The four isoforms of the RNA-binding protein hnRNPD/AUF1 have been proposed to limit the use of inflammatory mRNAs in innate immune cells. Mice engineered to lack AUF1s in all tissues are sensitive to acute inflammatory assaults; however, they also manifest complex degenerations obscuring assessment of AUF1s' roles in innate immune cells. Here, we restricted a debilitating AUF1 mutation to the mouse myeloid lineage and performed disease-oriented phenotypic analyses to assess the requirement of AUF1s in variable contexts of innate immune reactivity. Contrary to the whole-body mutants, the myeloid mutants of AUF1s did not show differences in their susceptibility to cytokine storms occurring during endotoxemia; neither in type-I cell-mediated reactions driving intestinal inflammation by chemical irritants. Instead, they were resistant to allergic airway inflammation and displayed reductions in inflammatory infiltrates and an altered T-helper balance. The ex-vivo analysis of macrophages revealed that the loss of AUF1s had a minimal effect on their proinflammatory gene expression. Moreover, AUF1s were dispensable for the classical polarization of cultured macrophages by LPS & IFNγ correlating with the unchanged response of mutant mice to systemic and intestinal inflammation. Notably, AUF1s were also dispensable for the alternative polarization of macrophages by IL4, TGFβ and IL10, known to be engaged in allergic reactions. In contrast, they were required to switch proinflammatory macrophages towards a pro-angiogenic phenotype induced by adenosine receptor signals. Congruent to this, the myeloid mutants of AUF1 displayed lower levels of vascular remodeling factors in exudates from allergen exposed lungs; were unable to support the growth and inflammatory infiltration of transplanted melanoma tumors; and failed to vascularize inert grafts unless supplemented with angiogenic factors. Mechanistically, adenosine receptor signals enhanced the association of AUF1s with the Vegfa, Il12b, and Tnf mRNAs to differentially regulate and facilitate the pro-angiogenic switch. Our data collectively demonstrates that AUF1s do not act as general anti-inflammatory factors in innate immune cells but have more specialized roles in regulons allowing specific innate immune cell transitions to support tissue infiltration and remodeling processes.

BACKGROUND AND AIMS:NAFLD is initiated by steatosis and can progress through fibrosis and cirrhosis to HCC. The RNA binding protein human antigen R (HuR) controls RNAs at the posttranscriptional level; hepatocyte HuR has been implicated in the regulation of diet-induced hepatic steatosis. The present study aimed to understand the role of hepatocyte HuR in NAFLD development and progression to fibrosis and HCC.APPROACH AND RESULTS:Hepatocyte-specific, HuR-deficient mice and control HuR-sufficient mice were fed either a normal diet or an NAFLD-inducing diet. Hepatic lipid accumulation, inflammation, fibrosis, and HCC development were studied by histology, flow cytometry, quantitative PCR, and RNA sequencing. The liver lipidome was characterized by lipidomics analysis, and the HuR-RNA interactions in the liver were mapped by RNA immunoprecipitation sequencing. Hepatocyte-specific, HuR-deficient mice displayed spontaneous hepatic steatosis and fibrosis predisposition compared to control HuR-sufficient mice. On an NAFLD-inducing diet, hepatocyte-specific HuR deficiency resulted in exacerbated inflammation, fibrosis, and HCC-like tumor development. A multi-omic approach, including lipidomics, transcriptomics, and RNA immunoprecipitation sequencing revealed that HuR orchestrates a protective network of hepatic-metabolic and lipid homeostasis-maintaining pathways. Consistently, HuR-deficient livers accumulated, already at steady state, a triglyceride signature resembling that of NAFLD livers. Moreover, up-regulation of secreted phosphoprotein 1 expression mediated, at least partially, fibrosis development in hepatocyte-specific HuR deficiency on an NAFLD-inducing diet, as shown by experiments using antibody blockade of osteopontin.CONCLUSIONS:HuR is a gatekeeper of liver homeostasis, preventing NAFLD-related fibrosis and HCC, suggesting that the HuR-dependent network could be exploited therapeutically.

