The intestinal porcine epithelial cell line (IPEC-J2) is a nontransformed jejunal model derived from unsuckled piglets. It is increasingly used in gut physiology research, yet its application to nutrient absorption remains underexplored. Four media types were assessed for their ability to support IPEC-J2 barrier maturation and permeability: Dulbecco's modified Eagle medium/Ham's F-12 with 10% fetal bovine serum (FBS10), 10% porcine serum (PS10), 5% porcine serum (PS5) supplemented with Wnt3a-R-spondin-Noggin conditioned medium (PS5+L-WRN), and PS5 with epidermal growth factor and insulin-transferrin-selenium (PS5+EGF+ITS). Culture medium influenced epithelial phenotype, with more than a 30-fold range in transepithelial electrical resistance (TEER), which plateaued by day 12. PS10 supported a balanced profile, characterized by intermediate TEER (2395.00 ± 485.51 Ω × cm2), lowest lactulose and mannitol permeability (p < 0.05), high lactate dehydrogenase activity (p < 0.05 vs. PS5+L-WRN and FBS10), enhanced membrane localization of tight junction proteins (p < 0.05 vs. PS5+EGF+ITS), and reduced fibroblast marker S100A4 (p < 0.05 vs. PS5+EGF+ITS). Treatment with digested infant milk formula did not compromise barrier integrity in PS10-cultured IPEC-J2 monolayers and enabled transport of free amino acids to the basolateral compartment. These findings support IPEC-J2 as an alternative to Caco-2 cells for applications in infant nutrition research in vitro.

Butyrate exhibits a dual effect on gut permeability, promoting epithelial health at low concentrations but disrupting barrier integrity at higher levels. This disruption at higher concentrations can be exploited, in vitro, to create gut barrier models with increased permeability. This study aimed to unravel the mechanism of action of sodium butyrate (NaB, 50–125 mM) on human intestinal enterocyte monolayers using the Caco-2 cell model. NaB reduced Trans-Epithelial Electrical Resistance (TEER) and increased lucifer yellow paracellular flux in a dose-dependent manner on Caco-2 monolayers, effects that persisted after NaB withdrawal. TEER reductions induced by NaB occurred under hyperosmotic conditions (430.5 ± 4.5 mOsm/kg). In contrast treatment with mannitol, a non-metabolizable osmotic agent used to mimic the osmotic effect, only caused a transient decrease in TEER. NaB was actively transported into cells, with increased mRNA expression of butyrate transporters MCT-1 and SMCT-1. However, NaB did not act as an energy substrate, as evidenced by decreased ATP levels and unchanged cellular reductase activity. Instead, NaB elevated Caspase-3 activity, induced stress markers (p21, HSP-70), and stimulated cytokine production (IL-6, IL-8, TNF). Most importantly, treatment with dexamethasone, a commonly used steroid, restored TEER and reduced stress and inflammation markers in Caco-2 monolayers disrupted by 75 mM NaB. Therefore, NaB treated Caco-2 monolayers are a suitable in vitro model for gut barrier dysfunction and inflammation such as Crohn's disease or necrotizing enterocolitis.

Early postnatal nutrition is critical for gut barrier maturation, yet infant formulas only partially replicate the functional benefits of human milk. Using infant- and adult-like Caco-2/HT29-MTX models, this in vitro study compared the effects of gastrointestinal digested human milk, dairy-, soya-, and amino acid-based infant formulas on gut barrier integrity and peptide transport. Barrier function was assessed by transepithelial electrical resistance and occludin-actin colocalization, while amino acid and peptide transport were profiled by liquid chromatography plus mass spectrometry. In the infant-like model, human milk uniquely enhanced occludin-actin co-localization (P < 0.05) and increased the abundance of basolateral bioactive peptides. Amino acid-based infant formula significantly increased occludin fluorescence intensity in infant-like monolayers but reduced transepithelial electrical resistance (TEER) in adult-like monolayers. For all digested foods, peptide transport and diversity were significantly greater in infant-like than in adult-like monolayers, with human milk promoting the highest basolateral peptide load. These findings demonstrate distinct functional differences between human milk and infant formulas in their interaction with the gut barrier. They also underscore the importance of using age-appropriate in vitro models for accurately evaluating dietary effects during early life, highlighting the need for further research to optimize infant nutrition strategies.

