Soluble factors released by Bifidobacterium breve C50 (Bb) alleviate the secretion of pro-inflammatory cytokines by immune cells, but their effect on intestinal epithelium remains elusive. To ...decipher the mechanisms accounting for the cross-talk between bacteria/soluble factors and intestinal epithelium, we measured the capacity of the bacteria, its conditioned medium (Bb-CM) and other Gram(+) commensal bacteria to dampen inflammatory chemokine secretion.
TNFalpha-induced chemokine (CXCL8) secretion and alteration of NF-kappaB and AP-1 signalling pathways by Bb were studied by EMSA, confocal microscopy and western blotting. Anti-inflammatory capacity was also tested in vivo in a model of TNBS-induced colitis in mice.
Bb and Bb-CM, but not other commensal bacteria, induced a time and dose-dependent inhibition of CXCL8 secretion by epithelial cells driven by both AP-1 and NF-kappaB transcription pathways and implying decreased phosphorylation of p38-MAPK and IkappaB-alpha molecules. In TNBS-induced colitis in mice, Bb-CM decreased the colitis score and inflammatory cytokine expression, an effect reproduced by dendritic cell conditioning with Bb-CM.
Bb and secreted soluble factors contribute positively to intestinal homeostasis by attenuating chemokine production. The results indicate that Bb down regulate inflammation at the epithelial level by inhibiting phosphorylations involved in inflammatory processes and by protective conditioning of dendritic cells.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Ciliopathies are a group of genetic multi-systemic disorders related to dysfunction of the primary cilium, a sensory organelle present at the cell surface that regulates key signaling pathways during ...development and tissue homeostasis. In order to identify novel genes whose mutations would cause severe developmental ciliopathies, >500 patients/fetuses were analyzed by a targeted high throughput sequencing approach allowing exome sequencing of >1200 ciliary genes. NEK8/NPHP9 mutations were identified in five cases with severe overlapping phenotypes including renal cystic dysplasia/hypodysplasia, situs inversus, cardiopathy with hypertrophic septum and bile duct paucity. These cases highlight a genotype-phenotype correlation, with missense and nonsense mutations associated with hypodysplasia and enlarged cystic organs, respectively. Functional analyses of NEK8 mutations in patient fibroblasts and mIMCD3 cells showed that these mutations differentially affect ciliogenesis, proliferation/apoptosis/DNA damage response, as well as epithelial morphogenesis. Notably, missense mutations exacerbated some of the defects due to NEK8 loss of function, highlighting their likely gain-of-function effect. We also showed that NEK8 missense and loss-of-function mutations differentially affect the regulation of the main Hippo signaling effector, YAP, as well as the expression of its target genes in patient fibroblasts and renal cells. YAP imbalance was also observed in enlarged spheroids of Nek8-invalidated renal epithelial cells grown in 3D culture, as well as in cystic kidneys of Jck mice. Moreover, co-injection of nek8 MO with WT or mutated NEK8-GFP RNA in zebrafish embryos led to shortened dorsally curved body axis, similar to embryos injected with human YAP RNA. Finally, treatment with Verteporfin, an inhibitor of YAP transcriptional activity, partially rescued the 3D spheroid defects of Nek8-invalidated cells and the abnormalities of NEK8-overexpressing zebrafish embryos. Altogether, our study demonstrates that NEK8 human mutations cause major organ developmental defects due to altered ciliogenesis and cell differentiation/proliferation through deregulation of the Hippo pathway.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mutations in UNC45A, a co-chaperone for myosins, were recently found causative of a syndrome combining cholestasis, diarrhea, loss of hearing and bone fragility. We generated induced pluripotent stem ...cells (iPSCs) from a patient with a homozygous missense mutation in UNC45A. Cells from this patient, which were reprogrammed using integration-free Sendaï virus, have normal karyotype, express pluripotency markers and are able to differentiate into the three germ cell layers.