As there is growing evidence for the tumor microenvironment’s role in tumorigenesis, we investigated the role of fibroblast-expressed kinases in triple-negative breast cancer (TNBC). Using a high-throughput kinome screen combined with 3D invasion assays, we identified fibroblast-expressed PIK3Cδ (f-PIK3Cδ) as a key regulator of cancer progression. Although PIK3Cδ was expressed in primary fibroblasts derived from TNBC patients, it was barely detectable in breast cancer (BC) cell lines. Genetic and pharmacological gain- and loss-of-function experiments verified the contribution of f-PIK3Cδ in TNBC cell invasion. Integrated secretomics and transcriptomics analyses revealed a paracrine mechanism via which f-PIK3Cδ confers its protumorigenic effects. Inhibition of f-PIK3Cδ promoted the secretion of factors, including PLGF and BDNF, that led to upregulation of NR4A1 in TNBC cells, where it acts as a tumor suppressor. Inhibition of PIK3Cδ in an orthotopic BC mouse model reduced tumor growth only after inoculation with fibroblasts, indicating a role of f-PIK3Cδ in cancer progression. Similar results were observed in the MMTV-PyMT transgenic BC mouse model, along with a decrease in tumor metastasis, emphasizing the potential immune-independent effects of PIK3Cδ inhibition. Finally, analysis of BC patient cohorts and TCGA data sets identified f-PIK3Cδ (protein and mRNA levels) as an independent prognostic factor for overall and disease-free survival, highlighting it as a therapeutic target for TNBC.

A translational discrepancy exists between animal models used for diabetes research and diabetes in man, thus human relevant models must become a priority. We showed that collectively human islet grafts from 26 donors statistically significantly adapted their function and endocrine and β-cell mass to HFD (high fat diet) in vivo in 233 RAG2KO mice. Human islet compensation occurs at 6 weeks and metabolic dysfunction upon chronic ≥10 weeks HFD. Herein, gene expression studies (GeneChip® Human Gene 2.0 ST Arrays) identified molecular pathways involved. Indeed, human islets compensate to HFD by upregulating hormones, transcription factors, antioxidants, and unfolded protein response genes, and then become dysfunctional at 10 wks -by downregulating these genes. Herein we show Krebs cycle and Mitochondrial electron transport chain was targeted with a transient increase at 6wk and then a drop-in expression at 10 wks; whereas uncouplinggene expression UCP2 and 3 increased. Glycolysis remained upregulated. Explanations for expanded islet and beta mass in HFD (vs. CTL) may be found in cell cycle heatmap. Lastly, a marker of activated stellate cells TAGLN, and matrix genes rose during islet dysfunction (fibrosis?). Conclusion: This human-based model system progressively depicts defects identified in human pre and T2DM that could serve as a “phase 0” in vivo model to test new targets and develop effective therapies in man. Disclosure J.A. Kerr-Conte: None. J. Thevenet: None. G. Pasquetti: None. P. Petit: None. C. Clabaut: None. V. Gmyr: None. C. Bonner: None. S. Gargani: None. F. Pattou: None. Funding JDRF; Programme d’investissements d’avenir; LabEx; European Genomic Institute for Diabetes (ANR-10-LABX-46)

As there is growing evidence for the tumor microenvironment’s (TME) role in tumorigenesis, we sought to investigate the role of fibroblast-expressed kinases in triple negative breast cancer (TNBC). Using a high-throughput kinome screen combined with 3D invasion assays, we identified fibroblast-expressed PIK3Cδ (f-PIK3Cδ) as a key regulator of progression. Although PIK3Cδ has been mainly described in leucocytes, we detected high expression in primary fibroblasts derived from TNBC patients, while PIK3Cδ was undetectable in cancer epithelial cell lines. Genetic and pharmacologic gain- and loss-of functions experiments verified the contribution of f-PIK3Cδ in TNBC cell invasion. By employing an integrated secretomics and transcriptomics analysis, we revealed a paracrine mechanism via which f-PIK3Cδ confers its pro-tumorigenic effects. Inhibition of f-PIK3Cδ promoted the secretion of factors, including PLGF and BDNF, which subsequently led to upregulation of NR4A1 in TNBC cells where it acts as a tumor suppressor. Inhibition of PIK3Cδ in an orthotopic BC mouse model reduced tumor growth only after inoculation with fibroblasts, indicating a role of f-PIK3Cδ in cancer progression. Similar results were observed in the MMTV-PyMT transgenic BC mouse model, in addition to a decrease on tumor metastasis emphasizing the potential immune-independent effects of PIK3Cδ inhibition. Finally, analysis of BC patient cohorts and TCGA datasets identified f-PIK3Cδ (protein and mRNA levels) as an independent prognostic factor for overall and disease free survival, highlighting it as a therapeutic target for TNBC.

