regulating factors regulated colonic ACE2. Inflammatory stimuli upregulated ACE2 in UC organoids [p = 1.7E-02], but not in non-IBD controls [p = 9.1E-01]. Anti-TNF therapy restored colonic ACE2 regulation in responders. CONCLUSIONS: Intestinal inflammation alters SARS-CoV-2 coreceptors in the intestine, with opposing dysregulations in ileum and colon. HNF4A, an IBD susceptibility gene, seems an important upstream regulator of ACE2 in ileum, whereas interferon signalling might dominate in colon.

Organoid-based Models to Study the Role of Host-microbiota Interactions in IBD.

The gut microbiota appears to play a central role in health, and alterations in the gut microbiota are observed in both forms of inflammatory bowel disease [IBD], namely Crohn's disease and ulcerative colitis. Yet, the mechanisms behind host-microbiota interactions in IBD, especially at the intestinal epithelial cell level, are not yet fully understood. Dissecting the role of host-microbiota interactions in disease onset and progression is pivotal, and requires representative models mimicking the gastrointestinal ecosystem, including the intestinal epithelium, the gut microbiota, and immune cells. New advancements in organoid microfluidics technology are facilitating the study of IBD-related microbial-epithelial cross-talk, and the discovery of novel microbial therapies. Here, we review different organoid-based ex vivo models that are currently available, and benchmark their suitability and limitations for specific research questions. Organoid applications, such as patient-derived organoid biobanks for microbial screening and 'omics technologies, are discussed, highlighting their potential to gain better mechanistic insights into disease mechanisms and eventually allow personalised medicine.

Expression Levels of 4 Genes in Colon Tissue Might Be Used to Predict Which Patients Will Enter Endoscopic Remission After Vedolizumab Therapy for Inflammatory Bowel Diseases.

BACKGROUND & AIMS: We aimed to identify biomarkers that might be used to predict responses of patients with inflammatory bowel diseases (IBD) to vedolizumab therapy. METHODS: We obtained biopsies from inflamed colon of patients with IBD who began treatment with vedolizumab (n = 31) or tumor necrosis factor (TNF) antagonists (n = 20) and performed RNA-sequencing analyses. We compared gene expression patterns between patients who did and did not enter endoscopic remission (absence of ulcerations at month 6 for patients with Crohn's disease or Mayo endoscopic subscore ≤1 at week 14 for patients with ulcerative colitis) and performed pathway analysis and cell deconvolution for training (n = 20) and validation (n = 11) datasets. Colon biopsies were also analyzed by immunohistochemistry. We validated a baseline gene expression pattern associated with endoscopic remission after vedolizumab therapy using 3 independent datasets (n = 66). RESULTS: We identified significant differences in expression levels of 44 genes between patients who entered remission after vedolizumab and those who did not; we found significant increases in leukocyte migration in colon tissues from patients who did not enter remission (P < .006). Deconvolution methods identified a significant enrichment of monocytes (P = .005), M1-macrophages (P = .05), and CD4+ T cells (P = .008) in colon tissues from patients who did not enter remission, whereas colon tissues from patients in remission had higher numbers of naïve B cells before treatment (P = .05). Baseline expression levels of PIWIL1, MAATS1, RGS13, and DCHS2 identified patients who did vs did not enter remission with 80% accuracy in the training set and 100% accuracy in validation dataset 1. We validated these findings in the 3 independent datasets by microarray, RNA sequencing and quantitative PCR analysis (P = .003). Expression levels of these 4 genes did not associate with response to anti-TNF agents. We confirmed the presence of proteins encoded by mRNAs using immunohistochemistry. CONCLUSIONS: We identified 4 genes whose baseline expression levels in colon tissues of patients with IBD associate with endoscopic remission after vedolizumab, but not anti-TNF, treatment. We validated this signature in 4 independent datasets and also at the protein level. Studies of these genes might provide insights into the mechanisms of action of vedolizumab.

