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Background: Atopic dermatitis (AD) is a chronic inflammatory skin disease resulting in decreased quality of life. Histamine and specifically the H 4 receptor play a key role in the inflammatory process in AD and serve as targets for novel therapeutic approaches., Objective: In the present study we aimed to elucidate the immunopathological mechanisms with which the H 4 receptor impacts TH2 cells and contributes to AD pathophysiology., Methods: Total CD4 + T cells obtained from healthy or AD individuals and in vitro differentiated TH2 cells were cultured under different conditions and the mRNA expression or protein production of target molecules were determined using quantitative real-time PCR and ELISA., Results: H 4 receptor mRNA expression was upregulated concentration dependent upon IL-4 stimulation in in vitro differentiated TH2 cells progressively during the differentiation. Transcriptomic analysis of in vitro differentiated TH2 versus TH1 cells revealed that the H 4 receptor among other genes represents one of the highly upregulated genes in TH2 cells. Most importantly, increased amounts of IL-5 and IL-13 mRNA expression were detected in in vitro differentiated TH2 cells as well as protein secretion in the presence of histamine or of the H 4 receptor-selective-agonist when compared to the untreated control., Conclusion: We show for the first time an H 4 receptor dependent upregulation of the pro-inflammatory cytokines IL-5 and IL-13 in human TH2 cells by histamine. This suggests that the blockade of the H 4 receptor may lead to downregulation of these cytokines and amelioration of AD symptoms as reported in first clinical studies., (© 2024 The Author(s). Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Modelling atopic dermatitis (AD) in vitro is paramount to understand the disease pathophysiology and identify novel treatments. Previous studies have shown that the Th2 cytokines IL-4 and IL-13 induce AD-like features in keratinocytes in vitro. However, it has not been systematically researched whether the addition of Th2 cells, their supernatants or a 3D structure is superior to model AD compared to simple 2D cell culture with cytokines. For the first time, we investigated what in vitro option most closely resembles the disease in vivo based on single-cell RNA sequencing data (scRNA-seq) obtained from skin biopsies in a clinical study and published datasets of healthy and AD donors. In vitro models were generated with primary fibroblasts and keratinocytes, subjected to cytokine treatment or Th2 cell cocultures in 2D/3D. Gene expression changes were assessed using qPCR and Multiplex Immunoassays. Of all cytokines tested, incubation of keratinocytes and fibroblasts with IL-4 and IL-13 induced the closest in vivo-like AD phenotype which was observed in the scRNA-seq data. Addition of Th2 cells to fibroblasts failed to model AD due to the downregulation of ECM-associated genes such as POSTN. While keratinocytes cultured in 3D showed better stratification than in 2D, changes induced with AD triggers did not better resemble AD keratinocyte subtypes observed in vivo. Taken together, our comprehensive study shows that the simple model using IL-4 or IL-13 in 2D most accurately models AD in fibroblasts and keratinocytes in vitro, which may aid the discovery of novel treatment options., (© 2024 Beiersdorf AG. Experimental Dermatology published by John Wiley & Sons Ltd.)