RNA binding proteins (RBP) play an essential role in the post-transcriptional control of RNAs. Less is known about the role of RBPs in the liver, particularly the hepatocyte-specific function. The RBP, HuR (encoded by Elavl1; embryonic lethal abnormal vision like 1) regulates mRNA splicing and stability. We found that HuR is diminished in human livers with steatosis and/or non-alcoholic steatohepatitis (NASH) and that it regulates a network of pathways that are essential in hepatic-lipid homeostasis maintenance. Hepatocyte-specific HuR deficient mice exhibited severe steatosis of the liver already at steady state, as compared to their wild type littermates. This phenotype was further enhanced in a model of diet-induced NASH, whereby HuR deficiency promoted inflammation and fibrosis development. Lipidomic analysis revealed that, HuR deficient livers at steady state accumulated a triglyceride-signature resembling that of NASH-livers of HuR sufficient mice. Moreover, hepatocyte-protective bile acid species were reduced, whereas, proinflammatory lipid mediators such as leukotrienes were induced in the absence of HuR, as studied by metabolomics. By studying HuR-RNA interactions in the liver, we found that HuR binds to targets involved in various lipid metabolic processes and that it prevents the hepatic-accumulation of adverse lipid species. The current study emphasizes how post-transcriptional processes control lipid metabolism to maintain homeostasis in the liver.

La capacité des ilots murins à se régénérer décline avec l'âge. L'âge est un facteur de risque quant au développement du diabète. Chez l'homme, la réplication des ilots pancréatiques décroit avec l'âge sur des coupes pancréatiques post mortem et en culture, autant que la détérioration progressive de la sécrétion d'insuline en réponse au glucose, et l'augmentation de l'apoptose. Du fait, le vieillissement limiterait l'avenir prometteur de la thérapie régénérative des cellules Bêta chez l'homme, ainsi que leur survie à long terme après allo-greffe. Notre laboratoire a développé précédemment (Diabétologie, 2013), un modèle chez la souris immunodéficiente, montrant que les ilots humains issus de donneurs jeunes (16 – 41 ans) étaient capables d'adaptation fonctionnelles (cpeptide humain) et en masse d'îlots à une obésité induite in vivo par régime Hyperlipidique (HFD). L'objectif de cette étude est de déterminer si les îlots issus de donneurs âgés (57-69 ans) ont la capacité de s'adapter à un régime HFD, et si l'IMC du donneur a une influence. Des souris Rag2-/- (n = 49) ont été transplantées avec 500 Ilots issus de pancréas de donneurs agés (n = 2) ou agés obèses (n = 5). Les animaux ont été soumis à un régime Contrôle ou Hyperlipidique (HFD) et suivis (Poids, glycémie, C-peptide humain) pendant sur 12 semaines. Après sacrifice, les greffons humains ont été analysés par morphométrie. Les îlots humains s'adaptent leur fonction (cpeptide humain) et leur masse d'îlots à l'obésité (p < 0,05) après 2 semaines de régime ; alors que les ilots de donneurs âgés nécessitent plus de temps (p < 0,05 à semaine 10). De plus, nous avons noté que les ilots de donneurs agés obèses sévères (IMC < 40) pouvait s'adapter (fonction et masse), contrairement aux ilots de donneurs atteints d'obésité morbide (IMC > 40). Les ilots issus de donneurs agés d'IMC normal ou obèses ont la capacité de s'adapter à un environnement obèsogène, que ce soit en fonction et en masse endocrine, contrairement à ceux issus de donneurs morbides. Ce travail montre l'importance de la sélection des donneurs pour l'allotransplantation d'ilots, ou la thérapie régénérative du diabète du type 2.

