Human Colonoids Research Articles

Catechol-O-Methyltransferase Loss Drives Cell-Specific Nociceptive Signaling via the Enteric Catechol-O-Methyltransferase/microRNA-155/Tumor Necrosis Factor α Axis

The etiology of abdominal pain in postinfectious, diarrhea-predominant irritable bowel syndrome (PI-IBS-D) is unknown, and few treatment options exist. Catechol-O-methyltransferase (COMT), an enzyme that inactivates and degrades biologically active catecholamines, plays an important role in numerous physiologic processes, including modulation of pain perception. Our objective was to determine the mechanism(s) of how decreased colonic COMT in PI-IBS-D patients contributes to the chronic abdominal pain phenotype after enteric infections. Colon neurons, epithelial cells, and macrophages were procured with laser capture microdissection from PI-IBS-D patients to evaluate cell-specific colonic COMT, microRNA-155 (miR-155), and tumor necrosis factor (TNF) α expression levels compared to recovered patients (infection cleared: did not develop PI-IBS-D) and control individuals. COMT-/-, colon-specific COMT-/-, and miR-155-/- mice and human colonoids were used to model phenotypic expression of COMT in PI-IBS-D patients and to investigate signaling pathways linking abdominal pain. Citrobacter rodentium and trinitrobenzene sulfonic acid animal models were used to model postinflammatory changes seen in PI-IBS-D patients. Colonic COMT levels were significantly decreased and correlated with increased visual analog scale abdominal pain ratings in PI-IBS-D patients compared to recovered patients and control individuals. Colonic miR-155 and TNF-α were increased in PI-IBS-D patients with diminished colonic COMT. COMT-/- mice had significantly increased expression of miR-155 and TNF-α in both colon tissues and dorsal root ganglia. Introduction of cV1q antibody (anti-TNF-α) into mice reversed visceral hypersensitivity after C rodentium and trinitrobenzene sulfonic acid. Decreased colonic COMT in PI-IBS-D patients drives abdominal pain phenotypes via the COMT/miR-155/TNF-α axis. These important findings will allow new treatment paradigms and more targeted and personalized medicine approaches for gastrointestinal disorders after enteric infections.

Autophagy Reduces the Degradation and Promotes Membrane Localization of Occludin to Enhance the Intestinal Epithelial Tight Junction Barrier against Paracellular Macromolecule Flux.

Functional loss of the gut epithelium's paracellular tight junction [TJ] barrier and defective autophagy are factors potentiating inflammatory bowel disease [IBD]. Previously, we showed the role of autophagy in enhancing the intestinal TJ barrier via pore-forming claudin-2 degradation. How autophagy regulates the TJ barrier-forming proteins remains unknown. Here, we investigated the role of autophagy in the regulation of occludin, a principal TJ component involved in TJ barrier enhancement. Autophagy induction using pharmacological activators and nutrient starvation increased total occludin levels in intestinal epithelial cells, mouse colonocytes and human colonoids. Autophagy induction enriched membrane occludin levels and reduced paracellular permeability of macromolecules. Autophagy-mediated TJ barrier enhancement was contingent on the presence of occludin as OCLN-/- nullified its TJ barrier-enhancing effect against macromolecular flux. Autophagy inhibited the constitutive degradation of occludin by preventing its caveolar endocytosis from the membrane and protected against inflammation-induced TJ barrier loss. Autophagy enhanced the phosphorylation of ERK-1/2 and inhibition of these kinases in Caco-2 cells and human colonic mucosa prevented the macromolecular barrier-enhancing effects of autophagy. In vivo, autophagy induction by rapamycin enhanced occludin levels in wild-type mouse intestines and protected against lipopolysaccharide- and tumour necrosis factor-α-induced TJ barrier loss. Disruption of autophagy with acute Atg7 knockout in adult mice decreased intestinal occludin levels, increasing baseline colonic TJ permeability and exacerbating the effect of experimental colitis. Our data suggest a novel role of autophagy in promoting the intestinal TJ barrier by increasing occludin levels in an ERK1/2 mitogen-activated protein kinase-dependent mechanism.

Gut Microbiota Alter Visceral Pain Sensation and Inflammation via Modulation of Synthesis of Resolvin D1 in Colonic Tuft Cells.

