Intestinal Stem Cell Compartment Research Articles

IL-33 Induces Paneth Cell Production of EGF and Soluble ST2, Regulating Epithelial Regeneration after Intestinal Injury

Crypt base intestinal stem cells (ISCs) maintain the epithelial lining of the intestines, but their frequencies are reduced in experimental models of graft vs. host disease (GVHD). Paneth cells provide an epithelial contribution to the stem cell niche, but their frequencies are also reduced in GVHD mouse models and in patients with GVHD. Mechanisms regulating ISCs and their niche after damage remain poorly understood. Interleukin-33 (IL-33) is an immunomodulatory alarmin typically thought to target lymphocytes such as T cells and innate lymphoid cells. In experimental models of allogeneic bone marrow transplantation, IL-33 can promote or attenuate GVHD, depending on the cellular target and the timing of the exposure. A secreted isoform of the IL-33 receptor, termed soluble ST2 (sST2), acts as a negative regulator by blocking IL-33 from binding to the membrane-bound isoform that mediates intracellular signaling. sST2 also has diagnostic and prognostic utility, serving as a biomarker for GVHD severity. While IL-33 has well-described effects on lymphocytes populations, there is limited understanding of the role of IL-33 signaling within the intestinal mucosa after damage. Utilizing experimental radiation injury to model conditioning-related intestinal pathology, we investigated the role of the IL-33/ST2 axis in epithelial damage and regeneration. Wild-type (WT) mice increased IL-33 mRNA and protein expression in the small intestine within days after 10 Gy total body irradiation (TBI). Intestinal sST2 expression also increased quickly in WT mice after TBI, but not in IL-33 -/- mice, suggesting that IL-33 itself was a regulator of sST2. While the ileal mucosa appeared similar at baseline in wild-type and IL-33 -/- mice, the IL-33 -/- mice demonstrated more severe radiation injury 5 days after 10 Gy TBI, with more crypt loss, loss of ISCs and Paneth cells, and an attenuated regenerative response with smaller crypts post-TBI containing fewer Ki67 + cells. 3-D whole-mount confocal microscopy of ileal tissue from IL-33-GFP reporter mice indicated that the crypt epithelium did not express IL-33 at baseline, but Olfm4 + ISCs adjacent to Paneth cells began to express IL-33 following radiation injury (Fig 1). Anti-Thy1-mediated lymphocyte depletion prior to irradiation of WT and IL-33 -/- mice did not abrogate the increased radiation sensitivity of IL-33 -/- mice, indicating a role for IL-33 beyond immunomodulation and suggesting that IL-33 could have direct effects within the epithelium. Intestinal organoid cultures confirmed epithelial expression of IL-33, confirmed increased radiation sensitivity of IL-33 -/- epithelium, and identified impaired epidermal growth factor (EGF) expression in the absence of IL-33. Furthermore, treatment of intestinal organoids with IL-33 induced EGF mRNA and protein expression, both of which were abrogated by the p38 inhibitor SB202190. Treatment of purified Lysozyme-DsRed + Paneth cells indicated that IL-33 directly promoted their production of EGF. In vivo, TBI-induced EGF upregulation was absent in IL-33 -/- mice. To further investigate this newly identified epithelial IL-33/ST2/EGF pathway in the ISC compartment, RiboTag mice, which facilitate analysis of lineage-specific proteome-relevant gene expression with minimal tissue processing and artifact introduction, were crossed to Olfm4-Cre and Lysozyme-Cre transgenics. Lineage-specific ribosome targeting was confirmed by immunostains and qPCR, and this approach also confirmed ISC upregulation of IL-33 following radiation injury and Paneth cell upregulation of EGF and sST2. Finally, to investigate the importance of this radiation-induced EGF upregulation, WT mice were treated with the EGFR inhibitor gefitinib following irradiation, which resulted in more severe radiation-induced ISC loss, similar to IL-33 deficiency. Furthermore, epithelial regeneration and ISC recovery were augmented in IL-33 -/- mice after TBI by treatment with exogenous EGF (Fig 2). These findings reveal an unknown pathway of stem cell niche regulation and crypt regeneration whereby the Paneth cell niche responds dynamically upon injury by upregulating EGF and the stem cells orchestrate regeneration by regulating their niche with IL-33. This regenerative circuit also highlights the breadth of IL-33 activity beyond immunomodulation and the therapeutic potential of EGF administration for treatment of intestinal injury.