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Variants in the UNC45A cochaperone have been recently associated with a syndrome combining diarrhea, cholestasis, deafness, and bone fragility. Yet the mechanism underlying intestinal failure in ...UNC45A deficiency remains unclear. Here, biallelic variants in UNC45A were identified by next-generation sequencing in 6 patients with congenital diarrhea. Corroborating in silico prediction, variants either abolished UNC45A expression or altered protein conformation. Myosin VB was identified by mass spectrometry as client of the UNC45A chaperone and was found misfolded in UNC45AKO Caco-2 cells. In keeping with impaired myosin VB function, UNC45AKO Caco-2 cells showed abnormal epithelial morphogenesis that was restored by full-length UNC45A, but not by mutant alleles. Patients and UNC45AKO 3D organoids displayed altered luminal development and microvillus inclusions, while 2D cultures revealed Rab11 and apical transporter mislocalization as well as sparse and disorganized microvilli. All those features resembled the subcellular abnormalities observed in duodenal biopsies from patients with microvillus inclusion disease. Finally, microvillus inclusions and shortened microvilli were evidenced in enterocytes from unc45a-deficient zebrafish. Taken together, our results provide evidence that UNC45A plays an essential role in epithelial morphogenesis through its cochaperone function of myosin VB and that UNC45A loss causes a variant of microvillus inclusion disease.
Background & Aims The transferrin receptor (CD71) is up-regulated in duodenal biopsy samples from patients with active celiac disease and promotes retrotransport of secretory immunolglobulin A ...(SIgA)-gliadin complexes. We studied intestinal epithelial cell lines that overexpress CD71 to determine how interactions between SIgA and CD71 promote transepithelial transport of gliadin peptides. Methods We analyzed duodenal biopsy specimens from 8 adults and 1 child with active celiac disease. Caco-2 and HT29-19A epithelial cell lines were transfected with fluorescence-labeled small interfering RNAs against CD71 . Interactions among IgA, CD71, and transglutaminase 2 (Tgase2) were analyzed by flow cytometry, immunoprecipitation, and confocal microscopy. Transcytosis of SIgA–CD71 complexes and intestinal permeability to the gliadin 3H-p31–49 peptide were analyzed in polarized monolayers of Caco-2 cells. Results Using fluorescence resonance energy transfer and in situ proximity ligation assays, we observed physical interactions between SIgA and CD71 or CD71 and Tgase2 at the apical surface of enterocytes in biopsy samples and monolayers of Caco-2 cells. CD71 and Tgase2 were co-precipitated with SIgA, bound to the surface of Caco-2 cells. SIgA–CD71 complexes were internalized and localized in early endosomes and recycling compartments but not in lysosomes. In the presence of celiac IgA or SIgA against p31–49, transport of intact 3H-p31–49 increased significantly across Caco-2 monolayers; this transport was inhibited by soluble CD71 or Tgase2 inhibitors. Conclusions Upon binding to apical CD71, SIgA (with or without gliadin peptides) enters a recycling pathway and avoids lysosomal degradation; this process allows apical–basal transcytosis of bound peptides. This mechanism is facilitated by Tgase2 and might be involved in the pathogenesis of celiac disease.
Celiac disease (CD) is an enteropathy resulting from an abnormal immune response to gluten-derived peptides in genetically susceptible individuals. This immune response is initiated by intestinal ...transport of intact peptide 31-49 (p31-49) and 33-mer gliadin peptides through an unknown mechanism. We show that the transferrin receptor CD71 is responsible for apical to basal retrotranscytosis of gliadin peptides, a process during which p31-49 and 33-mer peptides are protected from degradation. In patients with active CD, CD71 is overexpressed in the intestinal epithelium and colocalizes with immunoglobulin (Ig) A. Intestinal transport of intact p31-49 and 33-mer peptides was blocked by polymeric and secretory IgA (SIgA) and by soluble CD71 receptors, pointing to a role of SIgA-gliadin complexes in this abnormal intestinal transport. This retrotranscytosis of SIgA-gliadin complexes may promote the entry of harmful gliadin peptides into the intestinal mucosa, thereby triggering an immune response and perpetuating intestinal inflammation. Our findings strongly implicate CD71 in the pathogenesis of CD.