Background and AimsVisceral hypersensitivity and low grade mucosal inflammation are frequently observed in a subpopulation of irritable bowel syndrome (IBS) patients. The responsible mechanism is unclear. Resolvins are a novel class of anti-inflammatory lipid mediators that regulate resolution of inflammation and pain. We hypothesize that resolvin D1 (RvD1) synthesis is reduced in IBS-D colonic mucosa and contribute to the development of visceral hypersensitivity. MethodsWe used ELISA and qPCR to quantify the levels of RvD1 synthesis and the gene expression of LOX5 and LOX12 in colonic biopsy samples from healthy individuals (HC) or IBS-D patients. To evaluate the role of gut bacteria in regulating RvD1 synthesis, we colonized germ-free mice with different strains of bacteria, fecal microbiota from HC individuals or IBS-D patients. To evaluate the role of tuft cells in RvD1 synthesis, we examined the effect of fecal supernatant of IBS-D or HC subjects on human colonoids as well as colonoids from mice in which Chat-cre recombinase vector was used to knock-in diphtheria toxin sensitive receptor (DTRf/f) or knock-out the expression of Tlr4f/f or MyD88f/f. ResultsWe report that colonic biopsy samples from IBS-D patients generated significantly lower level of RvD1 and LOX5 mRNA. The conventionalization of germ-free mice with microbiota from IBS-D patients or gram-negative bacteria inhibited RvD1 biosynthesis and caused visceral hypersensitivity and mucosal inflammation. Colonic organoid studies demonstrate that Lps downregulated Lox5 mRNA expression and synthesis of RvD1 via Tlr4-MyD88 receptor signaling pathway in colonic tuft cells. ConclusionsOur findings indicate that RvD1 is generated in colonic tuft cells to regulate gut sensitivity to mechanical stimulation. Colonic commensal bacterial composition regulates the synthesis of RvD1 in colonic mucosa which is reduced in IBS-D patients. This appears to be mediated by elevated fecal lipopolysaccharide secondary to gram-negative gut dysbiosis.

Abstract 1474: Genome edited colorectal cancer organoid models reveal distinct microRNA activity patterns across different mutation profiles

Abstract Colorectal cancer (CRC) is the third most common cancer and second leading cause of cancer-related deaths worldwide. DNA mutations drive colon tumor formation by disrupting gene regulatory mechanisms that facilitate cell survival and growth. Distinct combinations of mutations can result in unique changes to these gene regulatory mechanisms leading to variability in responses to available therapeutics. MicroRNAs (miRNAs) are important regulators of gene expression in CRC and it is known that their expression can be altered by oncogenic mutations. However, it is unknown how distinct combinations of CRC-risk mutations differentially affect the regulation and expression of miRNAs. We hypothesize that some miRNAs will exhibit strong genotype-dependent changes in CRC whereas others will be altered irrespective of genotype. Utilizing sequencing data from The Cancer Genome Atlas (TCGA) and cutting-edge intestinal organoid (enteroid) models with different combinations of oncogenic mutations that commonly occur in CRC, we identified several distinct patterns of genotype-dependent miRNA expression. We also found a suite of miRNAs that are up-regulated in a mutation-independent manner. Among these, we demonstrated that miR-24-3p is a candidate master regulator of genes that are down-regulated in all mutational-contexts. RT-qPCR for miR-24-3p in genetically modified human colonoids with different combinations of oncogenic mutations further supports that miR-24-3p is elevated across various mutational contexts. Functional studies in the HCT116 cell line revealed that miR-24-3p controls the number of metabolically active, viable cells by regulating apoptosis but not proliferation. Moreover, follow-up ex vivo studies in mouse enteroids confirmed that miR-24 controls intestinal cell survival. To identify candidate gene targets that mediate the function of miR-24-3p, I performed an RNA-sequencing (RNA-seq) analysis and identified HMOX1 and PRSS8 as top candidate miR-24-3p targets. These findings suggest that miR-24-3p is a genotype-independent regulator of tumor cell apoptosis in CRC and merits further investigation. Overall, our findings provide a novel perspective on approaches for precision medicine in CRC. Citation Format: Jonathan W. Villanueva, Lawrence Kwong, Teng Han, Salvador Alonso Martinez, Fong Cheng Pan, Michael T. Shanahan, Matt Kanke, Shuibing Chen, Lukas E. Dow, Charles G. Danko, Praveen Sethupathy. Genome edited colorectal cancer organoid models reveal distinct microRNA activity patterns across different mutation profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1474.

P-305 Oxidized-LDL promotes colorectal cancer progression and growth of human colonoides

Expression levels of the oxidized-LDL receptor LOX-1 have been related to cancer progression in prostatic, gastric, pancreatic, and colorectal cancer (CRC); however, the relationship among pro-inflammatory markers, ox-LDL/LOX-1 and CRC are not completely elucidated. We tested the hypothesis that stimulation of CRC cell lines with TNF-α and ox-LDL promotes tumor progression. We also explore the effect of ox-LDL on human colonoids growth. The colorectal cancer (CRC) cell lines HCT116 and COLO320 were treated with TNF-α and ox-LDL and tested for LOX-1 expression. The effect on cell proliferation of this proinflammatory signals were assayed by MTT and cell count with the exclusion dye trypan blue. LOX-1-negative cell lines were obtained by transduction with lentiviral vectors encoding a shRNA specific for LOX-1. The expression of genes involved in tumor progression were analyzed by qRT-PCR and Western blot in wild type and LOX-1 negative cells treated with TNF-α and ox-LDL. Cell migration and invasion was assayed in ox-LDL and TNF-α treated cells. Human colonoids were stimulated with ox-LDL in the presence of the NADPH oxidase inhibitors ML171, VAS2870 and ROS scavenger NAC. Surface area and survival were assessed by contrast microscopy and MTT. Proliferation was assessed by click-chemistry. LOX-1 receptor is expressed in the CRC cell lines HCT116 and COLO320. Additionally, we found in microarrays data stored in public databases that LOX-1 mRNA is over-expressed in colorectal carcinomas and adenocarcinomas compared with normal colon tissue. Treatment of CRC cell lines with TNF-α and ox-LDL augmented LOX-1 expression and LOX-1 silencing abrogated this effect. Moreover, both cell lines treated with ox-LDL proliferated in a dose-dependent manner. Importantly, ox-LDL stimulated human organoids proliferation and viability also in a dose-dependent manner, an effect partly mediated by ROS. The activation of LOX-1 by ox-LDL induced the expression of angiogenic markers such as MMP2, MMP7, MMP9 and VEGF. LOX-1 activation by oxLDL also promotes epithelial to mesenchymal transition of human colorectal cancer cells, characterized by reduced expression of the epithelial marker E-cadherin and increased expression of mesenchymal markers such as Vimentin, N-Cadherin, TWIST, ZEB1 and ZEB2. In addition, we determined that ox-LDL promotes invasion and migration of colorectal cancer cells. Nevertheless, co-treatment of colorectal cancer cells with TNF-α and ox-LDL did not show an additive effect. LOX-1 activation by ox-LDL induces a transcription reprogramming of genes involved in angiogenesis, invasion, migration, and cell proliferation. In the context of inflammation and obesity, we speculate that overexpression of LOX-1 provides additional stimulation accelerating tumor progression.

