Ex vivo culture of intestinal crypt organoids as a model system for assessing cell death induction in intestinal epithelial cells and enteropathy

AbstractRewiring of host cytokine networks is a key feature of inflammatory bowel diseases (IBD) such as Crohn’s disease (CD). Th1-type cytokines—IFN-γ and TNF-α—occupy critical nodes within these networks and both are associated with disruption of gut epithelial barrier function. This may be due to their ability to synergistically trigger the death of intestinal epithelial cells (IECs) via largely unknown mechanisms. In this study, through unbiased kinome RNAi and drug repurposing screens we identified JAK1/2 kinases as the principal and nonredundant drivers of the synergistic killing of human IECs by IFN-γ/TNF-α. Sensitivity to IFN-γ/TNF-α-mediated synergistic IEC death was retained in primary patient-derived intestinal organoids. Dependence on JAK1/2 was confirmed using genetic loss-of-function studies and JAK inhibitors (JAKinibs). Despite the presence of biochemical features consistent with canonical TNFR1-mediated apoptosis and necroptosis, IFN-γ/TNF-α-induced IEC death was independent of RIPK1/3, ZBP1, MLKL or caspase activity. Instead, it involved sustained activation of JAK1/2-STAT1 signalling, which required a nonenzymatic scaffold function of caspase-8 (CASP8). Further modelling in gut mucosal biopsies revealed an intercorrelated induction of the lethal CASP8-JAK1/2-STAT1 module during ex vivo stimulation of T cells. Functional studies in CD-derived organoids using inhibitors of apoptosis, necroptosis and JAKinibs confirmed the causative role of JAK1/2-STAT1 in cytokine-induced death of primary IECs. Collectively, we demonstrate that TNF-α synergises with IFN-γ to kill IECs via the CASP8-JAK1/2-STAT1 module independently of canonical TNFR1 and cell death signalling. This non-canonical cell death pathway may underpin immunopathology driven by IFN-γ/TNF-α in diverse autoinflammatory diseases such as IBD, and its inhibition may contribute to the therapeutic efficacy of anti-TNFs and JAKinibs.

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Progress on isolation and short-term ex-vivo culture of highly purified non-apoptotic human intestinal epithelial cells (IEC)

European Journal of Cell Biology ◽

10.1078/0171-9335-00312 ◽

2003 ◽

Vol 82 (5) ◽

pp. 262-270 ◽

Cited By ~ 35

Author(s):

Johannes Grossmann ◽

Kathrin Walther ◽

Monika Artinger ◽

Stephan Kiessling ◽

Martin Steinkamp ◽

...

Keyword(s):

Epithelial Cells ◽

Intestinal Epithelial Cells ◽

Ex Vivo ◽

Intestinal Epithelial ◽

Short Term ◽

Human Intestinal Epithelial Cells ◽

Ex Vivo Culture

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Recognition of bacteria by intestinal epithelial cells and regulation of p53 signaling and cell death – interactions at the epithelial interface leading to spontaneous bacterial peritonitis in liver cirrhosis

10.1055/s-0038-1677101 ◽

2019 ◽

Author(s):

A Scholtis ◽

M Haderer ◽

K Gülow ◽

C Kunst ◽

H Gschwendtner ◽

...

Keyword(s):

Cell Death ◽

Liver Cirrhosis ◽

Epithelial Cells ◽

Spontaneous Bacterial Peritonitis ◽

Intestinal Epithelial Cells ◽

Intestinal Epithelial ◽

Bacterial Peritonitis ◽

P53 Signaling

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Decreased SLC26A3 expression and function in intestinal epithelial cells in response to Cryptosporidium parvum infection

AJP Cell Physiology ◽

10.1152/ajpcell.00278.2019 ◽

2019 ◽

Vol 317 (6) ◽

pp. C1205-C1212 ◽

Cited By ~ 2

Author(s):

Anoop Kumar ◽

Dulari Jayawardena ◽

Arivarasu N. Anbazhagan ◽

Ishita Chatterjee ◽

Shubha Priyamvada ◽

...

Keyword(s):