A24 INVESTIGATING THE IMPACT OF PARKINSON’S DISEASE-ASSOCIATED GENES ON INTESTINAL HOMEOSTASIS

Abstract Background Intestinal epithelial cells (IECs) provide an essential physical barrier between harsh luminal contents and underlying host tissue. The maintenance of intestinal homeostasis in this rapidly renewing tissue must be intricately regulated through the proliferation and differentiation of intestinal stem cells (ISCs). Dysregulation of this system results in the loss of barrier function, causing pathologies in both intestinal and extra-intestinal diseases. While Parkinson’s Disease (PD) is primarily a neurodegenerative disorder, there is increasing evidence linking PD progression and gastrointestinal dysfunction. For instance, constipation and increased bowel permeability are frequently observed years prior to development of motor dysfunction in PD, people with inflammatory bowel disease are more likely to develop PD, and a positive correlation exists between gastrointestinal infections and PD incidence. Our group recently developed a model to investigate the role of the gut in PD, demonstrating that mice with genetic ablation of the PD-associated gene Pink1 exhibited motor phenotypes only when previously infected with Gram-negative Citrobacter rodentium intestinal bacteria. As Pink1 and other PD-associated genes are expressed in IECs, we hypothesize that PD-associated gene mutations directly affect the epithelium and impact early PD pathophysiology. Purpose Investigate the impact of Pink1 and other PD-associated genes in IECs under steady state and infection. Method Single-cell RNA sequencing was performed on IECs isolated from Pink1 WT and KO mice, at steady state and following in vivo C. rodentium infection. Mice were sacrificed at an early timepoint of infection (day 6) to elucidate transcriptional differences between epithelial lineages of each genotype. Additionally, ex vivo colonoids were derived from primary mouse tissue and treated with lipopolysaccharide (LPS) to determine how PINK1 loss-of-function affects the inflammatory response of the epithelium. Result(s) Our data revealed that loss-of-function of PINK1 profoundly affected the ISC compartment and several epithelial lineages. Specifically, ISCs from infected Pink1 KO mice demonstrated differentially regulated proliferative and cell cycle genes, while transit amplifying cells showed dysregulated expression of tight junction genes, and enterocytes displayed differentially expressed oxidative damage and apoptotic genes. Preliminary data from colonoids showed that Pink1 KO mice, when stimulated with LPS, had increased pro-inflammatory cytokine gene expression. Conclusion(s) In Pink1 KO intestinal epithelial cells, there is indeed an altered cellular response upon infection in vivo and LPS treatment ex vivo. However, more information is needed to decern the mechanistic role of IECs in PD. By investigating the role of PD genes in the gastrointestinal tract, these studies carry important implications for understanding the initiation and progression of PD. Disclosure of Interest None Declared

IFN-Gamma Is Crucial for the Counterbalance of T Cell-Mediated Injury to the Intestinal Stem Cell Compartment By Regulatory T Cells in Mice and Humans

IFN-Gamma Is Crucial for the Counterbalance of T Cell-Mediated Injury to the Intestinal Stem Cell Compartment By Regulatory T Cells in Mice and Humans

Implications of Paneth cell dysfunction on gastrointestinal health and disease.

Paneth cells are specialized, secretory epithelial cells located in the small intestine. Although their existence was first described in 1872, their precise role in the gut remained unclear for over a century. Over the past few decades, elegant studies have shown Paneth cells play a key role enhancing gut barrier function, as niche cells for the intestinal stem cell compartment and via secreting antimicrobial peptides to establish an antimicrobial barrier at the epithelial surface. This review describes what is known about Paneth cell biology from human and animal studies with a focus on their putative role in clinical gastrointestinal disease. Recent work has demonstrated important associations of dysfunctional Paneth cells with several gastrointestinal disorders. These include Crohn's disease, enteric infections, graft-versus-host disease, necrotizing enterocolitis, and environmental enteric dysfunction. Ongoing studies are examining precisely how Paneth cell biology is altered in these various disease states. By understanding the mechanisms of Paneth cell regulation - and how these processes go awry in specific gastrointestinal diseases - we set the stage for using Paneth cells as biomarkers for disease progression and developing novel therapeutics that augment Paneth cell function to treat a spectrum of gastrointestinal disorders.

Modification of Diet to Reduce the Stemness and Tumorigenicity of Murine and Human Intestinal Cells.