Hypertrophic cardiomyopathy (HCM) is the most prevalent inherited cardiac disease. Up to 40% of cases are associated with heterozygous mutations in myosin binding protein C (cMyBP-C, MYBPC3). Most of these mutations lead to premature termination codons (PTC) and patients show reduction of functional cMyBP-C. This so-called haploinsufficiency most likely contributes to disease development. We analyzed mechanisms underlying haploinsufficiency using cardiac tissue from HCM-patients with truncation mutations in MYBPC3 (MYBPC3 trunc ). We compared transcriptional activity, mRNA and protein expression to donor controls. To differentiate between HCM-specific and general hypertrophy-induced mechanisms we used patients with left ventricular hypertrophy due to aortic stenosis (AS) as an additional control. We show that cMyBP-C haploinsufficiency starts at the mRNA level, despite hypertrophy-induced increased transcriptional activity. Gene set enrichment analysis (GSEA) of RNA-sequencing data revealed an increased expression of NMD-components. Among them, Up-frameshift protein UPF3B, a regulator of NMD was upregulated in MYBPC3 trunc patients and not in AS-patients. Strikingly, we show that in sarcomeres UPF3B but not UPF1 and UPF2 are localized to the Z-discs, the presumed location of sarcomeric protein translation. Our data suggest that cMyBP-C haploinsufficiency in HCM-patients is established by UPF3B-dependent NMD during the initial translation round at the Z-disc., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Safety assessment in retroviral vector-mediated gene therapy remains challenging. In clinical trials for different blood and immune disorders, insertional mutagenesis led to myeloid and lymphoid leukemia. We previously developed the In Vitro Immortalization Assay (IVIM) and Surrogate Assay for Genotoxicity Assessment (SAGA) for pre-clinical genotoxicity prediction of integrating vectors. Murine hematopoietic stem and progenitor cells (mHSPCs) transduced with mutagenic vectors acquire a proliferation advantage under limiting dilution (IVIM) and activate stem cell- and cancer-related transcriptional programs (SAGA). However, both assays present an intrinsic myeloid bias due to culture conditions. To detect lymphoid mutants, we differentiated mHSPCs to mature T cells and analyzed their phenotype, insertion site pattern, and gene expression changes after transduction with retroviral vectors. Mutagenic vectors induced a block in differentiation at an early progenitor stage (double-negative 2) compared to fully differentiated untransduced mock cultures. Arrested samples harbored high-risk insertions close to Lmo2 , frequently observed in clinical trials with severe adverse events. Lymphoid insertional mutants displayed a unique gene expression signature identified by SAGA. The gene expression-based highly sensitive molecular readout will broaden our understanding of vector-induced oncogenicity and help in pre-clinical prediction of retroviral genotoxicity., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

The kidney is a complex organ, which consists of multiple components with highly diverse cell types. A detailed understanding of these cell types in health and disease is crucial for the future development of preventive and curative treatment strategies. In recent years, single-cell RNA sequencing (scRNAseq) and single-nucleus RNA sequencing (snRNAseq) technology has opened up completely new possibilities in investigating the variety of renal cell populations in physiological and pathological states. Here, we systematically assessed differences between scRNAseq and snRNAseq approaches in transcriptome analysis of murine kidneys after ischemia-reperfusion injury. We included tissues from control kidneys and from kidneys harvested 1 wk after mild (17-min clamping time) and severe (27-min clamping time) transient unilateral ischemia. Our findings revealed important methodological differences in the discovery of inflammatory cells, tubular cells, and other specialized cell types. Although the scRNAseq approach was advantageous for investigating immune cells, the snRNAseq approach allowed superior insights into healthy and damaged tubular cells. Apart from differences in the quantitative discovery rate, we found important qualitative discrepancies in the captured transcriptomes with crucial consequences for the interpretation of cell states and molecular functions. Together, we provide an overview of method-dependent differences between scRNAseq and snRNAseq results from identical postischemic kidney tissues. Our results highlight the importance of choosing the right approach for specific research questions. NEW & NOTEWORTHY Single-cell and single-nucleus RNA sequencing technologies provide powerful new tools to examine complex tissues such as the kidney. This research reference paper provides practical information on the differences between the two technologies when examining murine kidneys after ischemia-reperfusion injury. The results will serve those who are debating which protocols to use in their given study.