Epithelial Cells ◽

Cryptosporidium Parvum ◽

Diarrheal Disease ◽

Intestinal Epithelial Cells ◽

Ex Vivo ◽

Jejunal Mucosa ◽

Intestinal Epithelial ◽

Chloride Absorption ◽

And Function

The protozoan parasite Cryptosporidium parvum (CP) causes cryptosporidiosis, a diarrheal disease worldwide. Infection in immunocompetent hosts typically results in acute, self-limiting, or recurrent diarrhea. However, in immunocompromised individuals infection can cause fulminant diarrhea, extraintestinal manifestations, and death. To date, the mechanisms underlying CP-induced diarrheal pathogenesis are poorly understood. Diarrheal diseases most commonly involve increased secretion and/or decreased absorption of fluid and electrolytes. We and others have previously shown impaired chloride absorption in infectious diarrhea due to dysregulation of SLC26A3 [downregulated in adenoma (DRA)], the human intestinal apical membrane Cl−/[Formula: see text] exchanger protein. However, there are no studies on the effects of CP infection on DRA activity. Therefore, we examined the expression and function of DRA in intestinal epithelial cells in response to CP infection in vitro and in vivo. CP infection (0.5 × 106 oocysts/well in 24-well plates, 24 h) of Caco-2 cell monolayers significantly decreased Cl−/[Formula: see text] exchange activity (measured as DIDS-sensitive 125I uptake) as well as DRA mRNA and protein levels. Substantial downregulation of DRA mRNA and protein was also observed following CP infection ex vivo in mouse enteroid-derived monolayers and in vivo in the ileal and jejunal mucosa of C57BL/6 mice for 24 h. However, at 48 h after infection in vivo, the effects on DRA mRNA and protein were attenuated and at 5 days after infection DRA returned to normal levels. Our results suggest that impaired chloride absorption due to downregulation of DRA could be one of the contributing factors to CP-induced acute, self-limiting diarrhea in immunocompetent hosts.

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Comparisons between Animal Models and Intestinal Crypt Cultures when Investigating the Impact of Fatty Acids on Gene Expression in Intestinal Epithelial Cells

The FASEB Journal ◽

10.1096/fasebj.2020.34.s1.06598 ◽

2020 ◽

Vol 34 (S1) ◽

pp. 1-1

Author(s):

Xiaojing Yang ◽

Elvira Mennillo ◽

Robert Tukey ◽

Shujuan Chen

Keyword(s):

Gene Expression ◽

Fatty Acids ◽

Animal Models ◽

Epithelial Cells ◽

Intestinal Epithelial Cells ◽

Intestinal Epithelial ◽

Intestinal Crypt ◽

The Impact

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Helicobacter hepaticusCytolethal Distending Toxin Causes Cell Death in Intestinal Epithelial Cells via Mitochondrial Apoptotic Pathway

Helicobacter ◽

10.1111/j.1523-5378.2010.00749.x ◽

2010 ◽

Vol 15 (2) ◽

pp. 98-107 ◽

Cited By ~ 30

Author(s):

Namal P. M. Liyanage ◽

Karoline C. Manthey ◽

Rohana P. Dassanayake ◽

Charles A. Kuszynski ◽

Gregory G. Oakley ◽

...

Keyword(s):

Cell Death ◽

Epithelial Cells ◽

Intestinal Epithelial Cells ◽

Intestinal Epithelial ◽

Apoptotic Pathway ◽

Mitochondrial Apoptotic Pathway

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Mucin 3 is involved in intestinal epithelial cell apoptosis viaN-(3-oxododecanoyl)-l-homoserine lactone-induced suppression of Akt phosphorylation

AJP Cell Physiology ◽

10.1152/ajpcell.00271.2013 ◽

2014 ◽

Vol 307 (2) ◽

pp. C162-C168 ◽

Cited By ~ 7

Author(s):

Ryoko Taguchi ◽

Shinya Tanaka ◽

Ga-Hyun Joe ◽

Hideaki Maseda ◽

Nobuhiko Nomura ◽

...

Keyword(s):

Cell Death ◽

Epithelial Cells ◽

Intestinal Epithelial Cells ◽

Virulence Gene ◽

Homoserine Lactone ◽

Intestinal Epithelial ◽

Cellular Functions ◽

Virulence Gene Expression ◽

Akt Phosphorylation ◽

Epithelial Cell Apoptosis

N-acyl-homoserine lactones (AHL) are quorum-sensing molecules in bacteria that play important roles in regulating virulence gene expression in pathogens such as Pseudomonas aeruginosa. The present study compared responses between undifferentiated and differentiated Caco-2 cells to N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL). A low concentration of 3-oxo-C12-HSL (30 μM) is sufficient to reduce viability accompanied by apoptosis via the suppression of phosphorylation by Akt in undifferentiated Caco-2 cells. The suppression of Akt phosphorylation appears specific in 3-oxo-C12-HSL, because other AHLs did not influence the phosphorylation status of Akt. The reduced viability induced by 3-oxo-C12-HSL was partially recovered by constitutively active Akt overexpression in undifferentiated Caco-2 cells. Since mucin is considered a vital component of the gut barrier, we investigated whether mucin protects cellular functions induced by 3-oxo-C12-HSL in undifferentiated Caco-2 cells. The results showed that mucin protected undifferentiated Caco-2 cells from apoptosis induced by 3-oxo-C12-HSL. 3-Oxo-C12-HSL did not induce cell death in differentiated Caco-2 cells that expressed higher levels of mucin 3 (MUC3) than undifferentiated Caco-2 cells. In addition, 3-oxo-C12-HSL promoted cell death in undifferentiated Caco-2 cells transfected with MUC3 siRNA and reduced MUC3 expression in undifferentiated Caco-2 cells. Therefore, MUC3 might be responsible for the survival of undifferentiated intestinal epithelial cells in the presence of 3-oxo-C12-HSL through regulating Akt phosphorylation. In conclusion, 3-oxo-C12-HSL might influence the survival of undifferentiated intestinal epithelial cells as well as interactions between these cells and pathogens.

