Human Colonic Organoids: An Epithelial Barrier Model of Crohn's Disease

It’s unknown if the increased intestinal permeability in patients with Crohn’s Disease (CD) is a cause or consequence of inflammation. In vivo, interactions between the microbiota, epithelium and immune system prevent investigation of the intrinsic properties of each component. Therefore, in this study colonic organoids have been used to first, determine their suitability as a model of the colonic epithelium, second to model the impact of bacterial components and pro-inflammatory cytokines on the paracellular barrier and thirdly to determine if the permeability of the intestinal epithelium is altered in patients with CD independent of the effects of the immune system and luminal bacteria Colonic organoids were grown from crypts isolated from transverse colonic biopsies from healthy and patients with CD. Ultrastructure of epithelial cells was determined by transmission electron microscopy. Transcript levels of proteins were determined by qPCR and the quantification and localisation of these proteins were determined by immunoblotting and immunofluorescent microscopy. Permeability of the epithelium was determined by FITC-dextran uptake into colonic organoids. Proliferation was analysed and measured by Ki67 staining and apoptosis by TUNEL staining. Statistical significance was determined by an unpaired or paired Student’s t-test or by a one-way ANOVA with a Bonferroni posthoc test. Spontaneously maturing day 15 colonoids have a polarized well-differentiated epithelium with well-developed tight junctions (TJs), similar to the native epithelium. Four-day-old colonospheres, in which the epithelium is functionally but not structurally polarised, had a cytoplasmic localisation of TJs. Exposure to lipopolysaccharide, but not lipoteichoic acid or muramyl dipeptide (20ng/mL) induced goblet cell development but had no effect on the TJs or epithelial permeability. However, pro-inflammatory cytokines, TNF-alpha (100ng/mL) and IFN-gamma (10ng/mL) increased the epithelial permeability and this was associated with loss of barrier-forming (occludin) and adaptor TJ proteins (ZO-1) and a parallel increase in the pore-forming TJ protein (claudin-2). Colonic organoids grown from patients with CD had an inherent defect in the development of the epithelium, with an apparent loss of polarity of the epithelium, evident as a redistribution of actin and E-cadherin from the membrane into the cytoplasm and a loss of NKCC1. This was associated with a redistribution of occludin and ZO-1, from the junction to the basolateral membrane and into the cytoplasm. Additionally, claudin-2 was concentrated in the TJ of the CD colonoids, whereas in control colonoids it was distributed throughout the basolateral membrane. Significantly, the epithelium of the CD colonoids was (p<0.05) more permeable than the epithelium of control colonoids. The native epithelium and colonic organoids derived from patients with CD had a significant downregulation of p120, a cytoplasmic regulatory protein associated with the stabilization of TJs, actin and E-cadherin. These data indicate that colonic organoids are a suitable model of the colonic epithelium and demonstrate that the colonic epithelium of patients with CD has an inherent permeability defect due to redistribution of the junctional proteins possibly as a consequence of the loss of p120. This occurs independent of the bacteria and immune system, suggesting that the increased epithelial permeability is a cause not a consequence of inflammation in CD