ScopeBlack raspberries (BRBs) have colorectal cancer (CRC) chemo‐preventative effects. As CRC originates from an intestinal stem cell (ISC) this study has investigated the impact of BRBs on normal and mutant ISCs.Methods and resultsMice with an inducible Apcfl mutation in either the ISC (Lgr5CreERT2 ) or intestinal crypt (AhCre/VillinCreERT2 ) are fed a control or 10% BRB‐supplemented diet. This study uses immunohistochemistry, gene expression analysis, and organoid culture to evaluate the effect of BRBs on intestinal homeostasis. RNAscope is performed for ISC markers on CRC adjacent normal colonic tissue pre and post BRB intervention from patients. 10% BRB diet has no overt effect on murine intestinal homeostasis, despite a reduced stem cell number. Following Apc ISC deletion, BRB diet extends lifespan and reduces tumor area. In the AhCre model, BRB diet attenuates the “crypt‐progenitor” phenotype and reduces ISC marker gene expression. In ex vivo culture BRBs reduce the self‐renewal capacity of murine and human Apc deficient organoids. Finally, the study observes a reduction in ISC marker gene expression in adjacent normal crypts following introduction of BRBs to the human bowel.ConclusionBRBs play a role in CRC chemoprevention by protectively regulating the ISC compartment and further supports the use of BRBs in CRC prevention.

Helminth-induced reprogramming of the stem cell compartment inhibits type 2 immunity.

Enteric helminths form intimate physical connections with the intestinal epithelium, yet their ability to directly alter epithelial stem cell fate has not been resolved. Here we demonstrate that infection of mice with the parasite Heligmosomoides polygyrus bakeri (Hpb) reprograms the intestinal epithelium into a fetal-like state marked by the emergence of Clusterin-expressing revival stem cells (revSCs). Organoid-based studies using parasite-derived excretory-secretory products reveal that Hpb-mediated revSC generation occurs independently of host-derived immune signals and inhibits type 2 cytokine-driven differentiation of secretory epithelial lineages that promote their expulsion. Reciprocally, type 2 cytokine signals limit revSC differentiation and, consequently, Hpb fitness, indicating that helminths compete with their host for control of the intestinal stem cell compartment to promote continuation of their life cycle.

Cell and chromatin transitions in intestinal stem cell regeneration.

The progeny of intestinal stem cells (ISCs) dedifferentiate in response to ISC attrition. The precise cell sources, transitional states, and chromatin remodeling behind this activity remain unclear. In the skin, stem cell recovery after injury preserves an epigenetic memory of the damage response; whether similar memories arise and persist in regenerated ISCs is not known. We addressed these questions by examining gene activity and open chromatin at the resolution of single Neurog3-labeled mouse intestinal crypt cells, hence deconstructing forward and reverse differentiation of the intestinal secretory (Sec) lineage. We show that goblet, Paneth, and enteroendocrine cells arise by multilineage priming in common precursors, followed by selective access at thousands of cell-restricted cis-elements. Selective ablation of the ISC compartment elicits speedy reversal of chromatin and transcriptional features in large fractions of precursor and mature crypt Sec cells without obligate cell cycle re-entry. ISC programs decay and reappear along a cellular continuum lacking discernible discrete interim states. In the absence of gross tissue damage, Sec cells simply reverse their forward trajectories, without invoking developmental or other extrinsic programs, and starting chromatin identities are effectively erased. These findings identify strikingly plastic molecular frameworks in assembly and regeneration of a self-renewing tissue.

38 - IL-22 Producing Innate Lymphocytes Traffic to the Intestinal Stem Cell Compartment after Conditioning-Related Radiation Injury

38 - IL-22 Producing Innate Lymphocytes Traffic to the Intestinal Stem Cell Compartment after Conditioning-Related Radiation Injury

Limitations to Understanding Intestinal Stem Cell Activity via Cre-Lox-Based Lineage Tracing.

The intestinal epithelium is a highly proliferative tissue with robust regenerative potential, governed by the intestinal stem cell (ISC) compartment. Our understanding of the cellular hierarchy within this compartment is drawn from Cre-lox–based lineage tracing studies, initially via the identification of crypt base columnar stem cells (CBCs) using an Lgr5eGFP-IRES-CreER allele and the R26LSL-LacZ reporter,1 showing that CBCs reconstitute all epithelial cell types during homeostasis.2 Despite their proliferative capacity, CBCs are exquisitely sensitive to injury, requiring facultative stem cells for postinjury regeneration.

