RESUMEN

Objectives: Crohn's disease (CD) initiation and pathogenesis are believed to involve an environmental trigger in a genetically susceptible person that results in an immune response against commensal gut bacteria, leading to a compromised intestinal epithelial barrier and a cycle of inflammation. However, it has been difficult to study the contribution of all factors together in a physiologically relevant model and in a heterogenous patient population. Methods: We developed an autologous colonic monolayer model that incorporated the immune response from the same donor and a commensal bacteria, Faecalibacterium prausnitzii. Two-dimensional monolayers were grown from three-dimensional organoids generated from intestinal biopsies, and the epithelial integrity of the epithelium was measured using transepithelial electrical resistance. We determined the effect of immune cells alone, bacteria alone and the co-culture of immune cells and bacteria on integrity. Results: Monolayers derived from CD donors had impaired epithelial integrity compared to those from non-inflammatory bowel disease (IBD) donors. This integrity was further impaired by culture with bacteria, but not immune cells, despite a higher frequency of inflammatory phenotype peripheral T cells in CD donors. Variability in epithelial integrity was higher in CD donors than in non-IBD donors. Conclusion: We have developed a new autologous model to study the complexity of CD, which allows for the comparison of the barrier properties of the colonic epithelium and the ability to study how autologous immune cells directly affect the colonic barrier and whether this is modified by luminal bacteria. This new model allows for the study of individual patients and could inform treatment decisions.

RESUMEN

OBJECTIVE: Human intestinal organoids (hIOs) have potential as a model for investigating intestinal diseases. The hIO system faces logistic challenges including limited access to biopsies or low expression of epithelial cell types. Previous research identified the feasibility of tissue from the transverse (TC) or sigmoid colon (SC), or from cryopreserved biopsies from regions of the gastrointestinal tract. We aimed to create a protocol for robust hIO generation that could be implemented across multiple centres, allowing for development of a consistent biobank of hIOs from diverse patients. RESULTS: TC and SC hIOs were expanded from fresh or frozen biopsies with standard or refined media. The expression of epithelial cells was evaluated via PCR. Growth of TC and SC hIO from healthy donors was reproducible from freshly acquired and frozen biopsies. A refined media including insulin-like growth factor (IGF)-1 and fibroblast growth factor (FGF)-2 enabled the expression of epithelial cells, including higher expression of goblet cells and enterocytes compared to standard organoid media. We identified a consistent time point where hIOs generated from frozen biopsies reflect similar hIO composition from freshly acquired samples. Feasibility of hIOs as a tool for research and clinical use, including the use of frozen biopsies, was demonstrated.

Asunto(s)

Mucosa Intestinal , Organoides , Células Epiteliales , Humanos , Intestinos

RESUMEN

Intestinal mucosal healing is the primary therapeutic goal of medical treatments for inflammatory bowel disease (IBD). Epithelial stem cells are key players in the healing process. Lgr5+ stem cells maintain cellular turnover during homeostasis in the colonic crypt. However, they are lost and dispensable for repair in a wide variety of injury models, including dextran sulfate sodium (DSS) colitis, radiation, helminth infection, and T-cell activation. The direct loss of Lgr5+ cells activates a plasticity response in the epithelium in which other cell types can serve as stem cells. Whether this paradigm applies to mouse models of IBD remains unknown. In contrast to previously tested models, IBD models involve an inflammatory response rooted in the loss of immunologic tolerance to intestinal luminal contents including the microbiome. Here, we show the persistence of Lgr5+ cells in oxazolone, 2,4,6-trinitrobenzene sulfonic acid (TNBS), and Il10-/-, and Il10-/- Tnfr1-/- IBD models. This contrasts with results obtained from DSS-induced injury. Through high-throughput expression profiling, we find that these colitis models were associated with distinct patterns of cytokine expression. Direct exposure of colonic epithelial organoids to DSS, oxazolone, or TNBS resulted in increased apoptosis and loss of Lgr5+ cells. Targeted ablation of Lgr5+ cells resulted in severe exacerbation of chronic, antibody-induced IL-10-deficient colitis, but had only modest effects in TNBS-induced colitis. These results show that distinct mouse models of IBD-like colitis induce different patterns of Lgr5+ stem cell retention and function.NEW & NOTEWORTHY Acute intestinal injury and epithelial repair are associated with the loss of fast-cycling Lgr5+ stem cells and plasticity in the activation of formerly quiescent cell populations. In contrast, here we show in murine inflammatory bowel disease the persistence of the Lgr5+ stem cell population and its essential role in restricting the severity of chronic colitis. This demonstrates a diversity of stem cell responses to colitis.