Expression and function of the P2X7 receptor on human malignant cell lines

The P2X7 receptor is a ligand-gated ion channel present on normal and malignant cells of hematopoietic and epithelial origins. Activation of the P2X7 receptor by extracellular adenosine 5’-triphosphate (ATP) causes the rapid flux of Na+, K+ and Ca2+. Continued activation of P2X7 allows the formation of large, non-specific pores, allowing the uptake of organic molecules including cation dyes. In addition, activation of P2X7 leads to a number of downstream signalling events which includes the maturation and release of interlukin (IL)-1β, as well as cell proliferation and death. As a result of these latter roles of P2X7 in cell survival, this receptor is attracting considerable interest for its growth-promoting and growth-inhibitory roles in cancer. Therefore, the aim of this project was to study the expression and function of the P2X7 receptor in myeloid leukemic and epithelial malignant cancer cell lines. Previous work in our laboratory demonstrated that the immunomodulatory cytokine, transforming growth factor (TGF)-β1 prevents the up-regulation of P2X7 in the human myeloid leukemic cell line, THP-1 by interferon (IFN)-γ and lipopolysaccharide (LPS). The mechanism of action of TGF-β1 in this process however was never elucidated. Therefore, the first part of my study aimed to determine the mechanism of action of TGF-β1 on the up-regulation of P2X7 expression and function in THP-1 cells differentiated with IFN-γ and LPS. Cell-surface molecules including P2X7 were examined by immunofluorescence staining. Total P2X7 protein and mRNA was assessed by immunoblotting and RT-PCR respectively. P2X7 function was evaluated by ATP-induced cation dye uptake measurements. IL-1β release was measured by ELISA. Cell-surface P2X7 was present on THP-1 cells differentiated for 3 days with IFN-γ and LPS but not on undifferentiated THP-1 cells. Similarly, ATP induced ethidium+ uptake into differentiated but not undifferentiated THP-1 cells. Coincubation of cells with TGF-β1 plus IFN-γ and LPS prevented the up-regulation of P2X7 expression and ATP-induced ethidium+ uptake Moreover, ATP-induced YOPRO- 12+ uptake and IL-1β release were abrogated in IFN-γ and LPS-treated cells coincubated with TGF-β1. Of note, TGF-β1 abrogated the amount of total P2X7 protein and mRNA induced by IFN-γ and LPS. Finally, TGF-β1 prevented the up-regulation of cell-surface CD86, but not other differentiation markers (CD14 and MHC class II), by IFN-γ and LPS. Collectively, these results indicate that TGF-β1 prevents the upregulation of P2X7 by IFN-γ and LPS in THP-1 monocytes by preventing P2X7 transcription and subsequent translation. Moreover, this effect of TGF-β1 is not due to general impairment of THP-1 cell differentiation by IFN-γ and LPS. This suggests that TGF-β1 may limit P2X7-mediated processes during inflammation and immunity, as well as in cancer cells. Previous work by others has indicated that ATP and 2(3’)-O-(4-benzoylbenzoyl) adenosine 5’-triphosphate (BzATP) induces the death of the human epithelial colon carcinoma cell lines, HCT-8 and Caco-2 in a manner characteristic of P2X7 activation. However a direct role for this receptor in this process was not established. Therefore, the second part of my study aimed to determine if HCT-8 and Caco-2 cells expressed functional P2X7. Cell-surface and total P2X7 was examined by immunofluorescence staining and immunoblotting respectively. P2X7 function was evaluated by ATP- and BzATP-induced ethidium+ uptake measurements. Reduction in cell numbers, as an indirect measure of cell death, was evaluated using a tetrazolium-based colorimetric assay. The human multiple myeloma cell line, RPMI 8226, was used as a positive control. HCT-8 and Caco-2 cells expressed low levels of cell-surface P2X7. Whole lysates of these cells expressed low levels of the full length (75 kDa) P2X7, but higher levels of a 42 kDa P2X7 variant. In contrast, RPMI 8226 cells expressed relatively high levels of cell-surface P2X7, as well as full-length P2X7. ATP and BzATP consistently failed to induce ethidium+ uptake in HCT-8 and Caco-2 despite extended incubation times of up to 30 min, and the use of several media known to potentiate P2X7 activation. ATP and BzATP also failed to cause significant death in both the cell lines. In contrast, ATP and BzATP induced ethidium+ uptake and death in RPMI 8226 cells. IFN-γ failed to upregulate P2X7 expression and function in HCT-8 cells. Collectively, these results indicate the absence of functional P2X7 in HCT-8 and Caco-2 cells despite the presence of low levels of cell-surface P2X7. The high proportion of a 42 kDa P2X7 variant provides a possible explanation for the lack of functional P2X7 in these cells. The presence of functional P2X7 in primary myeloid leukemias has been reported by others; however cell line models of myeloid leukemia that constitutively expresses functional P2X7 are limited. Therefore, the final part of my study aimed to determine if the human myeloid leukemic cell line KG-1 expresses functional P2X7. Cell-surface P2X7 was examined by immunofluorescence staining. Total P2X7 protein and mRNA was assessed by immunoblotting and RT-PCR respectively. P2X7 function was evaluated by ATP-induced ethidium+ uptake measurements. Reduction in cell numbers was evaluated using a tetrazolium-based colorimetric assay and cell death was confirmed using a trypan blue exclusion assay and morphological analysis. KG-1 cells expressed low levels of cell-surface P2X7, as well as low levels of P2X7 protein and mRNA. Both, ATP and BzATP consistently induced ethidium+ uptake in KCl and sucrose but not NaCl medium. ATP-induced ethidium+ uptake in a concentration dependant manner with a maximal response at 100 μM and with an EC50 of 8.5 μM which is ~ 10 fold lower than that reported for recombinant P2X7-induced cation fluxes. Moreover, the non-P2X7 agonist, α-β-methyl adenosine 5’-triphosphate (αβmeATP), as well as ATP, BzATP and adenosine 5\u27-O-[3-thiotriphosphate] (ATP-γ-S) induced ethidium+ uptake in KG-1 cells. The P2X7 antagonists, KN-62, AZ10606120 and A- 438079, each impaired ATP-induced ethidium+ uptake. ATP induced KG-1 cell death and morphological analysis indicated that this process was characteristic of apoptosis. ATP-induced apoptosis was impaired by KN-62 and AZ10606120. Collectively, these results indicate that KG-1 cells express functional P2X7 with an atypical pharmacological profile