Live-cell Ca2+ imaging is an important tool to detect activation of receptors by a putative ligand/drug and complements studies on transport processes, as intracellular Ca2+ changes provide direct evidence for substrate fluxes. Organoid-based systems offer numerous advantages over other in vitro systems such as cell lines, primary cells, or tissue explants, and in particular, intestinal organoid culture has revolutionized research on functional gastrointestinal processes. Calcium imaging using the fluorescent Ca2+ indicator Fura-2-AM can be applied to 3D intestinal organoids, which show an excellent dye-loading efficiency. Here we describe live-cell Ca2+ imaging in intestinal organoids, an important technique to improve research on malabsorption syndromes, secretory diarrhea, and metabolic disorders.

IntroductionIn newborns, the intestinal barrier is permeable but not inflamed. Understanding this unique state is essential for developing models relevant to infant gut physiology.MethodsThis study aimed to develop an in vitro model of the infant gut barrier treating Caco-2/HT29-MTX with 0.5, 0.8, and 1 mM sodium glycodeoxycholate (GDC).ResultsOur research demonstrates that GDC decreases Caco-2/HT29-MTX Trans-Epithelial Electrical Resistance (TEER) and increases paracellular permeability, without inflammation or cytotoxicity. Notably, the treatment with 0.8 mM GDC increased lactulose transport rate by 1.63-fold. The treatment also reduced the key tight junction protein, occludin, at the cell membrane, and increased acidic mucins and extracellular alkaline phosphatase activity. Additionally, GDC decreased cAMP, suggesting its mechanism of action was via activation of a G-protein coupled receptor. Of particular importance to nutrition studies, the GDC effect was reversible with TEER recovery within 4 h. Applying digested infant formula to 0.8 mM GDC-treated Caco-2/HT29-MTX monolayers resulted in a higher concentration of amino acids in the basolateral compartment compared to control monolayers.DiscussionThese findings suggest that GDC can modulate gut barrier properties in a controled, reversible manner, offering a valuable model for studying nutrient absorption and gut physiology in early life.

Interferometric experiments designed to detect the highly redshifted 21-cm signal from neutral hydrogen are producing increasingly stringent constraints on the 21-cm power spectrum, but some k-modes remain systematics-dominated. Mutual coupling is a major systematic that must be overcome in order to detect the 21-cm signal, and simulations that reproduce effects seen in the data can guide strategies for mitigating mutual coupling. In this paper, we analyse 12 nights of data from the Hydrogen Epoch of Reionization Array and compare the data against simulations that include a computationally efficient and physically motivated semi-analytic treatment of mutual coupling. We find that simulated coupling features qualitatively agree with coupling features in the data; however, coupling features in the data are brighter than the simulated features, indicating the presence of additional coupling mechanisms not captured by our model. We explore the use of fringe-rate filters as mutual coupling mitigation tools and use our simulations to investigate the effects of mutual coupling on a simulated cosmological 21-cm power spectrum in a "worst case" scenario where the foregrounds are particularly bright. We find that mutual coupling contaminates a large portion of the "EoR Window", and the contamination is several orders-of-magnitude larger than our simulated cosmic signal across a wide range of cosmological Fourier modes. While our fiducial fringe-rate filtering strategy reduces mutual coupling by roughly a factor of 100 in power, a non-negligible amount of coupling cannot be excised with fringe-rate filters, so more sophisticated mitigation strategies are required.

BACKGROUND & AIMS:Enteroendocrine cells (EECs) are known for their role in digestion and metabolism, yet their role in intestinal inflammation remains unclear. In inflammatory bowel diseases, a contribution of EECs to pathogenesis is indicated by autoantibodies affecting EEC function and general disease symptoms like insulin resistance and altered intestinal motility. Particularly, the L cell-derived hormone glucagon-like peptide 1 (GLP-1), suggested to orchestrate metabolic-inflammatory responses may influence inflammatory pathways in the intestine. METHODS:We quantified numbers of GLP-1+ cells in 4 different mouse models of intestinal inflammation and performed transcriptional analyses of colonic epithelial cells from inflamed interleukin-10-deficient mice. Using a publicly available single-cell RNA sequencing dataset including mucosal biopsies from patients with Crohn's disease, we confirmed findings from the murine models. A model of mitochondrial dysfunction (ClpPΔIEC mice) as well as murine and human intestinal organoids were used to study molecular mechanisms. RESULTS:Numbers of GLP-1 expressing cells are consistently reduced at the site of active disease in mouse models and patients with Crohn's disease. Despite this reduction, L cells from inflamed interleukin-10-deficient mice remained functional regarding GLP-1 secretion. Transcriptional analyses of intestinal epithelial cells indicate altered differentiation correlating with an inflammatory metabolic fingerprint. Reduced GLP-1+ cells in ClpPΔIEC mice and inhibition of respiration in organoid cultures supports a causative role for metabolism in steering differentiation. CONCLUSIONS:Reduction of GLP-1+ cells represents a general feature of ileal and colonic inflammation in mice and humans. Given the numerous properties of GLP-1, this reduction likely affects inflammatory processes in the mucosa and disease-related symptoms on multiple levels, and therefore, should be considered a therapeutic target in inflammatory bowel diseases.

