Perfusion System for Modification of Luminal Contents of Human Intestinal Organoids and Realtime Imaging Analysis of Microbial Populations

Intestinal organoids are 3D cell structures that replicate some aspects of organ function and are organized with a polarized epithelium facing a central lumen. To enable more applications, new technologies are needed to access the luminal cavity and apical cell surface of organoids. We developed a perfusion system utilizing a double-barrel glass capillary with a pressure-based pump to access and modify the luminal contents of a human intestinal organoid for extended periods of time while applying cyclic cellular strain. Cyclic injection and withdrawal of fluorescent FITC-Dextran coupled with real-time measurement of fluorescence intensity showed discrete changes of intensity correlating with perfusion cycles. The perfusion system was also used to modify the lumen of organoids injected with GFP-expressing E. coli. Due to the low concentration and fluorescence of the E. coli, a novel imaging analysis method utilizing bacteria enumeration and image flattening was developed to monitor E. coli within the organoid. Collectively, this work shows that a double-barrel perfusion system provides constant luminal access and allows regulation of luminal contents and luminal mixing.

Rare Trafficking CFTR Mutations Involve Distinct Cellular Retention Machineries and Require Different Rescuing Strategies

Most of the ~2100 CFTR variants so far reported are very rare and still uncharacterized regarding their cystic fibrosis (CF) disease liability. Since some may respond to currently approved modulators, characterizing their defect and response to these drugs is essential. Here we aimed characterizing the defect associated with four rare missense (likely Class II) CFTR variants and assess their rescue by corrector drugs. We produced CFBE cell lines stably expressing CFTR with W57G, R560S, H1079P and Q1100P, assessed their effect upon CFTR expression and maturation and their rescue by VX-661/VX-445 correctors. Results were validated by forskolin-induced swelling assay (FIS) using intestinal organoids from individuals bearing these variants. Finally, knock-down (KD) of genes previously shown to rescue F508del-CFTR was assessed on these mutants. Results show that all the variants preclude the production of mature CFTR, confirming them as Class II mutations. None of the variants responded to VX-661 but the combination rescued H1079P- and Q1100P-CFTR. The KD of factors that correct F508del-CFTR retention only marginally rescued R560S- and H1079P-CFTR. Overall, data evidence that Class II mutations induce distinct molecular defects that are neither rescued by the same corrector compounds nor recognized by the same cellular machinery, thus requiring personalized drug discovery initiatives.

Epithelial cells of the intestine acquire cell-intrinsic inflammation signatures during ageing

During ageing, cell-intrinsic and extrinsic factors lead to the decline of tissue function and organismal health. Disentangling these factors is important for developing effective strategies to prolong organismal healthspan. Here, we addressed this question in the mouse intestinal epithelium, which forms a dynamic interface with its microenvironment and receives extrinsic signals affecting its homeostasis and tissue ageing. We systematically compared transcriptional profiles of young and aged epithelial cells in vivo and ex vivo in cultured intestinal organoids. We found that all cell types of the aged epithelium exhibit an inflammation phenotype, which is marked by MHC class II upregulation and most pronounced in enterocytes. This was accompanied by elevated levels of the immune tolerance markers PD-1 and PD-L1 in the aged tissue microenvironment, indicating dysregulation of immunological homeostasis. Intestinal organoids from aged mice still showed an inflammation signature after weeks in culture, which was concurrent with increased chromatin accessibility of inflammation-associated loci. Our results reveal a cell-intrinsic, persistent inflammation phenotype in aged epithelial cells, which might contribute to systemic inflammation observed during ageing.