Interactions between Enterohemorrhagic Escherichia coli (EHEC) and Gut Commensals at the Interface of Human Colonoids.

ABSTRACTThe interactions between the gut microbiota and pathogens are complex and can determine the outcome of an infection. Enterohemorrhagic Escherichia coli (EHEC) is a major human enteric pathogen that colonizes the colon through attaching and effacing (AE) lesions and uses microbiota-derived molecules as cues to control its virulence. Different gut commensals can modulate EHEC virulence. However, the lack of an animal model that recapitulates the human pathophysiology of EHEC infection makes it challenging to investigate how variations in microbiota composition could affect host susceptibility to this pathogen. Here, we addressed these interactions building from simple to more complex in vitro systems, culminating with the use of the physiological relevant human colonoids as a model to study the interactions between EHEC and different gut commensals. We demonstrated that Bacteroides thetaiotaomicron and Enterococcus faecalis enhance virulence expression and AE lesion formation in cultured epithelial cells, as well as on the colonic epithelium, while commensal E. coli did not affect these phenotypes. Importantly, in the presence of these three commensals together, virulence and AE lesion are enhanced. Moreover, we identified specific changes in the metabolic landscape promoted by different members of the gut microbiota and showed that soluble factors released by E. faecalis can increase EHEC virulence gene expression. Our study highlights the importance of interspecies bacterial interactions and chemical exchange in the modulation of EHEC virulence.

Generation of CRISPR-Cas9-mediated genetic knockout human intestinal tissue-derived enteroid lines by lentivirus transduction and single-cell cloning.

Human intestinal tissue-derived enteroids (HIEs; also called organoids) are a powerful ex vivo model for gastrointestinal research. Genetic modification of these nontransformed cultures allows new insights into gene function and biological processes involved in intestinal diseases as well as gastrointestinal and donor segment-specific function. Here we provide a detailed technical pipeline and protocol for using the CRISPR-Cas9 genome editing system to knock out a gene of interest specifically in HIEs by lentiviral transduction and single-cell cloning. This protocol differs from a previously published alternative using electroporation of human colonoids to deliver piggyback transposons or CRISPR-Cas9 constructs, as this protocol uses a modified, fused LentiCRISPRv2-small-guiding RNA to express Cas9 and small-guiding RNA in a lentivirus. The protocol also includes the steps of gene delivery and subsequent single-cell cloning of the knockout cells as well as verification of clones and sequence identification of the mutation sites to establish knockout clones. An overview flowchart, step-by-step guidelines and troubleshooting suggestions are provided to aid the researcher in obtaining the genetic knockout HIE line within 2-3 months. In this protocol, we further describe how to use HIEs as an ex vivo model to assess host restriction factors for viral replication (using human norovirus replication as an example) by knocking out host attachment factors or innate immunity genes. Other applications are discussed to broaden the utility of this system, for example, to generate knockin or conditional knockout HIE lines to investigate the function of essential genes in many biological processes including other types of organoids.