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Suppressor of cytokine signaling-2 limits intestinal growth and enterotrophic actions of IGF-I in vivo

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00218.2005 ◽

2006 ◽

Vol 291 (3) ◽

pp. G472-G481 ◽

Cited By ~ 26

Author(s):

Carmen Z. Michaylira ◽

James G. Simmons ◽

Nicole M. Ramocki ◽

Brooks P. Scull ◽

Kirk K. McNaughton ◽

...

Keyword(s):

Small Intestine ◽

Epithelial Cells ◽

Intestinal Epithelial Cells ◽

Ex Vivo ◽

Cytokine Signaling ◽

Intestinal Epithelial ◽

Growth Responses ◽

Intestinal Growth ◽

Igf I

Suppressors of cytokine signaling (SOCS) typically limit cytokine receptor signaling via the JAK-STAT pathway. Considerable evidence demonstrates that SOCS2 limits growth hormone (GH) action on body and organ growth. Biochemical evidence that SOCS2 binds to the IGF-I receptor (IGF-IR) supports the novel possibility that SOCS2 limits IGF-I action. The current study tested the hypothesis that SOCS2 normally limits basal or IGF-I-induced intestinal growth and limits IGF-IR signaling in intestinal epithelial cells. Intestinal growth was assessed in mice homozygous for SOCS2 gene deletion (SOCS2 null) and wild-type (WT) littermates at different ages and in response to infused IGF-I or vehicle or EGF and vehicle. The effects of SOCS2 on IGF-IR signaling were examined in ex vivo cultures of SOCS2 null and WT intestine and Caco-2 cells. Compared with WT, SOCS2 null mice showed significantly enhanced small intestine and colon growth, mucosal mass, and crypt cell proliferation and decreases in radiation-induced crypt apoptosis in jejunum. SOCS2 null mice showed significantly greater growth responses to IGF-I in small intestine and colon. IGF-I-stimulated activation of IGF-IR and downstream signaling intermediates were enhanced in the intestine of SOCS2 null mice and were decreased by SOCS2 overexpression in Caco-2 cells. SOCS2 bound directly to the endogenous IGF-IR in Caco-2 cells. The intestine of SOCS2 null mice also showed enhanced growth responses to infused EGF. We conclude that SOCS2 normally limits basal and IGF-I- and EGF-induced intestinal growth in vivo and has novel inhibitory effects on the IGF-IR tyrosine kinase pathway in intestinal epithelial cells.

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Morphogenic protein epimorphin protects intestinal epithelial cells from oxidative stress by the activation of EGF receptor and MEK/ERK, PI3 kinase/Akt signals

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00181.2006 ◽

2007 ◽

Vol 292 (1) ◽

pp. G39-G52 ◽

Cited By ~ 32

Author(s):

Masahiro Iizuka ◽

Kenji Sasaki ◽

Yohei Hirai ◽

Kenichi Shindo ◽

Shiho Konno ◽

...

Keyword(s):

Oxidative Stress ◽

Hydrogen Peroxide ◽

Cell Death ◽

Epithelial Cells ◽

Egf Receptor ◽

Intestinal Epithelial Cells ◽

Pi3 Kinase ◽

Intestinal Epithelial ◽

Extracellular Signal Regulated Kinase ◽

Extracellular Signal

Epimorphin is a mesenchymal protein that regulates morphogenesis of epithelial cells. Our preliminary study suggested a novel function of epimorphin in enhancing survival of intestinal epithelial cells (IEC). Oxidative stress leads to cell injury and death and is suggested to be a key contributor to pathogenesis of inflammatory bowel disease. This study was conducted to determine whether epimorphin protects IEC from oxidative stress. Rat intestinal epithelial cell line IEC-6 was cultured with epimorphin (10 and 20 μg/ml), and the life span of IEC was assessed. The mean life span of IEC-6 cells was prolonged 1.9-fold ( P < 0.0006) by treatment with epimorphin. We then examined the epimorphin signaling pathways. Epimorphin phosphorylated epidermal growth factor (EGF) receptor, activated the MEK/extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase and phosphatidylinositol 3 (PI3) kinase/Akt pathways, phosphorylated Bad, and induced Bcl-XL and survivin. Hydrogen peroxide (1 mM) induced cell death in 92% of IEC-6 cells, but epimorphin dramatically diminished (88.7%) cell death induced by hydrogen peroxide ( P < 0.0001). This protective effect of epimorphin was significantly attenuated by inhibitors of MEK and PI3 kinase ( P < 0.0001) or EGF receptor-neutralizing antibody ( P = 0.0007). In wound assays, the number of migrated cells in the wound area decreased (72.5%) by treatment with 30 μM hydrogen peroxide, but epimorphin increased the number of migrated cells 3.18-fold ( P < 0.0001). These results support a novel function of epimorphin in protecting IEC from oxidative stress. This anti-oxidative function of epimorphin is dramatic and is likely mediated by the activation of EGF receptors and the MEK/extracellular signal-regulated kinase and PI3 kinase/Akt signaling pathways and through the induction of anti-apoptotic factors.

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