Dysfunctional microRNA (miRNA) networks contribute to inappropriate responses following pathological stress and are the underlying cause of several disease conditions. In pancreatic β cells, miRNAs have been largely unstudied and little is known about how specific miRNAs regulate glucose-stimulated insulin secretion (GSIS) or impact the adaptation of β cell function to metabolic stress. In this study, we determined that miR-7 is a negative regulator of GSIS in β cells. Using Mir7a2 deficient mice, we revealed that miR-7a2 regulates β cell function by directly regulating genes that control late stages of insulin granule fusion with the plasma membrane and ternary SNARE complex activity. Transgenic mice overexpressing miR-7a in β cells developed diabetes due to impaired insulin secretion and β cell dedifferentiation. Interestingly, perturbation of miR-7a expression in β cells did not affect proliferation and apoptosis, indicating that miR-7 is dispensable for the maintenance of endocrine β cell mass. Furthermore, we found that miR-7a levels are decreased in obese/diabetic mouse models and human islets from obese and moderately diabetic individuals with compensated β cell function. Our results reveal an interconnecting miR-7 genomic circuit that regulates insulin granule exocytosis in pancreatic β cells and support a role for miR-7 in the adaptation of pancreatic β cell function in obesity and type 2 diabetes.

Pax4 and MafA (v-maf musculoaponeurotic fibrosarcoma oncogene homolog A) are two transcription factors crucial for normal functions of islet beta cells in the mouse. Intriguingly, recent studies indicate the existence of notable difference between human and rodent islet in terms of gene expression and functions. To better understand the biological role of human PAX4 and MAFA, we investigated their expression in normal and diseased human islets, using validated antibodies. PAX4 was detected in 43.0±5.0% and 39.1±4.0% of normal human alpha and beta cells respectively. We found that MAFA, detected in 88.3±6.3% insulin+cells as in the mouse, turned out to be also expressed in 61.2±6.4% of human glucagons+ cells with less intensity than in insulin+ cells, whereas MAFB expression was found not only in the majority of glucagon+ cells (67.2±7.6%), but also in 53.6±10.5% of human insulin+ cells. Interestingly, MAFA nuclear expression in both alpha and beta cells, and the percentage of alpha cells expressing PAX4 were found altered in a substantial proportion of patients with type 2 diabetes. Both MAFA and PAX4 display, therefore, a distinct expression pattern in human islet cells, suggesting more potential plasticity of human islets as compared with rodent islets.

L'adaptation de la sécrétion d'insuline par les cellules béta-pancréatiques est requise pour maintenir l'équilibre glycémique au cours de la grossesse et de l'obésité. Une perte de cette adaptation peut entraîner le développement d'un diabète. La voie de signalisation JNK joue un rôle crucial dans la régulation de la fonction et la survie des cellules béta. L'objectif de ce travail fut de rechercher le rôle éventuel de « Dual Leucine Zipper Kinase » (DLK), une kinase conduisant la signalisation de JNK, dans les cellules béta au cours de la gestation et de l'obésité. Des îlots de Langerhans ont été prélevés sur des rates gestantes et des souris obèses maintenues sous un régime riche en graisse. L'expression de DLK a été soit augmentée soit invalidée par ARN interférence dans des îlots ou des cellules productrices d'insuline INS-1E ou MIN6. L'expression génique et protéique a été évaluée par PCR quantitative et Western Blotting. DLK est exprimée dans les cellules béta et dans les îlots humains et de rongeurs. L'expression de DLK augmente dans les îlots des rates gestantes de 14 jours. Cette augmentation corrèle avec une élévation de l'expression de l'isoforme prédominante et anti-apoptotique de JNK, JNK3. L'expression de DLK est aussi augmentée dans les îlots de souris obèses hyper-insulinémiques et non diabétiques. Cette augmentation pourrait résulter de l'effet du GLP-1. L'exendine 4 stimule la production de DLK dans les cellules béta en culture. La surexpression de DLK dans les cellules béta augmente la prolifération, la production et la sécrétion d'insuline en réponse au glucose. À l'inverse, la diminution de DLK réduit la production d'insuline et la survie des cellules en réponse au stress. Ce dysfonctionnement corrèle avec une réduction du contenu et de l'activation de JNK3. DLK contrôle la fonction et la survie des cellules béta en modulant l'activation de JNK3. Cette régulation pourrait être nécessaire à l'adaptation des cellules béta au cours de la gestation et de l'obésité.