OXPHOS DRIVES CRYPT FISSIONING REQUIRED FOR HEALING OF IBD ULCERS

Abstract INTRODUCTION The failure of ulcers healing is a characteristic feature of IBD. Mitochondrial dysfunction in intestinal epithelial cells (IEC) occurs in IBD and is linked to worse clinical outcomes in pediatric Crohn’s and ulcerative colitis (Kugrathasan et al Lancet 2017, Haberman et al Nat Comm 2019). Cellular polarization and maintenance of cell polarity are energy-dependent and highly regulated by mitochondrial biogenesis (Hardie DG et al Genes Dev 2011). Yet, it is unclear how mitochondria participate in mucosal repair. METHODS To model mitochondrial deficiency, TFAM, a transcriptional factor needed for mitochondrial DNA (mtDNA)-encoded genes was silenced in human colonoids (shTFAM) and VilCre/mTmG/TFAMfl/fl (IEC-TFAM-/-) mice. Levels of crypt branching and intestinal stem cell (ISC) and mitochondrial gene mRNA levels were assessed in colonoids cultured in Wnt-containing (WENR) media. Effects on IEC mitochondrial complex levels and oxygen consumption rates (OCR) were assessed in control IEC-TFAM-/- mice and effects on disease activity and ulcer healing assessed in DSS colitis IEC-TFAM-/- mice (fed DSS x 7d, peak ulcer healing-D13-15). Dual florescence confocal microscopy examined crypt fissioning and IHC in WT (Tom+) and Tfamfl/fl (GFP+) crypts during windows of peak ulcer healing. RESULTS shTFAM-treated colonoids expressed 55-92% lower mitochondrial gene mRNA (Cox6A1, mt-CO1, ATP5A1) levels (p<0.01) {A} and displayed reduced size (35%) and budding (86%) compared to WT cells (p<0.005) {B}. Studies on IEC from IEC-TFAM-/- mice revealed lower mitochondrial complex protein levels {C i-iv}, and >65% lower mtDNA (p<0.01) {D} compared to controls that correlated with 43 and 65% lower IEC OCR (using Oroboros). In DSS mice, levels of ulceration were greater in IEC-TFAM-/- mice (25%) compared to WT (7.5%) (P<0.005). More importantly, levels of crypt fissioning were 85% lower in TFAM-deficient (GFP+) compared to WT (Tom+) crypts {E}. WT fissioning crypt stained for SOX9 {F} and nuclear pβCat552 {G}. CONCLUSION Together, these results suggest that IEC mitochondrial deficiency as seen in IBD patients, impairs crypt fissioning during ulcer healing through a process that impairs cooperation between Wnt and PI3K signaling pathways. The data argue that therapeutic improvement of mitochondrial function may enhance mucosal repair in IBD by promoting crypt fissioning needed to re-establish barrier function.

Interleukin-37 regulates innate immune signaling in human and mouse colonic organoids

Intestinal epithelial cells (IEC) reside in close proximity to the gut microbiota and are hypo-responsive to bacterial products, likely to prevent maladaptive inflammatory responses. This is in part due to their strong expression of Single Ig IL-1 related receptor (SIGIRR), a negative regulator of interleukin (IL)-1 and toll-like receptor signaling. IL-37 is an anti-inflammatory cytokine that inhibits innate signaling in diverse cells by signaling through SIGIRR. Despite the strong expression of SIGIRR by IEC, few studies have examined whether IL-37 can suppress their innate immune signaling. We characterized innate immune responses of human and murine colonoids to bacteria (FliC, LPS) and host (IL-1β) products and the role of IL-37/SIGIRR in regulating these responses. We demonstrated that human colonoids responded only to FliC, but not to LPS or IL-1β. While colonoids derived from different donors displayed significant inter-individual variability in the magnitude of their innate responses to FliC stimulation, all colonoids released a variety of chemokines. Interestingly, IL-37 attenuated these responses through inhibition of p38 and NFκB signaling pathways. We determined that this suppression by IL-37 was SIGIRR dependent, in murine organoids. Along with species-specific differences in IEC innate responses, we show that IL-37 can promote IEC hypo-responsiveness by suppressing inflammatory signaling.

Fecal Microbiota Transplantation for Recurrent Clostridioides difficile Infection Associates With Functional Alterations in Circulating microRNAs

The molecular mechanisms underlying successful fecal microbiota transplantation (FMT) for recurrent Clostridioides difficile infection (rCDI) remain poorly understood. The primary objective of this study was to characterize alterations in microRNAs (miRs) following FMT for rCDI. Sera from 2 prospective multicenter randomized controlled trials were analyzed for miRNA levels with the use of the Nanostring nCounter platform and quantitative reverse-transcription (RT) polymerase chain reaction (PCR). In addition, rCDI-FMT and toxin-treated animals and exvivo human colonoids were used to compare intestinal tissue and circulating miRs. miR inflammatory gene targets in colonic epithelial and peripheral blood mononuclear cells were evaluated by quantitative PCR (qPCR) and 3'UTR reporter assays. Colonic epithelial cells were used for mechanistic, cytoskeleton, cell growth, and apoptosis studies. miRNA profiling revealed up-regulation of 64 circulating miRs 4 and 12 weeks after FMT compared with screening, of which the top 6 were validated in the discovery cohort by means of RT-qPCR. In a murine model of relapsing-CDI, RT-qPCR analyses of sera and cecal RNA extracts demonstrated suppression of these miRs, an effect reversed by FMT. In mouse colon and human colonoids, C difficile toxin B (TcdB) mediated the suppressive effects of CDI on miRs. CDI dysregulated DROSHA, an effect reversed by FMT. Correlation analyses, qPCR ,and 3'UTR reporter assays revealed that miR-23a, miR-150, miR-26b, and miR-28 target directly the 3'UTRs of IL12B, IL18, FGF21, and TNFRSF9, respectively. miR-23a and miR-150 demonstrated cytoprotective effects against TcdB. These results provide novel and provocative evidence that modulation of the gut microbiome via FMT induces alterations in circulating and intestinal tissue miRs. These findings contribute to a greater understanding of the molecular mechanisms underlying FMT and identify new potential targets for therapeutic intervention in rCDI.