Background: In hepatocellular carcinoma (HCC), histone deacetylases (HDACs) are frequently overexpressed. This results in chromatin compaction and silencing of tumor-relevant genes and microRNAs. Modulation of microRNA expression is a potential treatment option for HCC. Therefore, we aimed to characterize the epigenetically regulated miR-129-5p regarding its functional effects and target genes to understand its relevance for HCC tumorigenesis., Methods: Global miRNA expression of HCC cell lines (HLE, HLF, Huh7, HepG2, Hep3B) and normal liver cell lines (THLE-2, THLE-3) was analyzed after HDAC inhibition by miRNA sequencing. An in vivo xenograft mouse model and in vitro assays were used to investigate tumor-relevant functional effects following miR-129-5p transfection of HCC cells. To validate hepatoma-derived growth factor (HDGF) as a direct target gene of miR-129-5p, luciferase reporter assays were performed. Survival data and HDGF expression were analyzed in public HCC datasets. After siRNA-mediated knockdown of HDGF, its cancer-related functions were examined., Results: HDAC inhibition induced the expression of miR-129-5p. Transfection of miR-129-5p increased the apoptosis of HCC cells, decreased proliferation, migration and ERK signaling in vitro and inhibited tumor growth in vivo. Direct binding of miR-129-5p to the 3'UTR of HDGF via a noncanonical binding site was validated by luciferase reporter assays. HDGF knockdown reduced cell viability and migration and increased apoptosis in Wnt-inactive HCC cells. These in vitro results were in line with the analysis of public HCC datasets showing that HDGF overexpression correlated with a worse survival prognosis, primarily in Wnt-inactive HCCs., Conclusions: This study provides detailed insights into the regulatory network of the tumor-suppressive, epigenetically regulated miR-129-5p in HCC. Our results reveal for the first time that the therapeutic application of mir-129-5p may have significant implications for the personalized treatment of patients with Wnt-inactive, advanced HCC by directly regulating HDGF. Therefore, miR-129-5p is a promising candidate for a microRNA replacement therapy to prevent HCC progression and tumor metastasis., (© 2022. The Author(s).)

With an incidence of ~50%, the absence or reduced protein level of p53 is much more common than TP53 mutations in acute myeloid leukemia (AML). AML with FLT3-ITD (internal tandem duplication) mutations has an unfavorable prognosis and is highly associated with wt-p53 dysfunction. While TP53 mutation in the presence of FLT3-ITD does not induce AML in mice, it is not clear whether p53 haploinsufficiency or loss cooperates with FLT3-ITD in the induction of AML. Here, we generated FLT3-ITD knock-in; p53 knockout (heterozygous and homozygous) double-transgenic mice and found that both alterations strongly cooperated in the induction of cytogenetically normal AML without increasing the self-renewal potential. At the molecular level, we found the strong upregulation of Htra3 and the downregulation of Lin28a, leading to enhanced proliferation and the inhibition of apoptosis and differentiation. The co-occurrence of Htra3 overexpression and Lin28a knockdown, in the presence of FLT3-ITD, induced AML with similar morphology as leukemic cells from double-transgenic mice. These leukemic cells were highly sensitive to the proteasome inhibitor carfilzomib. Carfilzomib strongly enhanced the activity of targeting AXL (upstream of FLT3) against murine and human leukemic cells. Our results unravel a unique role of p53 haploinsufficiency or loss in the development of FLT3-ITD + AML., (© 2021. The Author(s).)

Objectives: Giant cell arteritis (GCA) is the most common primary vasculitis, preferentially affecting the aorta and its large-calibre branches. An imbalance between proinflammatory CD4 + T helper cell subsets and regulatory T cells (Tregs) is thought to be involved in the pathogenesis of GCA and Treg dysfunction has been associated with active disease. Our work aims to explore the aetiology of Treg dysfunction and the way it is affected by remission-inducing immunomodulatory regimens., Methods: A total of 41 GCA patients were classified into active disease (n=14) and disease in remission (n=27). GCA patients' and healthy blood donors' (HD) Tregs were sorted and subjected to transcriptome and phenotypic analysis., Results: Transcriptome analysis revealed 27 genes, which were differentially regulated between GCA-derived and HD-derived Tregs. Among those, we identified transcription factors, glycolytic enzymes and IL-2 signalling mediators. We confirmed the downregulation of forkhead box P3 (FOXP3) and interferon regulatory factor 4 (IRF4) at protein level and identified the ineffective induction of glycoprotein A repetitions predominant (GARP) and CD25 as well as the reduced T cell receptor (TCR)-induced calcium influx as correlates of Treg dysfunction in GCA. Inhibition of glycolysis in HD-derived Tregs recapitulated most identified dysfunctions of GCA Tregs, suggesting the central pathogenic role of the downregulation of the glycolytic enzymes. Separate analysis of the subgroup of tocilizumab-treated patients identified the recovery of the TCR-induced calcium influx and the Treg suppressive function to associate with disease remission., Conclusions: Our findings suggest that low glycolysis and calcium signalling account for Treg dysfunction and inflammation in GCA., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