IFN-Gamma Producing Regulatory T Cells Counterbalance T Cell-Mediated Injury to the Intestinal Stem Cell Compartment in Mice and Humans

Background:Graft-versus-host disease (GVHD) is a dreaded complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Such inflammatory diseases are fostered by damage to the intestinal barrier after transplantation. Consequently, the integrity and regeneration of the intestinal barrier is a key factor in the prevention of GVHD. On one side the main driver for regeneration of damaged gut epithelium are intestinal stem cells (ISC), but on the other side these cells are themselves primary targets of donor-derived T cells. One known mechanism of T cell mediated damage to the stem cell compartment is through IFN-γ dependent ISC toxicity. Yet, little is known about how T cells are contributing to the regeneration of damaged tissue after allo-HSCT and GVHD.Methods:To address this, we used preclinical models for allo-HSCT and GVHD including transplantation of recipient mice with escalating doses of Wildtype or IFN-γ-deficient allogeneic T cells and in the presence or absence of the JAK-1/2 inhibitor ruxolitinib. Intestinal regeneration was assessed by RNA-seq, flow cytometry and a newly established ex vivo organoid recovery assay. GVHD outcome was assessed by clinical scoring, histology and survival. Additionally, we established an allogeneic co-culture system of murine or human intestinal organoids with CD4+ conventional T cells or T regs -/+ Ruxolitinib. Effects on organoid growth and cell death were assessed by size measurements and manual counting after passaging.Results:We here demonstrate that recipient mice with increasingly dense intestinal infiltration by allogeneic T cells not only developed more severe GVHD (Fig. 1A), but also showed augmented recovery potential early after allo-HSCT (Fig. 1B). This was associated with intestinal gene signatures related to epithelial regeneration and protection from GVHD. Utilizing ex vivo cultures of intestinal organoids generated from murine allo-HSCT recipients, we found that development of GVHD but also regenerative capacity of ISCs were dependent on interferon (IFN)-γ-producing T cells in the intestine (Fig. 2A-B). Mice with fulminant GVHD and enhanced organoid recovery showed accumulation of intestinal regulatory T cells (Tregs) (Fig. 2C). Ex vivo, T regs nurtured growth of intestinal organoids in an IFN-γ dependent manner (Fig. 2D-E). This effect was diminished in intestinal organoids lacking IFNγR signaling, but was independent of T reg intrinsic IFNγR signaling (Fig. 2E-F). Intriguingly, treatment of murine allo-HSCT recipients with the JAK-1/2 inhibitor ruxolitinib enhanced epithelial organoid regeneration and numbers of intestinal Tregs (Fig. 3A-B). Similarily, growth of human intestinal organoids co-cultured with allogeneic T cells could be augmented by ruxolitinib treatment (Fig. 3C). We thus propose that the level and differentiation of infiltrating intestinal T cells determines both ISC damage and epithelial regeneration during immune-mediated tissue injury, leading to a sensitive equilibrium that can be modulated by therapeutic intervention. We also provide evidence that ruxolitinib improves ISC regeneration via IFNγ-producing Treg cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Apc-mutant cells act as supercompetitors in intestinal tumour initiation.

A delicate equilibrium of WNT agonists and antagonists in the intestinal stem cell (ISC) niche is critical to maintaining the ISC compartment, as it accommodates the rapid renewal of the gut lining. Disruption of this balance by mutations in the tumour suppressor gene APC, which are found in approximately 80% of all human colon cancers, leads to unrestrained activation of the WNT pathway1,2. It has previously been established that Apc-mutant cells have a competitive advantage over wild-type ISCs3. Consequently, Apc-mutant ISCs frequently outcompete all wild-type stem cells within a crypt, thereby reaching clonal fixation in the tissue and initiating cancer formation. However, whether the increased relative fitness of Apc-mutant ISCs involves only cell-intrinsic features or whether Apc mutants are actively involved in the elimination of their wild-type neighbours remains unresolved. Here we show that Apc-mutant ISCs function as bona fide supercompetitors by secreting WNT antagonists, thereby inducing differentiation of neighbouring wild-type ISCs. Lithium chloride prevented the expansion of Apc-mutant clones and the formation of adenomas by rendering wild-type ISCs insensitive to WNT antagonists through downstream activation of WNT by inhibition of GSK3β. Our work suggests that boosting the fitness of healthy cells to limit the expansion of pre-malignant clones may be a powerful strategy to limit the formation of cancers in high-risk individuals.