COLON SPECIFIC ROLE FOR SELENOPROTEIN P IN COLITIS AND COLITIS-ASSOCIATED CARCINOMA

Abstract Selenium (Se) is an essential trace element incorporated into selenoproteins (SEPs), which provide a broad scope of antioxidant functions. Se status inversely correlates with inflammatory bowel disease (IBD) severity, and animal models have shown that Se deficiency worsens colitis and colitis-associated cancer (CAC). One of the most highly expressed and functionally critical SEPs is Selenoprotein P (SEPP1/SELENOP). SELENOP serves two known roles: 1) distributing Se throughout the organism, and 2) acting as a lipid peroxidase. SELENOP is also markedly reduced in IBD. Global SELENOP reduction augmented experimental CAC. SELENOP is primarily produced in the liver for tissue distribution through the plasma; however, liver-specific SELENOP deletion failed to modify CAC, thus implicating epithelial SELENOP as a primary disease modifier. As the roles of intestinal epithelial SELENOP in colitis and CAC remain unknown, we investigated these roles using the Vil-CreERT2 driver to inducibly delete intestinal epithelial SELENOP in AOM/DSS inflammatory carcinogenesis models. First, Selenop deletion was confirmed in intestinal epithelial cells of tamoxifen-induced Vil-CreERT2; Selenopfl/fl mice (ΔIEC). Epithelial-specific SELENOP loss promoted tumor initiation, as evidenced by greater tumor multiplicity visible by colonoscopy in ΔIEC mice. Upon sacrifice, ΔIEC mice demonstrated increased colon tumor number and size. Additionally, ΔIEC colon tumors displayed greater apoptosis (cleaved caspase-3), DNA damage (pγH2AX), and dysplasia severity. Overall, these results are consistent with our model that epithelial SELENOP loss promotes tumor formation through increased intratumoral oxidative stress and resultant genomic instability. To further investigate SELENOP’s contributions in pure epithelial cultures, we determined that both CACO2 cells and human colonoids produce SELENOP in a sodium selenite-dependent fashion; furthermore, polarized CACO2 cells preferentially secrete SELENOP basolaterally, suggesting a local extracellular role for SELENOP in the immediate colonic epithelial microenvironment. Also, SELENOP knockdown in human intestinal organoids derived from ulcerative colitis (UC) patients increased oxidative stress, increased sensitivity to H2O2 treatment, and reduced organoid growth, implicating SELENOP in modulating the epithelial ROS-protective response. RNA-seq analysis from the PROTECT cohort revealed reduced SELENOP expression in pediatric UC biopsies that inversely correlated with disease activity and S100A8 (calprotectin) levels. Lastly, SELENOP RNA in situ hybridization of adult UC/Crohn’s Disease and CAC TMAs demonstrated decreased epithelial transcript in IBD and stage-dependent reductions in CAC. These data collectively indicate critical roles for SELENOP in epithelial and colonic microenvironment antioxidant defenses in IBD.

MESENCHYMAL TNFR1 EXPRESSION PRESERVES THE COLONIC EPITHELIAL STEM CELL NICHE

Abstract Tumor necrosis factor (TNF) is a highly expressed cytokine in inflammatory bowel disease (IBD). Although TNF can induce colonic epithelial dysfunction and apoptosis, recent studies suggest that TNF signalling promotes epithelial wound repair and stem cell function. Here we investigated the role of TNF receptor 1 (TNFR1) in mediating TNF’s effects on colonic epithelial stem cells, integral to mucosal healing in colitis. We demonstrate that Tnfr1-/- mice exhibit loss in Lgr5 expression (-52%, p<0.02; N=6) compared to wildtype (WT) controls. However, the opposite result was found in vitro, wherein murine Tnfr1-/- colonoids demonstrated a significant increase in Lgr5 expression (66%, p<0.007; N=6) compared to WT colonoids. Similarly, human colonoids treated with an anti-TNFR1 antibody also demonstrated an increase in Lgr5 expression, relative to IgG controls. To resolve the contradiction in the in vivo versus in vitro environment, we hypothesized that mesenchymal TNFR1 expression regulates the epithelial stem cell niche. To determine the relationships between these cell types, we co-cultured WT or Tnfr1-/- colonoids with WT or Tnfr1-/- colonic myofibroblasts (CMFs). We found that epithelial Lgr5 expression was significantly higher (by 52%, p<0.05; N=3) when co-cultured with WT compared to TNFR1-/- myofibroblasts. The loss of TNFR1 expression in vivo increases the number of αSMA+ mesenchymal cells by nearly 56% (N=6) but considerably reduces the pericryptal PDGFRα+ cells, suggesting modifications in mesenchymal populations that contribute to the epithelial stem cell niche. Functionally, primary Tnfr1-/--CMFs displayed PI3k (p<0.001; N=3) and MAPK (p<0.01; N=3)-dependent increases in migration, proliferation, and differentiation, but RNA profiling demonstrated by diminished levels of stem cell niche factors, Rspo3 (-80%, p<0.0001; N=6) and Wnt2b (-63%, p<0.008; N=6) compared to WT-CMFs. Supplementation with 50ng recombinant Rspo3 for 5 d to Lgr5-GFP organoids co-cultured with TNFR1-/--CMFs restored Lgr5 expression to wildtype levels. Therefore, TNFR1-mediated TNF signalling in mesenchymal cells promotes their ability to support an epithelial stem cell niche. These results should motivate future studies of the stem cell niche in the context of long-term treatment with anti-TNF therapies.