The 21 cm transition from neutral Hydrogen promises to be the best observational probe of the Epoch of Reionization (EoR). This has led to the construction of low-frequency radio interferometric arrays, such as the Hydrogen Epoch of Reionization Array (HERA), aimed at systematically mapping this emission for the first time. Precision calibration, however, is a requirement in 21 cm radio observations. Due to the spatial compactness of HERA, the array is prone to the effects of mutual coupling, which inevitably lead to non-smooth calibration errors that contaminate the data. When unsmooth gains are used in calibration, intrinsically spectrally smooth foreground emission begins to contaminate the data in a way that can prohibit a clean detection of the cosmological EoR signal. In this paper, we show that the effects of mutual coupling on calibration quality can be reduced by applying custom time-domain filters to the data prior to calibration. We find that more robust calibration solutions are derived when filtering in this way, which reduces the observed foreground power leakage. Specifically, we find a reduction of foreground power leakage by 2 orders of magnitude at k|| approximate to 0 . 5 h Mpc(-1).

Mitochondrial dysfunction is associated with inflammatory bowel diseases (IBDs). To understand how microbial-metabolic circuits contribute to intestinal injury, we disrupt mitochondrial function in the epithelium by deleting the mitochondrial chaperone, heat shock protein 60 (Hsp60Δ/ΔIEC). This metabolic perturbation causes self-resolving tissue injury. Regeneration is disrupted in the absence of the aryl hydrocarbon receptor (Hsp60Δ/ΔIEC;AhR-/-) involved in intestinal homeostasis or inflammatory regulator interleukin (IL)-10 (Hsp60Δ/ΔIEC;Il10-/-), causing IBD-like pathology. Injury is absent in the distal colon of germ-free (GF) Hsp60Δ/ΔIEC mice, highlighting bacterial control of metabolic injury. Colonizing GF Hsp60Δ/ΔIEC mice with the synthetic community OMM12 reveals expansion of metabolically flexible Bacteroides, and B. caecimuris mono-colonization recapitulates the injury. Transcriptional profiling of the metabolically impaired epithelium reveals gene signatures involved in oxidative stress (Ido1, Nos2, Duox2). These signatures are observed in samples from Crohn's disease patients, distinguishing active from inactive inflammation. Thus, mitochondrial perturbation of the epithelium causes microbiota-dependent injury with discriminative inflammatory gene profiles relevant for IBD.

Cells need to adapt constantly to internal and environmental changes ranging from normal physiological fluctuations to pathological alterations. Changes in the cellular demand can cause perturbations in different cellular compartments that in turn activate distinct signaling pathways to elicit transcriptional programs aiming at resolving the perturbation at the site of origin. In line, distinct adaptive responses to different types of “stresses” have been described for mitochondria. However, while responses to oxidative stress and hypoxia as well as events resulting in apoptosis are well understood, knowledge on signals, mediators, and targets employed in the response to disturbed mitochondrial proteostasis is still rudimentary (Ryan and Hoogenraad, 2007; Vogtle, 2021). Yet, protein aggregation has a significant impact on mitochondrial function and consequently, imbalances in mitochondrial proteostasis are implicated in ageing and are associated with a plethora of human diseases (Rath et al., 2018; Suomalainen and Battersby, 2018). The mitochondrial unfolded protein response (mtUPR or UPR) evoked by insufficient protein-folding capacity, accumulation of misfolded proteins or nondegradable protein aggregates in mitochondria, is a protective response to restore proteostasis. Upregulating nuclear-encoded mitochondrial chaperones and proteases as well as controlling mitochondrial RNA translation, mtUPR improves the mitochondrial folding environment, thus maintaining mitochondrial integrity (Munch, 2018). Although a growing number of players in mtUPR has been identified in the recent years, many open questions remain, including the identity of the initial signal, as well as unidentified molecular components to sense and mediate the retrograde signal to the nucleus (Vogtle, 2021). In 2011, we identified the double-stranded RNA (dsRNA)activated protein kinase (PKR) as a signaling component of the mammalian mtUPR and demonstrated its disease-relevance for inflammatory bowel diseases (Rath et al., 2011), findings that have been confirmed by us and others (Jackson et al., 2020; Khaloian et al., 2020). However, we were not able to identify the signal that leads to PKR activation upon induction of mtUPR by expression of a mutant protein, ornithine transcarbamylase (OTC)Δ, that accumulates in a misfolded state in the mitochondrial matrix (Ryan and Hoogenraad, 2007; Rath et al., 2011). New findings by Kim et al. now indicate that PKR can be activated by mitochondrial RNA that exist as intermolecular dsRNA, in particular under stress conditions (Kim et al., 2018). These results contribute to a more comprehensive understanding of mitochondrial stress signaling and make it OPEN ACCESS