Human Colonic Organoids: An Epithelial Barrier Model of Crohn's Disease

It’s unknown if the increased intestinal permeability in patients with Crohn’s Disease (CD) is a cause or consequence of inflammation. In vivo, interactions between the microbiota, epithelium and immune system prevent investigation of the intrinsic properties of each component. Therefore, in this study colonic organoids have been used to first, determine their suitability as a model of the colonic epithelium, second to model the impact of bacterial components and pro-inflammatory cytokines on the paracellular barrier and thirdly to determine if the permeability of the intestinal epithelium is altered in patients with CD independent of the effects of the immune system and luminal bacteria Colonic organoids were grown from crypts isolated from transverse colonic biopsies from healthy and patients with CD. Ultrastructure of epithelial cells was determined by transmission electron microscopy. Transcript levels of proteins were determined by qPCR and the quantification and localisation of these proteins were determined by immunoblotting and immunofluorescent microscopy. Permeability of the epithelium was determined by FITC-dextran uptake into colonic organoids. Proliferation was analysed and measured by Ki67 staining and apoptosis by TUNEL staining. Statistical significance was determined by an unpaired or paired Student’s t-test or by a one-way ANOVA with a Bonferroni posthoc test. Spontaneously maturing day 15 colonoids have a polarized well-differentiated epithelium with well-developed tight junctions (TJs), similar to the native epithelium. Four-day-old colonospheres, in which the epithelium is functionally but not structurally polarised, had a cytoplasmic localisation of TJs. Exposure to lipopolysaccharide, but not lipoteichoic acid or muramyl dipeptide (20ng/mL) induced goblet cell development but had no effect on the TJs or epithelial permeability. However, pro-inflammatory cytokines, TNF-alpha (100ng/mL) and IFN-gamma (10ng/mL) increased the epithelial permeability and this was associated with loss of barrier-forming (occludin) and adaptor TJ proteins (ZO-1) and a parallel increase in the pore-forming TJ protein (claudin-2). Colonic organoids grown from patients with CD had an inherent defect in the development of the epithelium, with an apparent loss of polarity of the epithelium, evident as a redistribution of actin and E-cadherin from the membrane into the cytoplasm and a loss of NKCC1. This was associated with a redistribution of occludin and ZO-1, from the junction to the basolateral membrane and into the cytoplasm. Additionally, claudin-2 was concentrated in the TJ of the CD colonoids, whereas in control colonoids it was distributed throughout the basolateral membrane. Significantly, the epithelium of the CD colonoids was (p<0.05) more permeable than the epithelium of control colonoids. The native epithelium and colonic organoids derived from patients with CD had a significant downregulation of p120, a cytoplasmic regulatory protein associated with the stabilization of TJs, actin and E-cadherin. These data indicate that colonic organoids are a suitable model of the colonic epithelium and demonstrate that the colonic epithelium of patients with CD has an inherent permeability defect due to redistribution of the junctional proteins possibly as a consequence of the loss of p120. This occurs independent of the bacteria and immune system, suggesting that the increased epithelial permeability is a cause not a consequence of inflammation in CD