Aberrant enteric neuromuscular system and dysbiosis in amyotrophic lateral sclerosis

ABSTRACT Amyotrophic Lateral Sclerosis is a neuromuscular disease characterized by the progressive death of motor neurons and muscle atrophy. The gastrointestinal symptoms in ALS patients were largely ignored or underestimated. The relationship between the enteric neuromuscular system and microbiome in ALS progression is unknown. We performed longitudinal studies on the enteric neuron system (ENS) and microbiome in the ALS human-SOD1G93A (Superoxide Dismutase 1) transgenic mice. We treated age-matched wild-type and ALS mice with butyrate or antibiotics to investigate the microbiome and neuromuscular functions. We examined intestinal mobility, microbiome, an ENS marker GFAP (Glial Fibrillary Acidic Protein), a smooth muscle marker (SMMHC, Smooth Muscle Myosin Heavy Chain), and human colonoids. The distribution of human-G93A-SOD1 protein was tested as an indicator of ALS progression. At 2-month-old before ALS onset, SOD1G93A mice had significantly lower intestinal mobility, decreased grip strength, and reduced time in the rotarod. We observed increased GFAP and decreased SMMHC expression. These changes correlated with consistent increased aggregation of mutated SOD1G93A in the colon, small intestine, and spinal cord. Butyrate or antibiotics treated SOD1G93A mice had a significantly longer latency to fall in the rotarod test, reduced SOD1G93A aggregation, and enhanced enteric neuromuscular function. Feces from 2-month-old SOD1G93A mice significantly enhanced SOD1G93A aggregation in human colonoids transfected with a SOD1G93A-GFP plasmid. Longitudinal studies of microbiome data further showed the altered bacterial community related to autoimmunity (e.g., Clostridium sp. ASF502, Lachnospiraceae bacterium A4), inflammation (e.g., Enterohabdus Muris,), and metabolism (e.g., Desulfovibrio fairfieldensis) at 1- and 2-month-old SOD1G93A mice, suggesting the early microbial contribution to the pathological changes. We have demonstrated a novel link between the microbiome, hSOD1G93A aggregation, and intestinal mobility. Dysbiosis occurred at the early stage of the ALS mice before observed mutated-SOD1 aggregation and dysfunction of ENS. Manipulating the microbiome improves the muscle performance of SOD1G93A mice. We provide insights into the fundamentals of intestinal neuromuscular function and microbiome in ALS.

Epithelial WNT2B and Desert Hedgehog Are Necessary for Human Colonoid Regeneration after Bacterial Cytotoxin Injury.

SummaryIntestinal regeneration and crypt hyperplasia after radiation or pathogen injury relies on Wnt signaling to stimulate stem cell proliferation. Mesenchymal Wnts are essential for homeostasis and regeneration in mice, but the role of epithelial Wnts remains largely uncharacterized. Using the enterohemorrhagic E. coli-secreted cytotoxin EspP to induce injury to human colonoids, we evaluated a simplified, epithelial regeneration model that lacks mesenchymal Wnts. Here, we demonstrate that epithelial-produced WNT2B is upregulated following injury and essential for regeneration. Hedgehog signaling, specifically activation via the ligand Desert Hedgehog (DHH), but not Indian or Sonic Hedgehog, is another driver of regeneration and modulates WNT2B expression. These findings highlight the importance of epithelial WNT2B and DHH in regulating human colonic regeneration after injury.

Aggregative Adherence Fimbriae II of Enteroaggregative Escherichia coli Are Required for Adherence and Barrier Disruption during Infection of Human Colonoids.

Symptomatic and asymptomatic infection with the diarrheal pathogen enteroaggregative Escherichia coli (EAEC) is associated with growth faltering in children in developing settings. The mechanism of this association is unknown, emphasizing a need for better understanding of the interactions between EAEC and the human gastrointestinal mucosa. In this study, we investigated the role of the aggregative adherence fimbriae II (AAF/II) in EAEC adherence and pathogenesis using human colonoids and duodenal enteroids. We found that a null mutant in aafA, the major subunit of AAF/II, adhered significantly less than wild-type (WT) EAEC strain 042, and adherence was restored in a complemented strain. Immunofluorescence confocal microscopy of differentiated colonoids, which produce an intact mucus layer comprised of the secreted mucin MUC2, revealed bacteria at the epithelial surface and within the MUC2 layer. The WT strain adhered to the epithelial surface, whereas the aafA deletion strain remained within the MUC2 layer, suggesting that the presence or absence of AAF/II determines both the abundance and location of EAEC adherence. In order to determine the consequences of EAEC adherence on epithelial barrier integrity, colonoid monolayers were exposed to EAEC constructs expressing or lacking aafA Colonoids infected with WT EAEC had significantly decreased epithelial resistance, an effect that required AAF/II, suggesting that binding of EAEC to the epithelium is necessary to impair barrier function. In summary, we show that production of AAF/II is critical for adherence and barrier disruption in human colonoids, suggesting a role for this virulence factor in EAEC colonization of the gastrointestinal mucosa.