IFN-λ therapy prevents severe gastrointestinal graft-versus-host disease

AbstractImmunopathology and intestinal stem cell (ISC) loss in the gastrointestinal (GI) tract is the prima facie manifestation of graft-versus-host disease (GVHD) and is responsible for significant mortality after allogeneic bone marrow transplantation (BMT). Approaches to prevent GVHD to date focus on immune suppression. Here, we identify interferon-λ (IFN-λ; interleukin-28 [IL-28]/IL-29) as a key protector of GI GVHD immunopathology, notably within the ISC compartment. Ifnlr1−/− mice displayed exaggerated GI GVHD and mortality independent of Paneth cells and alterations to the microbiome. Ifnlr1−/− intestinal organoid growth was significantly impaired, and targeted Ifnlr1 deficiency exhibited effects intrinsic to recipient Lgr5+ ISCs and natural killer cells. PEGylated recombinant IL-29 (PEG-rIL-29) treatment of naive mice enhanced Lgr5+ ISC numbers and organoid growth independent of both IL-22 and type I IFN and modulated proliferative and apoptosis gene sets in Lgr5+ ISCs. PEG-rIL-29 treatment improved survival, reduced GVHD severity, and enhanced epithelial proliferation and ISC-derived organoid growth after BMT. The preservation of ISC numbers in response to PEG-rIL-29 after BMT occurred both in the presence and absence of IFN-λ–signaling in recipient natural killer cells. IFN-λ is therefore an attractive and rapidly testable approach to prevent ISC loss and immunopathology during GVHD.

LINGO3 regulates mucosal tissue regeneration and promotes TFF2 dependent recovery from colitis

Aim: Recovery of damaged mucosal surfaces following inflammatory insult requires diverse regenerative mechanisms that remain poorly defined. Previously, we demonstrated that the reparative actions of Trefoil Factor 3 (TFF3) depend upon the enigmatic receptor, leucine rich repeat and immunoglobulin-like domain containing nogo receptor 2 (LINGO2). This study examined the related orphan receptor LINGO3 in the context of intestinal tissue damage to determine whether LINGO family members are generally important for mucosal wound healing and maintenance of the intestinal stem cell (ISC) compartment needed for turnover of mucosal epithelium. Methods and Results: We find that LINGO3 is broadly expressed on human enterocytes and sparsely on discrete cells within the crypt niche, that contains ISCs. Loss of function studies indicate that LINGO3 is involved in recovery of normal intestinal architecture following dextran sodium sulfate (DSS)-induced colitis, and that LINGO3 is needed for therapeutic action of the long acting TFF2 fusion protein (TFF2-Fc), including a number of signaling pathways critical for cell proliferation and wound repair. LINGO3-TFF2 protein-protein interactions were relatively weak however and LINGO3 was only partially responsible for TFF2 induced MAPK signaling suggesting additional un-identified components of a receptor complex. However, deficiency in either TFF2 or LINGO3 abrogated budding/growth of intestinal organoids and reduced expression of the intestinal ISC gene leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), indicating homologous roles for these proteins in tissue regeneration, possibly via regulation of ISCs in the crypt niche. Conclusion: We propose that LINGO3 serves a previously unappreciated role in promoting mucosal wound healing.

Volume-dependent dose–response of the intestinal stem cell niche and lymphoid tissue

Background and purposePlasticity of the intestinal stem cell compartment in response to radiation injury is regulated by a stem cell niche. We present here the first experimental observations of a dose-volume effect of the intestinal stem cell niche and of the solitary intestinal lymphoid tissues (SILT). Materials and methodsRegeneration of intestinal crypts in mice was studied following irradiation of millimetre-size jejunal sections with single doses of 6 to 24 Gy and compared to total body irradiation (TBI). The statistical distribution of cells per crypt was scored and regressed to a biomathematical model. The number of SILTs was scored for different doses and field sizes and crypt regeneration was correlated with SILT proximity. ResultsWe observed a differential dose–response of the intestinal stem cell niche at the centres of the irradiated sections, but only for field sizes below 10 mm. Irradiation of 5 mm jejunum results in an increase in crypt survival by up to an order of magnitude, compared to TBI. Distributions of cell-per-crypt numbers and comparison to biomathematical modelling suggest that these observations stem from a field size-dependent regeneration rate. The density of SILTs also exhibits a volume-dependent dose–response and increased crypt survival correlates with a proximity to SILTs. ConclusionOur findings present the first observation of a field-size dependent dose–response of the intestinal stem cell niche. Its regeneration process does apparently not rely on distant radiation-sensitive resources of the organism, such as the bone marrow. Yet, our observations suggest that the niche interacts with intact tissue in millimetres distance, leading to faster crypt regeneration. The field-size dependent dose–response of SILTs posits a role of the immune system on the dose-volume effect.

