Tezat

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Western Star (Corner Brook, N.L.), 1922-02-08

The Western Star began publication on Newfoundland's west coast on 4 April 1900, appearing weekly with brief semiweekly periods up to 1952, when it became a daily. The current collection contains 21 April 1900 - 31 December 1952

Type I and Type III Interferons Display Different Dependency on Mitogen-Activated Protein Kinases to Mount an Antiviral State in the Human Gut

Intestinal epithelial cells (IECs) are constantly exposed to commensal flora and pathogen challenges. How IECs regulate their innate immune response to maintain gut homeostasis remains unclear. Interferons (IFNs) are cytokines produced during infections. While type I IFN receptors are ubiquitously expressed, type III IFN receptors are expressed only on epithelial cells. This epithelium specificity strongly suggests exclusive functions at epithelial surfaces, but the relative roles of type I and III IFNs in the establishment of an antiviral innate immune response in human IECs are not clearly defined. Here, we used mini-gut organoids to define the functions of types I and III IFNs to protect the human gut against viral infection. We show that primary non-transformed human IECs, upon viral challenge, upregulate the expression of both type I and type III IFNs at the transcriptional level but only secrete type III IFN in the supernatant. However, human IECs respond to both type I and type III IFNs by producing IFN-stimulated genes that in turn induce an antiviral state. Using genetic ablation of either type I or type III IFN receptors, we show that either IFN can independently restrict virus infection in human IECs. Importantly, we report, for the first time, differences in the mechanisms by which each IFN establishes the antiviral state. Contrary to type I IFN, the antiviral activity induced by type III IFN is strongly dependent on the mitogen-activated protein kinases signaling pathway, suggesting a pathway used by type III IFNs that non-redundantly contributes to the antiviral state. In conclusion, we demonstrate that human intestinal epithelial cells specifically regulate their innate immune response favoring type III IFN-mediated signaling, which allows for efficient protection against pathogens without producing excessive inflammation. Our results strongly suggest that type III IFN constitutes the frontline of antiviral response in the human gut. We propose that mucosal surfaces, particularly the gastrointestinal tract, have evolved to favor type III IFN-mediated response to pathogen infections as it allows for spatial segregation of signaling and moderate production of inflammatory signals which we propose are key to maintain gut homeostasis

Initiation of an Inflammatory Response in Resident Intestinal Lamina Propria Cells -Use of a Human Organ Culture Model

<div><p>Resident human lamina propria immune cells serve as powerful effectors in host defense. Molecular events associated with the initiation of an intestinal inflammatory response in these cells are largely unknown. Here, we aimed to characterize phenotypic and functional changes induced in these cells at the onset of intestinal inflammation using a human intestinal organ culture model. In this model, healthy human colonic mucosa was depleted of epithelial cells by EDTA treatment. Following loss of the epithelial layer, expression of the inflammatory mediators <i>IL1B, IL6, IL8, IL23A, TNFA, CXCL2</i>, and the surface receptors CD14, TLR2, CD86, CD54 was rapidly induced in resident lamina propria cells <i>in situ</i> as determined by qRT-PCR and immunohistology. Gene microarray analysis of lamina propria cells obtained by laser-capture microdissection provided an overview of global changes in gene expression occurring during the initiation of an intestinal inflammatory response in these cells. Bioinformatic analysis gave insight into signalling pathways mediating this inflammatory response. Furthermore, comparison with published microarray datasets of inflamed mucosa <i>in vivo</i> (ulcerative colitis) revealed a significant overlap of differentially regulated genes underlining the <i>in vivo</i> relevance of the organ culture model. Furthermore, genes never been previously associated with intestinal inflammation were identified using this model. The organ culture model characterized may be useful to study molecular mechanisms underlying the initiation of an intestinal inflammatory response in normal mucosa as well as potential alterations of this response in inflammatory bowel disease.</p></div

Expression of surface receptors in resident lamina propria cells following loss of the epithelial layer.