Histone H3K9 methylation regulates chronic stress and IL-6-induced colon epithelial permeability and visceral pain.

Chronic stress is associated with activation of the HPA axis, elevation in pro-inflammatory cytokines, decrease in intestinal epithelial cell tight junction (TJ) proteins, and enhanced visceral pain. It is unknown whether epigenetic regulatory pathways play a role in chronic stress-induced intestinal barrier dysfunction and visceral hyperalgesia. Young adult male rats were subjected to water avoidance stress±H3K9 methylation inhibitors or siRNAs. Visceral pain response was assessed. Differentiated Caco-2/BBE cells and human colonoids were treated with cortisol or IL-6±antagonists. Expression of TJ, IL-6, and H3K9 methylation status at gene promoters was measured. Transepithelial electrical resistance and FITC-dextran permeability were evaluated. Chronic stress induced IL-6 up-regulation prior to a decrease in TJ proteins in the rat colon. The IL-6 level inversely correlated with occludin expression. Treatment with IL-6 decreased occludin and induced visceral hyperalgesia. Chronic stress and IL-6 increased H3K9 methylation and decreased transcriptional GR binding to the occludin gene promoter, leading to down-regulation of protein expression and increase in paracellular permeability. Intrarectal administration of a H3K9 methylation antagonist prevented chronic stress-induced visceral hyperalgesia in the rat. In a human colonoid model, cortisol decreased occludin expression, which was prevented by the GR antagonist RU486, and IL-6 increased H3K9 methylation and decreased TJ protein levels, which were prevented by inhibitors of H3K9 methylation. Our findings support a novel role for methylation of the repressive histone H3K9 to regulate chronic stress, pro-inflammatory cytokine-mediated reduction in colon TJ protein levels, and increase in paracellular permeability and visceral hyperalgesia.

Mucus layer modeling of human colonoids during infection with enteroaggragative E. coli

EAEC is a common cause of diarrheal illness worldwide. Pathogenesis is believed to occur in the ileum and colon, where the bacteria adhere and form a robust aggregating biofilm. Among the multiple virulence factors produced by EAEC, the Pic serine protease has been implicated in bacterial colonization by virtue of its mucinolytic activity. Hence, a potential role of Pic in mucus barrier disruption during EAEC infection has been long postulated. In this study, we used human colonoids comprising goblet cells and a thick mucin barrier as an intestinal model to investigate Pic’s roles during infection with EAEC. We demonstrated the ability of purified Pic, but not a protease defective Pic mutant to degrade MUC2. Western blot and confocal microscopy analysis revealed degradation of the MUC2 layer in colonoids infected with EAEC, but not with its isogenic EAECpic mutant. Wild-type and MUC2-knockdown colonoids infected with EAEC strains exposed a differential biofilm distribution, greater penetration of the mucus layer and increased colonization of the colonic epithelium by Wild-type EAEC than its isogenic Pic mutant. Higher secretion of pro-inflammatory cytokines was seen in colonoids infected with EAEC than EAECpic. Although commensal E. coli expressing Pic degraded MUC2, it did not show improved mucus layer penetration or colonization of the colonic epithelium. Our study demonstrates a role of Pic in MUC2 barrier disruption in the human intestine and shows that colonoids are a reliable system to study the interaction of pathogens with the mucus layer.

Enterohemorrhagic E. coli (EHEC) Secreted Serine Protease EspP‐Induced Injury and Regeneration is Guided by Desert Hedgehog and WNT2B in Human Colonoids

IntroductionEnterohemorrhagic E. coli (EHEC) is a major foodborne pathogen in developed countries. In humans, EHEC colonizes the colon and can cause life threatening hemorrhagic colitis and hemolytic uremic syndrome. The colon pathology typically shows hemorrhagic mucosa with apoptotic crypts; however, the epithelium regenerates after bacterial clearance. We have demonstrated that human colonoids are a relevant pathophysiological model to study early molecular events of EHEC infection. The goal of this study was to elucidate the mechanism of epithelial regeneration after EHEC‐induced injury.MethodsHuman colonoids derived from colonic biopsies obtained from healthy donors were treated overnight with a high dose of recombinant serine protease EspP (the major EHEC‐secreted cytotoxin) to induce epithelial apoptosis. Non‐treated cultures served as controls. Following incubation, EspP was washed from the cultures and both EspP‐treated and control populations were lysed. The protein signatures of the colonoids were identified and quantified with tandem mass spectrometry and iTRAQ. The proteome was validated by immunoblot or qRT‐PCR of selected genes in the absence of suitable antibodies.ResultsOvernight incubation of colonoids with EspP induced significant cell death, seen by propidium iodide staining. Based on the downstream targets that were upregulated, the proteome suggests that the WNT and Hedgehog pathways are essential for regeneration following EspP‐induced injury. Sonic and indian hedgehog are highly expressed under basal conditions in colonoids, but are downregulated during regeneration. Instead, desert hedgehog (DHH), which is below detection under basal conditions, was upregulated 3x fold in regenerating colonoids. Colonoids treated with the smoothened agonist (stimulator of hedgehog signaling) or recombinant DHH had increased survivability. WNT2B was also upregulated (2x fold) during regeneration. Inhibition of WNT secretion with IWP‐2 (porcupine inhibitor) had no effect on basal colonoid growth, but prevented regeneration of EspP‐injured colonoids. Knockout of WNT2B by CRISPR gene editing also halted regeneration.ConclusionsThe results of the current study demonstrate that 1) WNT and hedgehog pathways are important modulators of regeneration following EspP‐induced injury but 2) through Desert Hedgehog and WNT2B stimulation specifically, suggesting the various hedgehog ligands (sonic, indian, desert) are not redundant. 3) WNT2B is produced by the epithelia and is not essential for homeostasis, but is indispensable during regeneration. These signaling pathways are related, yet distinct from unchecked proliferation seen in colon cancers, suggesting that colonic stem cells specifically respond to bacterial‐mediated injury. This provides a new understanding of the mechanisms related to stem cell response in diarrheal diseases.Support or Funding InformationNIH grants: P01 AI125181, K01 DK106323, P30 DK089502, and U01 DK103168

