Genetic-Background Modulation of Core and Variable Autistic-Like Symptoms in Fmr1 Knock-Out Mice

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Modulation of Chromatin Remodelling Induced by the Freshwater Cyanotoxin Cylindrospermopsin in Human Intestinal Caco-2 Cells

International audienceCylindrospermopsin (CYN) is a cyanotoxin that has been recognised as an emerging potential public health risk. Although CYN toxicity has been demonstrated, the mechanisms involved have not been fully characterised. To identify some key pathways related to this toxicity, we studied the transcriptomic profile of human intestinal Caco-2 cells exposed to a sub-toxic concentration of CYN (1.6 mM for 24hrs) using a non-targeted approach. CYN was shown to modulate different biological functions which were related to growth arrest (with down-regulation of cdkn1a and uhrf1 genes), and DNA recombination and repair (with up-regulation of aptx and pms2 genes). Our main results reported an increased expression of some histone-modifying enzymes (histone acetyl and methyltransferases MYST1, KAT5 and EHMT2) involved in chromatin remodelling, which is essential for initiating transcription. We also detected greater levels of acetylated histone H2A (Lys5) and dimethylated histone H3 (Lys4), two products of these enzymes. In conclusion, CYN overexpressed proteins involved in DNA damage repair and transcription, including modifications of nucleosomal histones. Our results highlighted some new cell processes induced by CYN

Sweat Chloride Testing and Nasal Potential Difference (NPD) Are Primary Outcome Parameters in Treatment with Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Modulators

With the advent of CFTR modulators, surrogate outcome parameters that accurately quantify the improvement in CFTR activity are needed. In vivo biomarkers that reflect CFTR ion transport and can serve as outcomes in the treatment of CFTR modulators are the sweat Cl− test (SCT), the nasal potential difference (NPD) measurement or the intestinal current measurement (ICM). This review focus on the SCT and NPD. The SCT displays a low intra-patient variability in contrast to the NPD. It has been used extensively as a biomarker of CFTR function in clinical trials of CFTR modulator therapies and provides evidence for change in the short term. The level of functional rescue in the NPD increases up to 40% of normal CFTR in patients with a Gly551Asp treated with ivacaftor monotherapy, while in F508del homozygous patients treated with ivacaftor-lumacaftor, activity increased on average up to ~20% of normal activity. While both tests provide evidence of the effect on CFTR activity, they cannot be used at an individual level to predict the response to any CFTR modulators. Nevertheless, their rapid modification, reflecting electrophysiological properties, highlight their potential use in proof-of-concept studies for CFTR modulators

Pseudomonas aeruginosa LasB protease impairs innate immunity in mice and humans by targeting a lung epithelial cystic fibrosis transmembrane regulator–IL-6–antimicrobial–repair pathway

Background Pseudomonas aeruginosa lung infections are a huge problem in ventilator-associated pneumonia, cystic fibrosis (CF) and in chronic obstructive pulmonary disease (COPD) exacerbations. This bacterium secretes virulence factors that may subvert host innate immunity.Objective We evaluated the effect of P. aeruginosa elastase LasB, an important virulence factor secreted by the type II secretion system, on ion transport, innate immune responses and epithelial repair, both in vitro and in vivo.Methods Wild-type (WT) or cystic fibrosis transmembrane conductance regulator (CFTR)-mutated epithelial cells (cell lines and primary cells from patients) were treated with WT or Delta LasB pseudomonas aeruginosa O1 (PAO1) secretomes. The effect of LasB and PAO1 infection was also assessed in vivo in murine models.Results We showed that LasB was the most abundant protein in WT PAO1 secretomes and that it decreased epithelial CFTR expression and activity. In airway epithelial cell lines and primary bronchial epithelial cells, LasB degraded the immune mediators interleukin (IL)-6 and trappin-2, an important epithelial-derived antimicrobial molecule. We further showed that an IL-6/STAT3 signalling pathway was downregulated by LasB, resulting in inhibition of epithelial cell repair. In mice, intranasally instillated LasB induced significant weight loss, inflammation, injury and death. By contrast, we showed that overexpression of IL-6 and trappin-2 protected mice against WT-PAO1-induced death, by upregulating IL-17/IL-22 antimicrobial and repair pathways.Conclusions Our data demonstrate that PAO1 LasB is a major P. aeruginosa secreted factor that modulates ion transport, immune response and tissue repair. Targeting this virulence factor or upregulating protective factors such as IL-6 or antimicrobial molecules such as trappin-2 could be beneficial in P. aeruginosa-infected individuals