<p>(<b>A</b>) LEL induces gene expression of <i>CD14</i>, <i>CD86</i>, <i>TLR2</i>, and <i>CD54</i> in resident lamina propria cells <i>in situ</i>. Tissue samples of total mucosa (TM) were collected prior to culturing (0 h TM) and simultaneously from both TM and LEL mucosa after completion of epithelial cell release by EDTA treatment (5 h TM, 5 h LEL-M) (see Fig. 1a). Subsequently, transcript levels of <i>CD14</i>, <i>TLR2</i>, <i>CD54</i>, and <i>CD86</i> were determined by qRT-PCR. Transcript numbers (normalized to 1000 transcripts <i>PPIB</i>) of “5 h LEL-M” were set to 1, and those of all other conditions were calculated as a fraction/multiple of 1. Shown are the mean normalized transcript numbers ± SEM of three or four independent experiments. Numbers in brackets indicate the absolute transcript numbers normalized to 1000 transcripts <i>PPIB</i>. Gray bars represent transcript levels of TM (0 h, 5 h), black bars represent transcript levels of LEL-M (5 h). (<b>B</b>) LEL induces protein expression of CD14 and CD86 in resident lamina propria cells <i>in situ</i>. Double immunofluorescence staining of CD68 (green) and CD14 or CD86 (red) in healthy total mucosa prior to culturing (TM t = 0 h) and after 5 h of culture (TM t = 5 h) as well as in mucosa immediately after LEL (LEL-M t = 5 h). Colocalization of both antigens is shown by yellow signals in the overlay. Magnification: x40. LEL enhances the expression of CD14 and CD86 on some CD68⁺ and a large pool of CD68⁻ cells. The results represent one of three independent experiments. (<b>C</b>) Quantification of CD14⁺ and CD86⁺ cells in TM 5 h and LEL-M 5 h, respectively. The mean numbers (± SEM) of CD14⁺ and CD86⁺ cells, respectively, per area of 100 CD68⁺ cells were determined with three areas/experimental condition being evaluated. Shown are the results of two independent experiments.</p

Early inflammatory gene expression profile of resident lamina propria cells.

<p>Frozen tissue samples collected prior to culturing (TM 0 h) and immediately after release of the epithelial layer (LEL-M 5 h) were subjected to laser-capture microdissection of the lamina propria. Following RNA extraction, global gene expression profiles of lamina propria cells were obtained by microarray analysis. Shown is a heatmap of significantly regulated genes in LEL-LP 5 h vs. TM-LP 0 h (four matched replicates (R1–R4) for each time point replicates representing four independent experiments). Colors show expression relative to the average (black), higher (yellow), or lower (blue) relative expression, respectively. Data have been transformed to gene-wise zero mean and unit variance (z transform). Rows have been re-ordered by hierarchical clustering (complete linkage, Euclidean distance metrics).</p

Induction of inflammatory gene expression in resident lamina propria cells following loss of the epithelial layer.

<p>(<b>A</b>) Time scheme of the LEL model. Arrows indicate time points of tissue collection. Tissue samples were collected prior to culturing (TM t = 0 h), after washing (TM t = 2 h), as well during and after completion of epithelial cell release by EDTA treatment (LEL-M t = 3/4/5 h). As a control, tissue samples were collected from TM cultured for 5 h (TM t = 5 h). (<b>B</b>) LEL induces gene expression of <i>IL1B</i>, <i>IL6</i>, <i>IL8</i>, <i>IL23A</i>, <i>TNFA, IFNG</i>, and <i>CCL2</i> in resident lamina propria cells. Transcript levels of cytokines/chemokines were determined by qRT-PCR in tissue samples collected as described in (A). Shown are the mean normalized transcript numbers ± SEM of at least 4 independent experiments. Gray bars represent transcript levels of TM (t = 0/2/5 h), black bars represent transcript levels of LEL-M (t = 3/4/5h). (<b>C</b>) EDTA treatment does not induce inflammatory cytokines in PBMC or LPMC. PBMC and LPMC, respectively, were exposed to 0.7 mM EDTA/HBSS or medium (RPMI/2% FCS) for 3 h. Subsequently, transcript levels of <i>IL6</i>, <i>IL8</i>, <i>IL1B</i>, and <i>IL23A</i> were determined by qRT-PCR (<i>IL8</i> and <i>IL23A</i> not tested for LPMC). The results of two independent experiments are shown.</p

Top 40 upregulated genes in the LEL model.

1<p>log base 2 of fold change.</p>2<p>P value from moderated t test, adjusted by the Benjamini & Hochberg method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097780#pone.0097780-Benjamini1" target="_blank">[18]</a>.</p

Over-representation of GO terms in dataset of upregulated genes in the LEL model.

1<p>P value of hypergeometric test for over-representation (conditional on GO structure).</p>2<p>Count: number of GO term associated genes in the dataset of upregulated genes.</p>3<p>Size: total number of GO term associated genes.</p