Human Colonoids as a Model for Studying Host‐Pathogen Interactions with Campylobacter jejuni

BackgroundCampylobacter is a leading cause of bacterial enterocolitis in the U.S. and around the world. In addition to the acute morbidity, it is associated with delayed sequelae such as environmental enteropathy and post‐infection irritable bowel syndrome. Host responses to Campylobacter infection are poorly studied due to colonization resistance in mice thereby restricting virulence studies to immortalized cell lines. These models lack the complexity of the host environment. Recent studies have highlighted the use of organoids for studying enteric pathogens. We propose that human organoids provide a unique platform for studying pathogenic mechanisms incorporating unique host (human) and infection (strain) characteristics.ObjectiveTo demonstrate human colonic organoids (colonoids) as a model for studying C. jejuni infection.MethodsSigmoid colonic biopsies were collected from healthy volunteers between 18–35 years of age (n=3). Intestinal crypts were isolated, washed and plated in matrigel domes for colonoid formation. Mature colonoids were harvested after 7 days, and dissociated using a 0.05% trypsin solution. These were plated on collagen IV coated plates, generating 2D‐monolayers for infection experiments. Three different C. jejuni strains (Sequence Types 45, 22, 8) were grown to an OD600 of 1 and used to infect the colonoid monolayers. To assess adhesion and internalization, colonoids were infected for a total of 3 hours after which monolayers were either washed and subsequently lysed (to determine attached plus invaded bacteria) or treated with gentamycin and then lysed (to determine invaded bacteria). Bacterial quantification was done by plating on Mueller Hinton agar. Additional colonoid monolayers were grown on permeable transwell inserts. Baseline transepithelial resistance (TER) of confluent monolayers was recorded and then continuously assessed every hour after the addition of the C. jejuni strains (using impedance spectroscopy in cellZscope) to evaluate changes in barrier integrity.ResultsAll C. jejuni strains attached and invaded colonoid monolayers derived from all three volunteers. Two strains (ST 45 and 22) caused greater adhesion and invasion in all three colonoids compared to the third strain (Adhesion mean (SEM): 4.32 x 10−2 (2.33 x 10−3), 9.36 x 10−3 (4.82 x 10−4) vs 7.79 x 10−3 (3.51 x 10−3); Invasion mean (SEM) 3.47 x 10−4 (2.13 x 10−5), 2.33 x 10−4 (1.36 x 10−4) vs 4.57 x 10−5 (1.73 x 10−5) (ANOVA, p<0.05, n=3/group for both analyses). Additionally, greater drop in TER was seen in colonoid monolayers exposed to the two strains with higher adhesion and invasion (Mean % of baseline TER at 30 hr post‐infection 2.58% (1.21), 14.14% (3.39) vs 42.29% (18.49), p<0.01). These results correlated with virulence of these strains in conventional T84 monolayers. Transcriptomic and proteomic characterization of the C. jejuni infection in these colonoids is in progress.ConclusionsHuman colonoids are useful to assess the role of the host in infectivity and epithelial barrier responses to C. jejuni infection. Future work involves determining responses in colonoids derived from volunteers with specific characteristics to create unique host‐bacterial infection models.Support or Funding InformationSupported by funding from Department of Defense W81XWH‐18‐1‐0074

Epithelial WNT2B and Desert Hedgehog are Necessary for Human Colonoid Regeneration after Bacterial Cytotoxin Injury

Intestinal regeneration and crypt hyperplasia after radiation or pathogen injury relies on Wnt signaling to stimulate stem cell proliferation. Mesenchymal Wnts are essential for homeostasis and regeneration in mice, but the role of epithelial Wnts remains largely uncharacterized. Using the enterohemorrhagic E. coli secreted cytotoxin, EspP, to induce injury to human colonoids, we evaluated a simplified, epithelial regeneration model that lacks mesenchymal Wnts. To identify key regeneration-associated pathways, proteomics was performed on the colonoids after injury. Here, we demonstrate that epithelial-produced WNT2B is upregulated following injury and essential for regeneration. Hedgehog signaling, specifically activation via the ligand Desert Hedgehog (DHH), but not Indian or Sonic Hedgehog, is another driver of regeneration and modulates WNT2B expression. These findings highlight the importance of epithelial WNT2B and DHH in regulating human colonic regeneration after injury.