Relative gene expression of <i>polr2d</i>, <i>polr2l</i>, <i>med6</i>, <i>ddx20</i>, <i>kat5</i>, <i>myst1</i> and <i>ehmt2</i> in differentiated Caco-2 cells after 24 hrs exposure to 1.6 µM CYN.

<p>Values are presented as means ± SE, and normalised to the reference gene <i>RPLP0</i>. Six independent experiments were performed.</p><p>^{*, **}: significantly different from the control group (respectively <i>P</i><0.005 and <i>P</i><0.001).</p

Tree of the biological processes up-regulated in differentiated Caco-2 cells after CYN exposure.

<p>The 522 genes showing up-regulation after 24 hrs exposure to 1.6 µM CYN were annotated within 22 biological processes (GO terms) with an enrichment score greater than 1.5. GO terms had a false discovery rate score of between 0.05 and 0.01 (in orange) or less than 0.01 (in red); white GO terms were ancestors.</p

Associated network functions in differentiated Caco-2 cells after 24 hrs exposure to 1.6 µM CYN.

<p>The 2911 genes showing differential regulation were associated within networks using Ingenuity Pathway Analysis. In this figure the two networks with the highest score are presented. They are respectively associated with gene expression and RNA Post-Transcriptional Modification (A), or with gene expression (B) biological functions. Lines between gene products represent known interactions, with solid lines representing direct interactions and dashed lines representing indirect interactions. Genes showing up-regulation of expression levels in response to CYN exposure are in red, while down-regulated genes are in green.</p

Cytotoxic effects of CYN in differentiated Caco-2 cells treated for 24 hrs.

<p>Cytotoxicity effect was measured by neutral red uptake assay. Values are presented as means ± SE, and expressed as percentages of the vehicle control. Three independent experiments were performed. *, $: values are (*, <i>P</i><0.05) or tended ($, <i>P</i><0.1) to be different from 100.</p

An unexpected effect of TNF-α on F508del-CFTR maturation and function

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An unexpected effect of TNF-α on F508del-CFTR maturation and function [v1; ref status: indexed, http://f1000r.es/5jf]

Cystic fibrosis (CF) is a multifactorial disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR), which encodes a cAMP-dependent Cl- channel. The most frequent mutation, F508del, leads to the synthesis of a prematurely degraded, otherwise partially functional protein. CFTR is expressed in many epithelia, with major consequences in the airways of patients with CF, characterized by both fluid transport abnormalities and persistent inflammatory responses. The relationship between the acute phase of inflammation and the expression of wild type (WT) CFTR or F508del-CFTR is poorly understood. The aim of the present study was to investigate this effect. The results show that 10 min exposure to TNF-alpha (0.5-50ng/ml) of F508del-CFTR-transfected HeLa cells and human bronchial cells expressing F508del-CFTR in primary culture (HBE) leads to the maturation of F508del-CFTR and induces CFTR chloride currents. The enhanced CFTR expression and function upon TNFα is sustained, in HBE cells, for at least 24 h. The underlying mechanism of action involves a protein kinase C (PKC) signaling pathway, and occurs through insertion of vesicles containing F508del-CFTR to the plasma membrane, with TNFα behaving as a corrector molecule. In conclusion, a novel and unexpected action of TNFα has been discovered and points to the importance of systematic studies on the roles of inflammatory mediators in the maturation of abnormally folded proteins in general and in the context of CF in particular