BACKGROUND: Patients with hepatocellular carcinoma (HCC) have very limited treatment options. For the last fourteen years, the multi-tyrosine kinase inhibitor sorafenib has been used as standard-of-care therapeutic agent in advanced HCC. Unfortunately, drug resistance develops in many cases. Therefore, we aimed to find a way to mitigate drug resistance and to improve the sorafenib efficacy in HCC cells. MicroRNAs play a significant role in targeting genes involved in tumor control suggesting microRNA/sorafenib combination therapy as a promising treatment option in advanced HCC. METHODS: MiR-449a-5p target genes were identified by Ago-RIP sequencing and validated by luciferase reporter assays and expression analyses . Target gene expression and survival data were analyzed in public HCC datasets. Tumor-relevant functional effects of miR-449a-5p and its target genes as well as their impact on the effects of sorafenib were analyzed using in vitro assays. An indirect transwell co-culture system was used to survey anti-angiogenic effects of miR-449a-5p. RESULTS: PEA15, PPP1CA and TUFT1 were identified as direct target genes of miR-449a-5p. Overexpression of these genes correlated with a poor outcome of HCC patients. Transfection with miR-449a-5p and repression of miR-449a-5p target genes inhibited cell proliferation and angiogenesis, induced apoptosis and reduced AKT and ERK signaling in HLE and Huh7 cells. Importantly, miR-449a-5p potentiated the efficacy of sorafenib in HCC cells via downregulation of PEA15, PPP1CA and TUFT1. CONCLUSIONS: This study provides detailed insights into the targetome and regulatory network of miR-449a-5p. Our results demonstrate for the first time that targeting PEA15, PPP1CA and TUFT1 via miR-449a overexpression could have significant implications in counteracting sorafenib resistance suggesting miR-449a-5p as a promising candidate for a microRNA/sorafenib combination therapy., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Hematopoietic stem cell gene therapy is emerging as a promising therapeutic strategy for many diseases of the blood and immune system. However, several individuals who underwent gene therapy in different trials developed hematological malignancies caused by insertional mutagenesis. Preclinical assessment of vector safety remains challenging because there are few reliable assays to screen for potential insertional mutagenesis effects in vitro. Here we demonstrate that genotoxic vectors induce a unique gene expression signature linked to stemness and oncogenesis in transduced murine hematopoietic stem and progenitor cells. Based on this finding, we developed the surrogate assay for genotoxicity assessment (SAGA). SAGA classifies integrating retroviral vectors using machine learning to detect this gene expression signature during the course of in vitro immortalization. On a set of benchmark vectors with known genotoxic potential, SAGA achieved an accuracy of 90.9%. SAGA is more robust and sensitive and faster than previous assays and reliably predicts a mutagenic risk for vectors that led to leukemic severe adverse events in clinical trials. Our work provides a fast and robust tool for preclinical risk assessment of gene therapy vectors, potentially paving the way for safer gene therapy trials., Competing Interests: Declaration of interests A patent application has been filed under registration number EP3394286A1 (Analytical process for genotoxicity assessment)., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Invariant natural killer T (iNKT) cells develop in thymus before emigrating and settling peripheral tissues and organs. In contrast to regular naïve T cells, most iNKT cells do not continuously recirculate but are rather sessile and can adopt phenotypically as well as functionally to their tissue environment. To explore this in more detail, we focused on the most widely distributed CD4 + iNKT1 cells and compared the transcriptome of cells isolated from liver and spleen. Whereas there are only very few genuine differences in the transcriptomes of CD4 + iNKT1 cells of these two organs, the mode of cell isolation left clear marks in the transcriptomic signature. In contrast to liver cell isolated in the cold, cells prepared by enzymatic tissue digestion upregulated quickly a series of genes known to respond to stress. Therefore, to avoid erroneous conclusions, a comparison of expression profiles must take into consideration the history of cell preparation., Competing Interests: The authors declare no competing financial interests., (© 2021 The Authors.)