Butyrate Does Not Protect Against Inflammation-induced Loss of Epithelial Barrier Function and Cytokine Production in Primary Cell Monolayers From Patients With Ulcerative Colitis.

BACKGROUND AND AIMS: In vitro studies using immortalised cancer cell lines showed that butyrate has an overall positive effect on epithelial barrier integrity, but the physiological relevance of cancer cell lines is limited. We developed epithelial monolayers from human tissue samples of patients with ulcerative colitis [UC] to assess the effect of butyrate on epithelial barrier function. METHODS: A protocol to establish monolayers from primary epithelial cells of UC patients [n = 10] and non-UC controls [n = 10] was optimised. The monolayers were treated with 8 mM sodium butyrate ± tumour necrosis factor alpha [TNFα] and type II interferon [IFNγ] for 48 h. Changes in transepithelial electrical resistance were monitored. Barrier gene expression levels were measured. Inflammatory proteins in the supernatant of the cells were quantified with OLINK. RESULTS: We demonstrated that primary monolayer cultures can be grown within 1 week of culture with robust resistance values and polarised tight junction expression. Butyrate treatment of the cultures increased resistance but was detrimental in combination with TNFα and IFNγ. The combined treatment further induced even higher IL8 mRNA and inflammatory protein secretion than for the inflammatory mediators alone. The observed effects were similar in cultures from patients and non-UC controls, suggesting that there were no patient-specific responses responsible for these findings. CONCLUSIONS: We found that butyrate does not protect against inflammation-induced barrier dysfunction and even worsens its effects in primary epithelial monolayers of UC patients and controls. The basic mechanisms of butyrate should therefore be reconsidered in future studies, in particular in patients with active inflammation and pre-existing barrier defects as is known for UC.

Human intestinal epithelium in a dish: Current models for research into gastrointestinal pathophysiology.

Determining the exact pathogenesis of chronic gastrointestinal diseases remains difficult due to the complex in vivo environment. In this review we give an overview of the available epithelial cell culture systems developed to investigate pathophysiology of gastrointestinal diseases. Traditionally used two-dimensional (2D) immortalised (tumour) cell lines survive long-term, but are not genetically stable nor represent any human in particular. In contrast, primary cultures are patient unique, but short-lived. Three-dimensional (3D) organoid cultures resemble the crypt-villus domain and contain all cell lineages, are long-lived and genetically stable. Unfortunately, manipulation of the 3D organoid system is more challenging. Combining the 3D and 2D technologies may overcome limitations and offer the formation of monolayers on permeable membranes or flow-chambers. Determining the right model to use will depend on the pathology of interest and the focus of the research, defining which cell types need to be included in the model.

Age-specific function of α5ß1 integrin in microglial migration during early colonization of the developing mouse cortex.

Microglia, the immune cells of the central nervous system, take part in brain development and homeostasis. They derive from primitive myeloid progenitors that originate in the yolk sac and colonize the brain mainly through intensive migration. During development, microglial migration speed declines which suggests that their interaction with the microenvironment changes. However, the matrix-cell interactions allowing dispersion within the parenchyma are unknown. Therefore, we aimed to better characterize the migration behavior and to assess the role of matrix-integrin interactions during microglial migration in the embryonic brain ex vivo. We focused on microglia-fibronectin interactions mediated through the fibronectin receptor α5ß1 integrin because in vitro work indirectly suggested a role for this ligand-receptor pair. Using 2-photon time-lapse microscopy on acute ex vivo embryonic brain slices, we found that migration occurs in a saltatory pattern and is developmentally regulated. Most importantly, there is an age-specific function of the α5ß1 integrin during microglial cortex colonization. At embryonic day (E) 13.5, α5ß1 facilitates migration while from E15.5, it inhibits migration. These results indicate a developmentally regulated function of α5ß1 integrin in microglial migration during colonization of the embryonic brain.