High-Throughput Functional Analysis of CFTR and Other Apically Localized Proteins in iPSC-Derived Human Intestinal Organoids

Induced Pluripotent Stem Cells (iPSCs) can be differentiated into epithelial organoids that recapitulate the relevant context for CFTR and enable testing of therapies targeting Cystic Fibrosis (CF)-causing mutant proteins. However, to date, CF-iPSC-derived organoids have only been used to study pharmacological modulation of mutant CFTR channel activity and not the activity of other disease-relevant membrane protein constituents. In the current work, we describe a high-throughput, fluorescence-based assay of CFTR channel activity in iPSC-derived intestinal organoids and describe how this method can be adapted to study other apical membrane proteins. Specifically, we show how this assay can be employed to study CFTR and ENaC channels and an electrogenic acid transporter in the same iPSC-derived intestinal tissue. This phenotypic platform promises to expand CF therapy discovery to include strategies that target multiple determinants of epithelial fluid transport.

Harnessing the Biology of Canine Intestinal Organoids to Heighten Understanding of Inflammatory Bowel Disease Pathogenesis and Accelerate Drug Discovery: A One Health Approach

In a recent issue of the Lancet, the prevalence of Inflammatory Bowel Disease (IBD) was estimated at 7 million worldwide. Overall, the burden of IBD is rising globally, with direct and indirect healthcare costs ranging between $14.6 and $31.6 billion in the U.S. alone in 2014. There is currently no cure for IBD, and up to 40% of patients do not respond to medical therapy. Although the exact determinants of the disease pathophysiology remain unknown, the prevailing hypothesis involves complex interplay among host genetics, the intestinal microenvironment (primarily bacteria and dietary constituents), and the mucosal immune system. Importantly, multiple chronic diseases leading to high morbidity and mortality in modern western societies, including type II diabetes, IBD and colorectal cancer, have epidemiologically been linked to the consumption of high-calorie, low-fiber, high monosaccharide, and high-fat diets (HFD). More specifically, data from our laboratory and others have shown that repeated consumption of HFD triggers dysbiotic changes of the gut microbiome concomitant with a state of chronic intestinal inflammation and increased intestinal permeability. However, progress in our understanding of the effect of dietary interventions on IBD pathogenesis has been hampered by a lack of relevant animal models. Additionally, current in vitro cell culture systems are unable to emulate the in vivo interplay between the gut microbiome and the intestinal epithelium in a realistic and translatable way. There remains, therefore, a critical need to develop translatable in vitro and in vivo models that faithfully recapitulate human gut-specific physiological functions to facilitate detailed mechanistic studies on the impact of dietary interventions on gut homeostasis. While the study of murine models has been pivotal in advancing genetic and cellular discoveries, these animal systems often lack key clinical signs and temporal pathological changes representative of IBD. Specifically, some limitations of the mouse model are associated with the use of genetic knockouts to induce immune deficiency and disease. This is vastly different from the natural course of IBD developing in immunologically competent hosts, as is the case in humans and dogs. Noteworthily, abundant literature suggests that canine and human IBD share common clinical and molecular features, such that preclinical studies in dogs with naturally occurring IBD present an opportunity to further our understanding on disease pathogenesis and streamline the development of new therapeutic strategies. Using a stepwise approach, in vitro mechanistic studies investigating the contribution of dietary interventions to chronic intestinal inflammation and “gut leakiness” could be performed in intestinal organoids and organoid derived monolayers. The biologic potential of organoids stems from the method’s ability to harness hard-wired cellular programming such that the complexity of the disease background can be reflected more accurately. Likewise, the effect of therapeutic drug candidates could be evaluated in organoids prior to longitudinal studies in dog and human patients with IBD. In this review, we will discuss the value (and limitations) of intestinal organoids derived from a spontaneous animal disease model of IBD (i.e., the dog), and how it can heighten understanding of the interplay between dietary interventions, the gut microbiota and intestinal inflammation. We will also review how intestinal organoids could be used to streamline the preclinical development of therapeutic drug candidates for IBD patients and their best four-legged friends.

Microbial-Derived Metabolites Induce Epithelial Recovery Via the Sting Pathway in Mice and Men and Protect from Graft-Versus-Host Disease

Background: Graft-versus-host disease (GVHD) is a dreaded complication after allogeneic stem-cell transplantation (ASCT). Previously, we and others showed that activation of type I interferon (IFN-I) inducing pathways such as RIG-I/MAVS or cGAS-STING can promote the integrity of the intestinal barrier and limit GVHD (Fischer et al., Sci Transl Med, 2017). However, the signals that drive these protective IFN-I responses are poorly understood. Commensal microbiota can (i) have distant effects on immune responses through modulation of IFN-I signaling (Steed et al, Science, 2017; Swimm et al, Blood, 2018) and (ii) predict mortality in ASCT patients (Peled et al., N Engl J Med, 2020). We hypothesized that microbiota-derived products such as microbial metabolites engage IFN-I signaling in immune and non-immune cells poising them for induction of protective responses. We established a prospective, multi-centric clinical study in patients newly diagnosed with acute leukemia and performed longitudinal stool sampling to track changes in microbial community composition and metabolites expression levels. Submitted as a separate abstract to ASH 2021 (Orberg & Meedt et al.), we show that patients with high metabolite expression going into ASCT are less likely to develop GVHD. In this study, we translate our clinical observations to mouse models of acute GVHD and human and mouse intestinal organoids to uncover the molecular mechanisms via which metabolites protect the intestinal barrier during ASCT. Methods: Stool samples from ASCT patients were obtained in Munich and at Regensburg in accordance to IRB-approved study protocols. Patients were sampled at initial diagnosis (Dx), prior to conditioning and weekly after ASCT up to day 28. We analyzed samples by 16S rRNA sequencing and mass spectrometry to obtain a complete picture of microbiome composition and function. Next, we tested metabolites which we detected in patients (desaminotyrosine [DAT], indole-3-carboxaldehyde [ICA]) as treatment in preclinical models in ASCT and GVHD mouse models. Outcomes were assessed by a novel organoid recovery assay in addition to established read-outs. To obtain a mechanistic understanding of the signaling pathways involved, we stimulated WT or IFN-I-signaling-impaired mouse (incl. STING -/-, MAVS -/-, IFNαR -/-) as well as human intestinal crypt-derived organoids with metabolites. Results: Here, we present a 64-year-old female patient diagnosed with AML who received a 9/10 HLA-matched ASCT. At day 7, i.v. antibiotics were started due to fever and Enterococcus bacteraemia. At day 15, the patient developed skin and GI GVHD (Glucksberg III). At the timepoint initial diagnosis (Dx), i.e. the timepoint when we diagnosed AML but before therapy was initiated, we detected rich alpha diversity (Panel 1a) in the patient's stool. Flavonifractor plautii, a producer of the metabolite DAT, was detectable (Panel 1b). We observed high-level expression of metabolites including short-chain fatty acids, DAT, ICA and secondary bile acids (Panel 1c). Following ASCT, and especially at the early time-points day 0 and day 7, alpha diversity and metabolite expression declined drastically. We confirmed this trend in our multi-centric cohort of ASCT patients by comparing levels of DAT and ICA sampled at admission to the transplantation ward (Conditioning) versus at clinical diagnosis of GVHD: in patients with GVHD, metabolite levels were drastically reduced (Panel 2). Next, we prophylactically administered metabolites in a major mismatch mouse ASCT model. Metabolite-treated mice showed significantly showed better outcomes in our organoid recovery assay, which measures the ability of intestinal stem cells to recover after allogeneic injury (Panel 3). This effect that was abrogated in STING -/- recipients. Metabolite stimulation of mouse small intestinal organoids promoted organoid numbers and size, and required intact STING signaling (Panel 4a). Human colon organoids also responded to DAT and ICA, however the metabolite effect was lost when co-administered with the STING-inhibitor H151. Conclusions: We identify that microbial-derived metabolites detected in patients can engage the STING pathway in humans and mice to confer resistance from immune damage. Thus, prophylactic administration of metabolite cocktails or bacterial consortia that can produce these metabolites may reduce occurrence of GVHD in ASCT patients. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.