IκBα deficiency imposes a fetal phenotype to intestinal stem cells.

The intestinal epithelium is a paradigm of adult tissue in constant regeneration that is supported by intestinal stem cells (ISCs). The mechanisms regulating ISC homeostasis after injury are poorly understood. We previously demonstrated that IκBα, the main regulator of NF-κB, exerts alternative nuclear functions as cytokine sensor in a subset of PRC2-regulated genes. Here, we show that nuclear IκBα is present in the ISC compartment. Mice deficient for IκBα show altered intestinal cell differentiation with persistence of a fetal-like ISC phenotype, associated with aberrant PRC2 activity at specific loci. Moreover, IκBα-deficient intestinal cells produce morphologically aberrant organoids carrying a PRC2-dependent fetal-like transcriptional signature. DSS treatment, which induces acute damage in the colonic epithelium of mice, results in a temporary loss of nuclear P-IκBα and its subsequent accumulation in early CD44-positive regenerating areas. Importantly, IκBα-deficient mice show higher resistance to damage, likely due to the persistent fetal-like ISC phenotype. These results highlight intestinal IκBα as a chromatin sensor of inflammation in the ISC compartment.

The ErbB3 receptor tyrosine kinase regulates expression of Notch target genes and intestinal stem cell markers.

BackgroundStem cell‐driven regeneration of the intestinal epithelium is important for recovery of damage from insults including inflammation, chemotherapy, and radiation. This process is controlled through tight regulation of intestinal stem cells (ISCs). Our lab has previously reported that intestine‐specific knockout of the receptor tyrosine kinase ErbB3 leads to precocious development of Paneth cells and increased Paneth cell numbers in mice the mouse intestinal epithelium. Since Paneth cells can play a supportive role for ISCs, we tested the hypothesis that altered ErbB3 signaling results in changes in the ISC compartment.MethodsWe crossed ErbB3flox/flox mice with mice expressing villin promoter‐driven Cre to generate an intestinal epithelial‐specific ErbB3 knockout mouse model (ErbB3‐IEKO). Ileal samples were collected from these mice, fixed, and embedded in paraffin for immunostaining. Tissue samples from the ileum were also processed for RNA and protein for qPCR and immunoblots. Samples were probed for Notch target genes and Notch ligands due to the importance of Notch signaling in maintaining the intestinal stem cell compartment. Enteroids were generated from ileum of C57Bl/6 mice and treated with the ErbB3 ligand Nrg‐1β. To activate and inactivate Notch experimentally, enteroids and HT29 cells were treated with valproic acid (VPA) and the γ‐secretase inhibitor DAPT respectively.ResultsqPCR of ErbB3‐IEKO ileum showed increased expression of Lgr5 (rapid‐cycling ISCs) and Bmi1 (facultative ISCs or secretory progenitors capable of regenerating ISCs). Ileal sections also showed increased numbers of Lgr5+ cells by in situ hybridization, and western blots showed increased protein levels of Bmi1 in ErbB3‐IEKO ileum. qPCR of ErbB3‐IEKO ileum showed increased expression of Notch target genes Hey1, Hey2, and Heyl, as well as expression of Notch ligands Dll1, Jag1, and Jag2. Enteroids treated with Nrg‐1β had decreased expression of Hey1, Lgr5, and Bmi1. Enteroids and HT29 cells treated with VPA to activate Notch had decreased expression of ErbB3 RNA; this was abrogated by DAPT treatment.ConclusionErbB3 negatively regulates expression of Lgr5 and Bmi1, markers for fast‐cycling and quiescent ISCs, respectively, indicating a possible role for ErbB3 in the ISC niche. Furthermore, ErbB3 and Notch appear to negatively regulate one another, suggesting a possible mechanism for ErbB3’s effects on ISCs and intestinal cell differentiation.Support or Funding InformationThis work was supported by NIH award R01DK095004.