P2X7 receptor activation mediates organic cation uptake into human myeloid leukaemic KG-1 cells

The P2X7 purinergic receptor is an ATP-gated cation channel with an emerging role in neoplasia. In this study we demonstrate that the human KG-1 cell line, a model of acute myelogenous leukaemia, expresses functional P2X7. RT-PCR and immunochemical techniques demonstrated the presence of P2X7 mRNA and protein respectively in KG-l cells, as well as in positive control multiple myeloma RPMI 8226 cells. Flow cytometric measurements demonstrated that ATP induced ethidium(+) uptake into KG-l cells suspended in sucrose medium (EC(50) of ∼3 μM), but not into cells in NaCl medium. In contrast, ATP induced ethidium(+) uptake into RPMI 8226 cells suspended in either sucrose or NaCl medium (EC(50) of ∼3 or ∼99 μM, respectively), as well as into RPMI 8226 cells in KCl medium (EC(50) of ∼18 μM). BzATP and to a lesser extent ATPγS and αβ-methylene ATP, but not ADP or UTP, also induced ethidium(+) uptake into KG-1 cells. ATP-induced ethidium(+) uptake was completely impaired by the P2X7 antagonists, AZ10606120 and A-438079. ATP-induced ethidium(+) uptake was also impaired by probenecid but not by carbenoxolone, both pannexin-1 antagonists. ATP induced YO-PRO-1(2+) and propidium(2+) uptake into KG-1 cells. Finally, sequencing of full-length P2X7 cDNA identified several single nucleotide polymorphisms (SNPs) in KG-1 cells including H155Y, A348T, T357S and Q460R. RPMI 8226 cells contained A348T, A433V and H521Q SNPs. In conclusion, the KG-1 cell line expresses functional P2X7. This cell line may help elucidate the signalling pathways involved in P2X7-induced survival and invasiveness of myeloid leukaemic cells

TGF-beta1 prevents up-regulation of the P2X7 receptor by IFN-gamma and LPS inleukemic THP-1 monocytes

AbstractThe P2X7 receptor is an extracellular ATP-gated cation channel critical in inflammation and immunity, and can be up-regulated by IFN-γ and LPS. This study aimed to examine the effect of TGF-β1 on the up-regulation of P2X7 function and expression in leukemic THP-1 monocytes differentiated with IFN-γ and LPS. Cell-surface molecules including P2X7 were examined by immunofluorescence staining. Total P2X7 protein and mRNA was assessed by immunoblotting and RT-PCR respectively. P2X7 function was evaluated by ATP-induced cation dye uptake measurements. Cell-surface P2X7 was present on THP-1 cells differentiated for 3days with IFN-γ and LPS but not on undifferentiated THP-1 cells. ATP induced ethidium+ uptake into differentiated but not undifferentiated THP-1 cells, and the P2X7 antagonist, KN-62, impaired ATP-induced ethidium+ uptake. Co-incubation of cells with TGF-β1 plus IFN-γ and LPS prevented the up-regulation of P2X7 expression and ATP-induced ethidium+ uptake in a concentration-dependent fashion with a maximum effect at 5ng/ml and with an IC50 of ~0.4ng/ml. Moreover, ATP-induced YO-PRO-12+ uptake and IL-1β release were abrogated in cells co-incubated with TGF-β1. TGF-β1 also abrogated the amount of total P2X7 protein and mRNA induced by IFN-γ and LPS. Finally, TGF-β1 prevented the up-regulation of cell-surface CD86, but not CD14 and MHC class II, by IFN-γ and LPS. These results indicate that TGF-β1 prevents the up-regulation of P2X7 function and expression by IFN-γ and LPS in THP-1 monocytes. This suggests that TGF-β1 may limit P2X7-mediated processes in inflammation and immunity

Sprouty2 limits intestinal tuft and goblet cell numbers through GSK3β-mediated restriction of epithelial IL-33

Dynamic regulation of colonic secretory cell numbers is a critical component of the response to intestinal injury and inflammation. Here, the authors show that loss of the intracellular signalling regulator Sprouty2 in the intestinal epithelial cells is a protective response to injury that leads to increased secretory cell numbers, thus limiting colitis severity