Eating healthy foods supplies your body with nutrients to stay strong. But did you ever wonder how nutrients enter your body? After chewing and swallowing, your food is digested, and enters the gut as mush. If you picture your intestine as a tube, the food is on the inside and your body is around the tube. The inner layer of the tube that touches the food is formed by special cells that can transport nutrients like sugar and protein. Some people cannot properly absorb nutrients. The molecules that transport nutrients also transport certain drugs. Thus, investigating intestinal transport is very important to help people with absorption issues and to design better drugs. We used a new scientific model called organoids to study intestinal transport processes. Organoids are tiny “mini-guts” grown in the lab from human cells. Organoids have many advantages over other models used by scientists to study the gut.

The intestinal epithelium represents the most regenerative tissue in the human body, located in proximity to the dense and functionally diverse microbial milieu of the microbiome. Episodes of tissue injury and incomplete healing of the intestinal epithelium are a prerequisite for immune reactivation and account for recurrent, chronically progressing phenotypes of inflammatory bowel diseases (IBD). Mitochondrial dysfunction and associated changes in intestinal epithelial functions are emerging concepts in the pathogenesis of IBD, suggesting impaired metabolic flexibility of epithelial cells affects the regenerative capacity of the intestinal tissue. Next to rendering the intestinal mucosa susceptible to inflammatory triggers, metabolic reprogramming of the epithelium is implicated in shaping adverse microbial environments. In this review, we introduce the concept of "metabolic injury" as a cell autonomous mechanism of tissue wounding in response to mitochondrial perturbation. Furthermore, we highlight epithelial metabolism as intersection of microbiome, immune cells and epithelial regeneration.

AbstractMitochondrial dysfunction in intestinal epithelial cells (IECs) is associated with chronic inflammation. To understand how microbial-metabolic circuits contribute to tissue injury, we disrupt mitochondrial function in IECs by deleting heat shock protein 60 (Hsp60Δ/ΔIEC). While metabolic perturbation causes self-resolving tissue injury, regeneration is disrupted in the susceptible host (Hsp60Δ/ΔIEC;Il10-/-). Interestingly, metabolic injury induces microbial dysbiosis, includingBacteroidesspp. expansion. Tissue pathology is absent in the distal colon of germfree (GF) Hsp60Δ/ΔIECmice, highlighting bacterial control of metabolic injury. In line, host tryptophan metabolism is rewired, and the absence of bacterial-mediated AhR signaling (Hsp60Δ/ΔIEC;AhR-/-) causes severe acceleration of injury independent of IL-22. Furthermore, selective colonization of GF Hsp60Δ/ΔIECmice with OMM12induces metabolic injury andBacteroides caecimurisexpansion, which generates metabolic injury in mono-colonized mice. In conclusion, mitochondrial perturbation of the epithelium causes dysbiotic expansion ofBacteroidesspp., supporting the concept that microbe-host cross-talk contributes to metabolic injury in intestinal inflammation.Graphical Abstract: Control of metabolic injury by microbial signalsHsp60 deletion is induced in intestinal epithelial cells leading to mitochondrial perturbation, causing a shift in colonocyte metabolism (1, 2)Intestinal crypts display loss of stemness and weakening of the mucus barrier accompanied by dysbiotic changes, notably expansion ofBacteroidesspp. (3 – 6)The injury phenotype is accompanied by immune cell recruitment and loss of regulatory mechanisms in a colitis-susceptible model (7)AhR-dependent signals are critical to maintain balanced bacterial-metabolic circuits for tissue healing and homeostasis (8)Created withBioRender.com.D: day; Hsp60+: heat shock protein 60-positive; Lgr5+: leucine-rich repeat-containing G-protein coupled receptor 5-positive; AhR: aryl hydrocarbon receptor; OXPHOS: oxidative phosphorylation; MT-UPR: mitochondrial unfolded protein response; Trb3: tribbles pseudokinase 3; Ido1: indoleamine 2, 3-dioxygenase 1.