Asunto(s)

Colitis/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células Madre/citología , Animales , Proliferación Celular/fisiología , Modelos Animales de Enfermedad , Epitelio/metabolismo , Homeostasis/fisiología , Mucosa Intestinal/metabolismo , Ratones , Regeneración/fisiología , Células Madre/metabolismo

RESUMEN

Dynamic regulation of intestinal cell differentiation is crucial for both homeostasis and the response to injury or inflammation. Sprouty2, an intracellular signaling regulator, controls pathways including PI3K and MAPKs that are implicated in differentiation and are dysregulated in inflammatory bowel disease. Here, we ask whether Sprouty2 controls secretory cell differentiation and the response to colitis. We report that colonic epithelial Sprouty2 deletion leads to expanded tuft and goblet cell populations. Sprouty2 loss induces PI3K/Akt signaling, leading to GSK3ß inhibition and epithelial interleukin (IL)-33 expression. In vivo, this results in increased stromal IL-13+ cells. IL-13 in turn induces tuft and goblet cell expansion in vitro and in vivo. Sprouty2 is downregulated by acute inflammation; this appears to be a protective response, as VillinCre;Sprouty2F/F mice are resistant to DSS colitis. In contrast, Sprouty2 is elevated in chronic colitis and in colons of inflammatory bowel disease patients, suggesting that this protective epithelial-stromal signaling mechanism is lost in disease.

Asunto(s)

Colitis/genética , Glucógeno Sintasa Quinasa 3 beta/genética , Homeostasis/genética , Interleucina-33/genética , Proteínas de la Membrana/genética , Proteínas Serina-Treonina Quinasas/genética , Animales , Recuento de Células , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Niño , Colitis/inducido químicamente , Colitis/metabolismo , Colitis/patología , Colon/efectos de los fármacos , Colon/metabolismo , Colon/patología , Femenino , Regulación de la Expresión Génica , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Células Caliciformes/efectos de los fármacos , Células Caliciformes/metabolismo , Células Caliciformes/patología , Células HT29 , Homeostasis/efectos de los fármacos , Humanos , Interleucina-33/metabolismo , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Dodecil Sulfato de Sodio/administración & dosificación

RESUMEN

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 αß-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.

Asunto(s)

Adenosina Trifosfato/metabolismo , Células Mieloides/metabolismo , Antagonistas del Receptor Purinérgico P2X/farmacología , Receptores Purinérgicos P2X7/metabolismo , Cationes/metabolismo , Línea Celular , Humanos , Polimorfismo de Nucleótido Simple/genética

RESUMEN

The 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-ß1 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-ß1 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 IC(50) of ~0.4ng/ml. Moreover, ATP-induced YO-PRO-1(2+) uptake and IL-1ß release were abrogated in cells co-incubated with TGF-ß1. TGF-ß1 also abrogated the amount of total P2X7 protein and mRNA induced by IFN-γ and LPS. Finally, TGF-ß1 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-ß1 prevents the up-regulation of P2X7 function and expression by IFN-γ and LPS in THP-1 monocytes. This suggests that TGF-ß1 may limit P2X7-mediated processes in inflammation and immunity.

Asunto(s)

Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Monocitos/efectos de los fármacos , Receptores Purinérgicos P2/efectos de los fármacos , Factor de Crecimiento Transformador beta1/farmacología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Adenosina Trifosfato/farmacología , Antígenos CD/análisis , Apirasa/análisis , Antígeno B7-2/análisis , Células Cultivadas , Humanos , Receptores de Lipopolisacáridos/análisis , Monocitos/química , Receptores Purinérgicos P2/análisis , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/fisiología , Receptores Purinérgicos P2X7 , Regulación hacia Arriba

RESUMEN

BACKGROUND: The extracellular ATP-gated cation channel, P2X7 receptor, has an emerging role in neoplasia, however progress in the field is limited by a lack of malignant cell lines expressing this receptor. METHODS: Immunofluorescence labelling and a fixed-time ATP-induced ethidium+ uptake assay were used to screen a panel of human malignant cell lines for the presence of functional P2X7. The presence of P2X7 was confirmed by RT-PCR, immunoblotting and pharmacological approaches. ATP-induced cell death was measured by colourimetric tetrazolium-based and cytofluorometric assays. ATP-induced CD23 shedding was measured by immunofluorescence labelling and ELISA. RESULTS: RPMI 8226 multiple myeloma cells expressed P2X7 mRNA and protein, as well as P2X1, P2X4 and P2X5 mRNA. ATP induced ethidium+ uptake into these cells with an EC50 of ~116 µM, and this uptake was reduced in the presence of extracellular Ca²+ and Mg²+. The P2X7 agonist 2'- and 3'-0(4-benzoylbenzoyl) ATP, but not UTP, induced ethidium+ uptake. ATP-induced ethidium+ uptake was impaired by the P2X7 antagonists, KN-62 and A-438079. ATP induced death and CD23 shedding in RPMI 8226 cells, and both processes were impaired by P2X7 antagonists. The metalloprotease antagonists, BB-94 and GM6001, impaired ATP-induced CD23 shedding but not ethidium+ uptake. CONCLUSIONS: P2X7 receptor activation induces cell death and CD23 shedding in RPMI 8226 cells. GENERAL SIGNIFICANCE: RPMI 8226 cells may be useful to study the role of P2X7 in multiple myeloma and B-lymphocytes.

Asunto(s)

Apoptosis , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Receptores de IgE/metabolismo , Receptores Purinérgicos P2/metabolismo , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Etidio/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Mieloma Múltiple/genética , ARN Mensajero/genética , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X7 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas