Systems analysis of the transcriptional response of human ileocecal epithelial cells to Clostridium difficile toxins and effects on cell cycle control

<p>Abstract</p> <p>Background</p> <p>Toxins A and B (TcdA and TcdB) are <it>Clostridium difficile</it>'s principal virulence factors, yet the pathways by which they lead to inflammation and severe diarrhea remain unclear. Also, the relative role of either toxin during infection and the differences in their effects across cell lines is still poorly understood. To better understand their effects in a susceptible cell line, we analyzed the transciptome-wide gene expression response of human ileocecal epithelial cells (HCT-8) after 2, 6, and 24 hr of toxin exposure.</p> <p>Results</p> <p>We show that toxins elicit very similar changes in the gene expression of HCT-8 cells, with the TcdB response occurring sooner. The high similarity suggests differences between toxins are due to events beyond transcription of a single cell-type and that their relative potencies during infection may depend on differential effects across cell types within the intestine. We next performed an enrichment analysis to determine biological functions associated with changes in transcription. Differentially expressed genes were associated with response to external stimuli and apoptotic mechanisms and, at 24 hr, were predominately associated with cell-cycle control and DNA replication. To validate our systems approach, we subsequently verified a novel G₁/S and known G₂/M cell-cycle block and increased apoptosis as predicted from our enrichment analysis.</p> <p>Conclusions</p> <p>This study shows a successful example of a workflow deriving novel biological insight from transcriptome-wide gene expression. Importantly, we do not find any significant difference between TcdA and TcdB besides potency or kinetics. The role of each toxin in the inhibition of cell growth and proliferation, an important function of cells in the intestinal epithelium, is characterized.</p

Effect of clostridium toxins on intestinal epithelial cell morphology

[ES] El trabajo aquí descrito se denomina “Efecto de las toxinas de Clostridium sobre la morfología de las células epiteliales intestinales”. Clostridium difficile es una bacteria gram positiva que causa trastornos intestinales en humanos. Entran en el cuerpo humano a través de los alimentos y se dirigen el epitelio intestinal para invadir los tejidos. Producen dos toxinas (Toxina A y Toxina B) que les ayudan a entrar a través de la barrera epitelial intestinal. Las células Caco-2 se usan comúnmente como sistemas de células modelo para imitar enterocitos intestinales. Así mismo, se está estudiando el efecto de las toxinas de Clostridium usando Caco-2 y ciego de ratón como modelos de epitelios intestinales. El objetivo de esta investigación es comparar estos sistemas modelo y centrarse en las vías conocidas como la ruta de señalización de las Rho GTPasas (mediante Ingenuity Pathway Analysis software), junto con otros genes y vías importantes que afecten la morfología celular. Respecto a esto, la línea de trabajo consiste en realizar una búsqueda bibliográfica para obtener un conjunto de genes que estén afectados por los efectos de las toxinas de Clostridium difficile. A continuación, se procederá a utilizar R (lenguaje de escritura estadístico) y paquetes de Bioconductor (maSigPro) para comprender los cambios que se producen en estos genes. Se manejarán 2 conjuntos de datos diferentes en total: un conjunto de datos de puntos de tiempo (ratones) y datos de un microarray de genes de Caco-2. Comparando la diferencia en la expresión de los genes bajo el efecto de las toxinas y en condiciones basales después de 2 horas y 6 horas en el ciego de ratones y 4,5 horas en Caco2, se han obtenido los primeros reguladores afectados por la infección de toxinas. El siguiente paso fue relacionar estos genes significativos con sus vías de señalización para ver cuáles son los más afectados. Este enfoque de sistemas permitió una visión amplia del efecto de la infección por Clostridium difficile en dos sistemas modelo, ratones y Caco-2. Por otra parte, la comparación del ciego de ratón con Caco2, que son células similares a las del colon, mostraron diversos efectos de las toxinas que son consecuencia de las diferencias entre estos sistemas modelo.[EN] The thesis explained here is called the “Effect of Clostridium Toxins on Intestinal Epithelial Cell Morphology”. Clostridium difficile is a gram-positive bacterium that causes bowel disorders in humans. They enter human body through food and target the intestinal epithelia to gain entry. They produce two toxins (Toxin A and Toxin B) that help them gain entry through the intestinal epithelial barrier. Caco-2 cells are commonly used as model cell systems to mimic intestinal enterocytes. Thus, it is being studied the effect of the clostridium toxins using Caco-2 and mouse cecum as intestinal epithelia models. The aim of this research is to cross compare these model systems and focus on known pathways like the Rho GTPases signaling (from Ingenuity Pathway Analysis software) along with other important genes and pathways that affect cell morphology. In this regard, the work line is a literature search for genes affected by the effect of Clostridium difficile toxins. Then proceed to use R (statistical scripting language) and Bioconductor packages (maSigPro) to understand the changes occurring in these genes. 2 different datasets will be handled in total: one time points data (mice) and one Caco-2 gene microarray data. Comparing the difference in expression of the genes under the effect of the toxins and under basal conditions after 2 hours and 6 hours in mice cecum and 4.5 hours in Caco2 have revealed the first regulators to be affected after toxins infection. Then, we proceed to relate these significant genes to their pathways to see which ones where the most affected. This systems approach allowed a broad view of the effect of Clostridium difficile infection in two model systems, mice and Caco2. Moreover, the comparison of mice cecum and Caco2 colon-like cells showed diverse toxins effects that are a consequence of differences between these systems.Cantó Santos, J. (2017). Effect of clostridium toxins on intestinal epithelial cell morphology. http://hdl.handle.net/10251/86542TFG

The role of intestinal epithelium in inflammatory bowel disease and inflammation related intestinal cancer

The intestinal epithelial cells(IEC) are indispensable factors in the host protection against the harmful luminal content. In this thesis we aimed to gain further insight in the role of IEC in the Inflammatory Bowel Disease(IBD) aetiology, since it is an important mediator between the already known factors that give onset of IBD. To investigate the IEC involvement in IBD we stimulated freshly isolated biopsies from CD patients with PXR ligands to inhibit the NF-κB(inflammatory key regulator) signalling. We have found that the presence of PXR was more important than the actual PXR activation to inhibit the NF-κB activity. It becomes increasingly clear that the function of PXR goes beyond its primary function in the xenobiotic detoxification pathways. We found that high PXR expression is present exclusively in the neoplastic intestinal epithelium, whereas no PXR protein could be detected in normal or inflamed intestinal tissue. In vitro experiments uncovered that this high PXR expression reduces the growth speed of the cells but increases the high cell density survival. Proper functioning IEC are depending on correctly folded (inside the endoplasmic reticulum(ER)) and expressed proteins. We postulate that inflammation related conditions affect ER-dependent proteins in expression, e.g. ABCG2 and initiate ER-stress. As such, ABCG2 expression and function is impeded due to ER-protein folding difficulties. Besides, ER stress in Paneth cells is found in a subset of CD patients carrying an ATG16L1 risk allele(G). This indicates that multiple factors IBD related factors can provoke protein-folding difficulties. Overall we conclude that, the intestinal epithelium has a distinct function in the aetiology of IBD patients on many disease-associated factors. This thesis justifies the extensive investigation of IEC driven therapeutic approaches for IBD patients