Mitochondrial proteostasis, regulated by the mitochondrial unfolded protein response (UPRmt), is crucial for maintenance of cellular functions and survival. Elevated oxidative and proteotoxic stress in mitochondria must be attenuated by the activation of a ubiquitous UPRmt to promote prostate cancer (PCa) growth. Here we show that the 2 key components of the UPRmt, heat shock protein 60 (HSP60, a mitochondrial chaperonin) and caseinolytic protease P (ClpP, a mitochondrial protease), were required for the development of advanced PCa. HSP60 regulated ClpP expression via c-Myc and physically interacted with ClpP to restore mitochondrial functions that promote cancer cell survival. HSP60 maintained the ATP-producing functions of mitochondria, which activated the β-catenin pathway and led to the upregulation of c-Myc. We identified a UPRmt inhibitor that blocked HSP60's interaction with ClpP and abrogated survival signaling without altering HSP60's chaperonin function. Disruption of HSP60-ClpP interaction with the UPRmt inhibitor triggered metabolic stress and impeded PCa-promoting signaling. Treatment with the UPRmt inhibitor or genetic ablation of Hsp60 inhibited PCa growth and progression. Together, our findings demonstrate that the HSP60-ClpP–mediated UPRmt is essential for prostate tumorigenesis and the HSP60-ClpP interaction represents a therapeutic vulnerability in PCa.

The intestinal epithelium critically contributes to oral bioavailability of drugs by constituting an important site for drug absorption and metabolism. In particular, intestinal epithelial cells (IEC) actively serve as gatekeepers of drug and nutrient availability. IECs' transport processes and metabolism are interrelated to the whole-body metabolic state and represent potential points of origin as well as therapeutic targets for a variety of diseases. Human intestinal organoids represent a superior model of the intestinal epithelium, overcoming limitations of currently used in vitro models. Caco-2 cells or rodent explant models face drawbacks such as their cancer and non-human origin, respectively, but are commonly used to study intestinal nutrient absorption, enterocyte metabolism and oral drug bioavailability, despite poorly correlative data. In contrast, intestinal organoids allow investigating distinct aspects of bioavailability including spatial resolution of transport, inter-individual differences and high-throughput screenings. As several countries have already developed strategic roadmaps to phase out animal experiments for regulatory purposes, intestinal organoid culture and organ-on-a-chip technology in combination with in silico approaches are roads to go in the preclinical and regulatory setup and will aid implementing the 3Rs (reduction, refinement and replacement) principle in basic science.

Aim: We aimed to determine whether the sodium/glucose cotransporter family member SGLT3, a proposed glucose sensor, is expressed in the intestine and/or kidney, and if its expression is altered in mouse models of obesity and in humans before and after weight-loss surgery. Main methods: We used in situ hybridization and quantitative PCR to determine whether the Sglt3 isoforms 3a and 3b were expressed in the intestine and kidney of C57, leptin-deficient ob/ob, and diabetic BTBR ob/ob mice. Western blotting and immunohistochemistry were also used to assess SGLT3 protein levels in jejunal biopsies from obese patients before and after weight-loss Roux-en-Y gastric bypass surgery (RYGB), and in lean healthy controls. Key findings: Sglt3a/3b mRNA was detected in the small intestine (duodenum, jejunum and ileum), but not in the large intestine or kidneys of mice. Both isoforms were detected in epithelial cells (confirmed using intestinal organoids). Expression of Sglt3a/3b mRNA in duodenum and jejunum was significantly lower in ob/ob and BTBR ob/ob mice than in normal-weight littermates. Jejunal SGLT3 protein levels in aged obese patients before RYGB were lower than in lean individuals, but substantially upregulated 6 months post-RYGB. Significance: Our study shows that Sglt3a/3b is expressed primarily in epithelial cells of the small intestine in mice. Furthermore, we observed an association between intestinal mRNA Sglt3a/3b expression and obesity in mice, and between jejunal SGLT3 protein levels and obesity in humans. Further studies are required to determine the possible role of SGLT3 in obesity.