In recent years, stem cell-derived organoids have become a cell culture standard that is widely used for studying various scientific issues that were previously investigated through animal experiments and using common tumor cell lines. After their initial hype, concerns regarding their standardization have been raised. Here, we aim to provide some insights into our experience in standardizing murine colonic epithelial organoids, which we use as a replacement method for research on inflammatory bowel disease. Considering good scientific practice, we examined various factors that might challenge the design and outcome of experiments using these organoids. First, to analyze the impact of antibiotics/antimycotics, we performed kinetic experiments using ZellShield® and measured the gene expression levels of the tight junction markers Ocln , Zo-1 , and Cldn4 , the proliferation marker Ki67 , and the proinflammatory cytokine Tnfα . Because we found no differences between cultivations with and without ZellShield®, we then performed infection experiments using the probiotic Escherichia coli Nissle 1917 as an already established model setup to analyze the impact of technical, interexperimental, and biologic replicates. We demonstrate that interexperimental differences pose the greatest challenge for reproducibility and explain our strategies for addressing these differences. Additionally, we conducted infection experiments using freshly isolated and cryopreserved/thawed organoids and found that cryopreservation influenced the experimental outcome during early passages. Formerly cryopreserved colonoids exhibited a premature appearance and a higher proinflammatory response to bacterial stimulation. Therefore, we recommend analyzing the growth characteristics and reliability of cryopreserved organoids before to their use in experiments together with conducting several independent experiments under standardized conditions. Taken together, our findings demonstrate that organoid culture, if standardized, constitutes a good tool for reducing the need for animal experiments and might further improve our understanding of, for example, the role of epithelial cells in inflammatory bowel disease development., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 Talke F. zur Bruegge et al.)

Preeclampsia is associated with an increased cardiovascular morbidity of mother and offspring, thus contributing to a substantial burden in women and children's health. It has been proven that endothelial progenitor cell (EPC) numbers and functional characteristics are impaired in cardiovascular disease and preeclampsia, although causative factors for the latter have remained elusive. MicroRNA (miRNA) modifications are a potential mechanism through which exposure to an altered environment translates into the development of chronic disease. In this study, we examined whether development of preeclampsia corresponds to alterations of miRNAs in maternal- and cord-blood-derived EPC. To test this end, we analyzed maternal and neonatal miRNAs via RNA sequencing from endothelial cells of preeclamptic and healthy controls in different cell culture passages. We were able to demonstrate differentially represented miRNAs in all groups. Hsa-miR-1270 showed significantly different levels in cord blood EPC from preeclampsia versus control and was negatively correlated with mRNA levels of its predicted targets ANGPTL7 and TFRC . Transfection with an hsa-miR-1270 inhibitor decreased the tube formation capacity and chemotactic motility but did not change proliferation in vitro. Target predictions and gene set enrichment analyses identified alternative splicing as a significantly enriched pathway for hsa-miR-1270. The top miRNAs in three other groups were predicted to target transcriptional and developmental pathways. Here, we showed for the first time significantly different levels of miRNAs and differently represented mRNA levels of predicted target genes in EPC derived from preeclampsia. Understanding the effects of preeclampsia on the epigenetic mechanisms of EPC will be crucial and may provide initial insights for further evaluation of the benefits of therapies targeting this cell population.