In this study, we used an immunodeficient mouse model to explore, in vivo, the longitudinal adaptation of human islets to an obesogenic environment.

Aims/hypothesis Transcription factor 7-like 2 (TCF7L2) is a Wnt-signalling-associated transcription factor. Genetic studies have clearly demonstrated that DNA polymorphisms within TCF7L2 confer the strongest known association with increased risk of type 2 diabetes. However, the impact of the TCF7L2 type-2-diabetes-associated rs7903146 T allele on biological function and morphology of human pancreatic islets is unknown. Methods Paraffin sections of pancreases from 187 brain-deceased donors (HbA 1c <6.5% [48 mmol/mol]) were used to genotype the TCF7L2 variant rs7903146 and evaluate its impact on islet morphology and alpha and beta cell subpopulations following immunostaining for glucagon and C-peptide. Following islet isolation, we investigated the correlation between TCF7L2 genotype and in vitro islet functional variables from our in-house pancreatic database. Results TCF7L2 rs7903146 (T/T) was associated with reduced basal and glucose-stimulated insulin secretion in isolated human islets, and reduced islet density in whole pancreas. Morphological analysis demonstrated islet size was increased in T/T carriers. Furthermore, rs7903146 was associated with an increased glucagon/C-peptide ratio, especially in bigger islets. Conclusion/interpretation The TCF7L2 variant rs7903146 risk allele is associated with impaired insulin secretion, reduction of total islet number and quantitative as well as qualitative morphological changes in human islets. Understanding how the TCF7L2 genotype modulates its activity and how TCF7L2 impacts the islet morphology may aid the design of new therapeutic approaches for the treatment of type 2 diabetes.

The Wharton's Jelly (WJ) of the umbilical cord (UC) is an excellent source of mesenchymal stem cells (MSCs) with a range of potential therapeutic applications. The present study was conducted to demonstrate the efficiency of the protocols used by Biogenea-Cellgenea Ltd. for isolation and expansion of WJ MSCs from donors across Greece. Umbilical cord samples were collected from 599 females following childbirth and processed for WJ MSC isolation. Stem cells were expanded using DMEM-based media and cell counts and overall viability figures derived using Trypan blue exclusion. To investigate the application of isolation and expansion protocols on samples received 1, 2, 3, 4 and 5 d after their collection, ten fresh samples were processed at these time intervals and evaluated. The cellular yield of most WJ samples was 1.1–5.0×10(6) cells at 21–30 d after processing. As culture time increased, cell counts decreased. Statistical analysis of mean cell counts showed a significant reduction after 21 d. Finally, we demonstrate for the first time that it is possible to obtain satisfactory cell numbers from samples processed 1, 2, 3, 4 and even 5 d after collection. We have derived favourable data on the protocols used at Biogenea-Cellgenea Ltd. to isolate and culture MSCs from the WJ. Protocol choice is crucial when handling large numbers of samples on a daily basis and should be made to ensure the best possible outcome.

The umbilical cord blood (UCB) is a major source of hematopoietic progenitor cells. These cells can be cryopreserved for possible future use. In order to evaluate the viability of cryopreserved progenitor cells, a small amount of cells is thawed and subjected to the trypan blue exclusion test in order to count the dead (blue) cells and the live (white) cells.We used the Promega Glo-Max luminometer along with the CytoTox-Glo Cytotoxicity kit and the manufacturer's protocol as an alternative and rapid assay compared to the traditional trypan blue exclusion test.The calculated viability percentages were similar between the 2 assays, but the counting time for a whole 96-well plate is only 30 min.The CytoTox-Glo kit is a time-saving assay for the accurate evaluation of progenitor cell viability following cryopreservation and thawing.