Mitochondrial metabolism, dynamics, and stress responses in the intestinal stem cell niche play a pivotal role in regulating intestinal epithelial cell homeostasis, including self-renewal and differentiation. In addition, mitochondria are increasingly recognized for their involvement in sensing the metabolic environment and their capability of integrating host and microbial-derived signals. Gastrointestinal diseases such as inflammatory bowel diseases and colorectal cancer are characterized by alterations of intestinal stemness, the microbial milieu, and mitochondrial metabolism. Thus, mitochondrial function emerges at the interface of determining health and disease, and failure to adapt mitochondrial function to environmental cues potentially results in aberrant tissue responses. A mechanistic understanding of the underlying role of mitochondrial fitness in intestinal pathologies is still in its infancy, and therapies targeting mitochondrial (dys)function are currently lacking. This review discusses mitochondrial signaling and metabolism in intestinal stem cells and Paneth cells as critical junction translating host- and microbe-derived signals into epithelial responses. Consequently, we propose mitochondrial fitness as a hallmark for intestinal epithelial cell plasticity, determining the regenerative capacity of the epithelium.

Intestinal transport and sensing processes and their interconnection to metabolism are relevant to pathologies such as malabsorption syndromes, inflammatory diseases, obesity and type 2 diabetes. Constituting a highly selective barrier, intestinal epithelial cells absorb, metabolize, and release nutrients into the circulation, hence serving as gatekeeper of nutrient availability and metabolic health for the whole organism. Next to nutrient transport and sensing functions, intestinal transporters including peptide transporter 1 (PEPT1) are involved in the absorption of drugs and prodrugs, including certain inhibitors of angiotensin-converting enzyme, protease inhibitors, antivirals, and peptidomimetics like β-lactam antibiotics. Here, we verify the applicability of 3D organoids for in vitro investigation of intestinal biochemical processes related to transport and metabolism of nutrients and drugs. Establishing a variety of methodologies including illustration of transporter-mediated nutrient and drug uptake and metabolomics approaches, we highlight intestinal organoids as robust and reliable tool in this field of research. Currently used in vitro models to study intestinal nutrient absorption, drug transport and enterocyte metabolism, such as Caco-2 cells or rodent explant models are of limited value due to their cancer and non-human origin, respectively. Particularly species differences result in poorly correlative data and findings obtained in these models cannot be extrapolated reliably to humans, as indicated by high failure rates in drug development pipelines. In contrast, human intestinal organoids represent a superior model of the intestinal epithelium and might help to implement the 3Rs (Reduction, Refinement and Replacement) principle in basic science as well as the preclinical and regulatory setup.

OBJECTIVE:Reduced Paneth cell (PC) numbers are observed in inflammatory bowel diseases and impaired PC function contributes to the ileal pathogenesis of Crohn's disease (CD). PCs reside in proximity to Lgr5+ intestinal stem cells (ISC) and mitochondria are critical for ISC-renewal and differentiation. Here, we characterise ISC and PC appearance under inflammatory conditions and describe the role of mitochondrial function for ISC niche-maintenance.DESIGN:Ileal tissue samples from patients with CD, mouse models for mitochondrial dysfunction (Hsp60Δ/ΔISC) and CD-like ileitis (TNFΔARE), and intestinal organoids were used to characterise PCs and ISCs in relation to mitochondrial function.RESULTS:In patients with CD and TNFΔARE mice, inflammation correlated with reduced numbers of Lysozyme-positive granules in PCs and decreased Lgr5 expression in crypt regions. Disease-associated changes in PC and ISC appearance persisted in non-inflamed tissue regions of patients with CD and predicted the risk of disease recurrence after surgical resection. ISC-specific deletion of Hsp60 and inhibition of mitochondrial respiration linked mitochondrial function to the aberrant PC phenotype. Consistent with reduced stemness in vivo, crypts from inflamed TNFΔARE mice fail to grow into organoids ex vivo. Dichloroacetate-mediated inhibition of glycolysis, forcing cells to shift to mitochondrial respiration, improved ISC niche function and rescued the ability of TNFΔARE mice-derived crypts to form organoids.CONCLUSION:We provide evidence that inflammation-associated mitochondrial dysfunction in the intestinal epithelium triggers a metabolic imbalance, causing reduced stemness and acquisition of a dysfunctional PC phenotype. Blocking glycolysis might be a novel drug target to antagonise PC dysfunction in the pathogenesis of CD.