An organoid is a three-dimensional multicellular structure that shows realistic micro-anatomy of an organ. This
model mimics the
environment, architecture and multi-lineage differentiation of the ...original organs and allows to answer many interesting biological questions. For these reasons, they are widely used in stem cell, regenerative medicine, toxicology, pharmacology, and host-microbe interactions research. In order to study organoids, microscopy is very useful: It is possible to make three-dimensional reconstruction of serial sections but it is time consuming and error-prone. Here we propose an alternative solution: Tissue clearing reduces the dispersion of light because it homogenizes the refractive index of the tissue, allowing sample observation throughout its thickness. We have compared different clearing techniques on mouse intestinal organoids using different acquisition methods.
Coeliac disease is a gut disease driven by an abnormal immune response towards dietary gluten in genetically susceptible individuals. Whether and, if so, how abnormal transport of gluten across the ...gut epithelium may participate in the pathogenesis of coeliac disease remains debatable. This paper summarises the interactions of gluten-derived peptides with the intestinal epithelium and discusses the mechanisms that control their transport across the epithelium. It shows how recent data point to a key role for the transcellular pathway and highlights the 'Trojan horse' role of secretory IgA which can hijack the transferrin receptor and allow the rapid translocation of intact gluten peptides into the mucosa. These recent findings might be useful for the design of new treatments.
: Physical forces play an important role in regulating cell functions. We applied mechanical strain to human fibroblasts by magnetic attraction of superparamagnetic arginine‐glycine‐aspartic acid ...(RGD)‐coated beads. We confirmed that the MAP kinases Erk and p38 are activated by mechanical strain, and went further by demonstrating the activation of Elk‐1 by mechanical strain, mainly through a MEK‐Erk pathway. Transfection of a dominant negative form of the G protein rac‐1 (rac T17N), and inhibition of PI3K, an effector of rac‐1, efficiently prevented Elk‐1 activation by mechanical forces. Transfection with C3 transferase, known to inhibit rhoA, and inhibition of rock (a downstream effector of rhoA), gave similar results. However, contrary to the active form of rhoA (rho G14V), transfection of the active form of rac‐1 (rac G12V) induced Elk activation and mimicked the effects of mechanical strain. These results point out that the two small G proteins rhoA and rac‐1 participate in cell sensitivity to mechanical strain and lead to the modulation of the Erk pathway.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
The sun is the most important and universal source of non-ionizing radiation shed on human populations. Life evolved on Earth bathed by this radiation. Solar UV damages cells, leading to deleterious ...conditions such as photoaging and carcinogenesis in human skin. During the process of evolution, the cells selected dark- and light-dependent repair mechanisms as a defence against these hazardous effects. This study describes the induction by non-coherent infrared radiation (700–2000 nm), in the absence of rising temperature, of a strong cellular defense against solar UV cytotoxicity as well as induction of cell mitosis. Blocking mitoses with arabinoside-cytosine or protein synthesis with cycloheximide did not abolish the protection, leading to the conclusion that this protection is independent of cell division and of protein neosynthesis. The protection provided by infrared radiation against solar UV radiation is shown to be a long-lasting (at least 24 h) and cumulatif phenomenon. Infrared radiation does not protect the lipids in cellular membranes against UVA induced peroxidation. The protection is not mediated by heat shock proteins. Living organisms on the Earth’s surface are bathed by infrared radiation every day, before being submitted to solar UV. Thus, we propose that this as yet undescribed natural process of cell protection against solar UV, acquired and preserved through evolutional selection, plays an important role in life maintenance. Understanding and controlling this mechanism could provide important keys to the prevention of solar UV damage of human skin.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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