Ascl2-Dependent Cell Dedifferentiation Drives Regeneration of Ablated Intestinal Stem Cells.

Ablation of LGR5+ intestinal stem cells (ISCs) is associated with rapid restoration of the ISC compartment. Different intestinal crypt populations dedifferentiate to provide new ISCs, but the transcriptional and signaling trajectories that guide this process are unclear, and a large body of work suggests that quiescent "reserve" ISCs contribute to regeneration. By timing the interval between LGR5+ lineage tracing and lethal injury, we show that ISC regeneration is explained nearly completely by dedifferentiation, with contributions from absorptive and secretory progenitors. The ISC-restricted transcription factor ASCL2 confers measurable competitive advantage to resting ISCs and is essential to restore the ISC compartment. Regenerating cells re-express Ascl2 days before Lgr5, and single-cell RNA sequencing (scRNA-seq) analyses reveal transcriptional paths underlying dedifferentiation. ASCL2 target genes include the interleukin-11 (IL-11) receptor Il11ra1, and recombinant IL-11 enhances crypt cell regenerative potential. These findings reveal cell dedifferentiation as the principal means for ISC restoration and highlight an ASCL2-regulated signal that enables this adaptive response.

Protecting intestinal stem cells

Mucosal Immunology The intestinal epithelium is replaced every week. Thus, maintenance of this tissue requires rapid self-renewal driven by intestinal stem cells. This homeostasis is disrupted in a number of settings, including allogeneic bone marrow transplantation. After bone marrow transplantation, allogeneic T cells often attack and kill intestinal cells in an interferon-γ (IFN-γ)–dependent manner. Takashima et al. used in vivo transplant models and in vitro organoid systems to define the targets of IFN-γ in the mouse intestine. They found that IFN-γ directly targets intestinal stem cells. Furthermore, inhibitors of JAK-STAT signaling could be used to protect the intestinal stem cell compartment from T cell–mediated damage. Sci. Immunol. 4 , eaay8556 (2019).

Krüppel-like Factor 5 Regulates Stemness, Lineage Specification, and Regeneration of Intestinal Epithelial Stem Cells

Background & AimsSelf-renewal and multipotent differentiation are cardinal properties of intestinal stem cells (ISCs), mediated in part by WNT and NOTCH signaling. Although these pathways are well characterized, the molecular mechanisms that control the ‘stemness’ of ISCs are still not well defined. Here, we investigated the role of Krüppel-like factor 5 (KLF5) in regulating ISC functions.MethodsWe performed studies in adult Lgr5EGFP-IRES-creERT2;Rosa26LSLtdTomato (Lgr5Ctrl) and Lgr5EGFP-IRES-creERT2;Klf5fl/fl;Rosa26LSLtdTomato (Lgr5ΔKlf5) mice. Mice were injected with tamoxifen to activate Cre recombinase, which deletes Klf5 from the intestinal epithelium in Lgr5ΔKlf5 but not Lgr5Crtl mice. In experiments involving irradiation, mice were subjected to 12 Gy total body irradiation (TBI). Tissues were collected for immunofluorescence (IF) analysis and next generation sequencing. Oganoids were derived from fluoresecence activated cell sorted- (FACS-) single cells from tamoxifen-treated Lgr5ΔKlf5 or Lgr5Crtl mice and examined by immunofluorescence stain.ResultsLgr5+ ISCs lacking KLF5 proliferate faster than control ISCs but fail to self-renew, resulting in a depleted ISC compartment. Transcriptome analysis revealed that Klf5-null Lgr5+ cells lose ISC identity and prematurely differentiate. Following irradiation injury, which depletes Lgr5+ ISCs, reserve Klf5-null progenitor cells fail to dedifferentiate and regenerate the epithelium. Absence of KLF5 inactivates numerous selected enhancer elements and direct transcriptional targets including canonical WNT- and NOTCH-responsive genes. Analysis of human intestinal tissues showed increased levels of KLF5 in the regenerating epithelium as compared to those of healthy controls.ConclusionWe conclude that ISC self-renewal, lineage specification, and precursor dedifferentiation require KLF5, by its ability to regulate epigenetic and transcriptional activities of ISC-specific gene sets. These findings have the potential for modulating ISC functions by targeting KLF5 in the intestinal epithelium.

T-Cell Derived Interferon Gamma Directly Targets Intestinal Epithelium to Induce Stem Cell Apoptosis in GI Gvhd

Crypt base intestinal stem cells (ISCs) marked by Lgr5 and Olfm4 maintain the intestinal epithelium, and Paneth cells (PCs) provide an epithelial niche for ISCs in the small bowel. ISCs are reduced during gastrointestinal (GI) GVHD, but the precise mechanisms including the role of niche injury are unknown. Additionally, the specific effects of Interferon-γ (IFNγ) on intestinal epithelium in GVHD remain ill-defined. We evaluated kinetics of ISC loss by histology after allogeneic (allo) BMT using Lgr5-LacZ reporter mice. In both MHC- and miHA-mismatched models (LP>B6 and B6>BDF1), ISC numbers quickly recovered from pretransplant TBI conditioning in recipients of T-cell-depleted (TCD) BMT by day +10, but ISCs failed to recover in recipients of allo T cells. T-cell-induced ISC reduction was functionally validated by genetic marking of stem cell progeny and by culturing intestinal organoids from crypts isolated post-BMT. Similar to the kinetics of ISC loss, ISC-dependent organoid-forming capacity was impaired in recipients of allo T cells compared with TCD BMT recipients on day +10 (p<0.05). Likewise, BMT into Olfm4 reporter mice showed significantly reduced lineage tracing from ISCs in recipients of allo T cells (Fig. 1). To better understand the potential for T cells to interact with the ISC compartment, we performed whole-mount 3-D microscopy to distinguish T cell localization within the intraepithelial and lamina propria compartments post-transplant. We found that donor T cells invading the small bowel after BMT were mostly in the crypt region, and infiltration within the lamina propria adjacent to the ISC compartment was much greater than invasion within the epithelium itself (Fig. 2). We next established an ex vivo co-culture system, to model interactions between intestinal epithelium and donor T cells and investigate mechanisms of T-cell-mediated ISC injury. Screening of effector pathways revealed no impact of perforin or FasL, but identified IFNγ as a principal mediator of ISC injury. Culture with allo T cells significantly reduced viable human and mouse intestinal organoid numbers, and this was inhibited by IFNγ neutralization. IFNγ receptor knockout (IFNγR-/-) organoids were resistant to T cells. IFNγ increased expression of Bak1 and decreased expression of Bcl2 in organoids, and induced ISC apoptosis defined by Annexin+Dapi-Lgr5+ phenotype. ISC killing was mediated by intraepithelial JAK/STAT signaling, as JAK1- and STAT1-deficient organoids were resistant, and it was inhibited by Ruxolitinib. Investigating the role of IFNγ in vivo, FACS analysis confirmed donor T cells to be the primary producers of IFNγ in crypt lamina propria, and BMT with IFNγ-/- donor T cells reduced crypt apoptosis, and preserved ISC frequencies. Moreover, BMT with recipients lacking IFNγR specifically in the intestinal epithelium significantly protected ISCs, reduced crypt apoptosis, and ameliorated GI GVHD pathology (Fig.3). Furthermore, ISCs were also protected by epithelial deletion of STAT1 and by Ruxolitinib treatment. As specific genetic manipulation of ISCs in vivo is not possible because genetic targeting of ISCs results in the same changes in their progeny, we utilized ex vivo models to determine if IFNγ kills ISCs by directly inducing their apoptosis or by damaging the PC niche. Manipulation of the niche by culturing wild-type ISCs with IFNγR-/- PCs was not protective to allo T cells. Using a niche-independent high-purity Lgr5+ ISC culture system based on combined GSK3β and HDAC inhibition, IFNγ directly induced cleaved-caspase-3+ ISC apoptosis and substantial ISC colony death, which were inhibited by Bak/Bax deficiency and by the pan-caspase inhibitor QVD. These results confirmed that IFNγ can directly induce ISC apoptosis independent of other cytotoxic effector molecules and independent of injury to the PC niche. In summary, T cells migrating to the GI tract primarily infiltrated the lamina propria adjacent to the ISC compartment, and T-cell-derived IFNγ directly targeted intestinal epithelium via JAK1/STAT1 signaling to induce ISC apoptosis in a PC-independent manner. ISC reduction and GI GVHD pathology were prevented by inhibiting the IFNγR/JAK1/STAT1 axis within the intestinal epithelium, indicating that in addition to their effects on T cells, JAK inhibitors may treat GVHD by inhibiting pathologic cytokine signaling within target organs and shielding them from allo T cells. Disclosures Hanash: Nexus Global Group LLC: Consultancy.