Background and Aims: Programmed death 1 (PD-1) checkpoint inhibition has shown promising results in patients with hepatocellular carcinoma, inducing objective responses in approximately 20% of treated patients. The roles of other coinhibitory molecules and their individual contributions to T-cell dysfunction in liver cancer, however, remain largely elusive., Approach and Results: We performed a comprehensive mRNA profiling of cluster of differentiation 8 (CD8) T cells in a murine model of autochthonous liver cancer by comparing the transcriptome of naive, functional effector, and exhausted, tumor-specific CD8 T cells. Subsequently, we functionally validated the role of identified genes in T-cell exhaustion. Our results reveal a unique transcriptome signature of exhausted T cells and demonstrate that up-regulation of the inhibitory immune receptor T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitor motif domains (TIGIT) represents a hallmark in the process of T-cell exhaustion in liver cancer. Compared to PD-1, expression of TIGIT more reliably identified exhausted CD8 T cells at different stages of their differentiation. In combination with PD-1 inhibition, targeting of TIGIT with antagonistic antibodies resulted in synergistic inhibition of liver cancer growth in immunocompetent mice. Finally, we demonstrate expression of TIGIT on tumor-infiltrating CD8 T cells in tissue samples of patients with hepatocellular carcinoma and intrahepatic cholangiocarcinoma and identify two subsets of patients based on differential expression of TIGIT on tumor-specific T cells., Conclusions: Our transcriptome analysis provides a valuable resource for the identification of key pathways involved in T-cell exhaustion in patients with liver cancer and identifies TIGIT as a potential target in checkpoint combination therapies., (© 2020 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Morbidity and mortality rates in acute lung injury (ALI) increase with age. As alveolar epithelial type II cells (AE2) are crucial for lung function and repair, we hypothesized that aging promotes senescence in AE2 and contributes to the severity and impaired regeneration in ALI. ALI was induced with 2.5 μg lipopolysaccharide/g body weight in young (3 mo) and old (18 mo) mice that were euthanized 24 h, 72 h, and 10 days later. Lung function, pulmonary surfactant activity, stereology, cell senescence, and single-cell RNA sequencing analyses were performed to investigate AE2 function in aging and ALI. In old mice, surfactant activity was severely impaired. A 60% mortality rate and lung function decline were observed in old, but not in young, mice with ALI. AE2 of young mice adapted to injury by increasing intracellular surfactant volume and proliferation rate. In old mice, however, this adaptive response was compromised, and AE2 of old mice showed signs of cell senescence, increased inflammatory signaling, and impaired surfactant metabolism in ALI. These findings provide evidence that ALI promotes a limited proliferation rate, increased inflammatory response, and surfactant dysfunction in old, but not in young, mice, supporting an impaired regenerative capacity and reduced survival rate in ALI with advancing age.

Invariant natural killer T (iNKT) cells represent a subclass of T cells possessing a restricted repertoire of T cell receptors enabling them to recognize lipid derived ligands. iNKT cells are continuously generated in thymus and differentiate into three main subpopulations: iNKT1, iNKT2, and iNKT17 cells. We investigated the transcriptomes of these subsets comparing cells isolated from young adult (6-10 weeks old) and aged BALB/c mice (25-30 weeks of age) in order to identify genes subject to an age-related regulation of expression. These time points were selected to take into consideration the consequences of thymic involution that radically alter the existing micro-milieu. Significant differences were detected in the expression of histone genes affecting all iNKT subsets. Also the proliferative capacity of iNKT cells decreased substantially upon aging. Several genes were identified as possible candidates causing significant age-dependent changes in iNKT cell generation and/or function such as genes coding for granzyme A, ZO-1, EZH2, SOX4, IGF1 receptor, FLT4, and CD25. Moreover, we provide evidence that IL2 differentially affects homeostasis of iNKT subsets with iNKT17 cells engaging a unique mechanism to respond to IL2 by initiating a slow rate of proliferation., (Copyright © 2020 Papadogianni, Ravens, Dittrich-Breiholz, Bernhardt and Georgiev.)

Ischemia reperfusion injury (IRI) is linked with inflammation in kidney transplantation (ktx). The chemokine CXCL13, also known as B lymphocyte chemoattractant, mediates recruitment of B cells within follicles of lymphoid tissues and has recently been identified as a biomarker for acute kidney allograft rejection. The goal of this study was to explore whether IRI contributes to the up-regulation of CXCL13 levels in ktx. It is demonstrated that systemic levels of CXCL13 were increased in mouse models of uni- and bilateral renal IRI, which correlated with the duration of IRI. Moreover, in unilateral renal IRI CXCL13 expression in ischemic kidneys was up-regulated. Immunohistochemical studies revealed infiltration of CD22+ B-cells and, single-cell RNA sequencing analysis a higher number of cells expressing the CXCL13 receptor CXCR5, in ischemic kidneys 7 days post IRI, respectively. The potential relevance of these findings was also evaluated in a mouse model of ktx. Increased levels of serum CXCL13 correlated with the lengths of cold ischemia times and were further enhanced in allogenic compared to isogenic kidney transplants. Taken together, these findings indicate that IRI is associated with increased systemic levels of CXCL13 in renal IRI and ktx., (Copyright © 2020 Kreimann, Jang, Rong, Greite, von Vietinghoff, Schmitt, Bräsen, Schiffer, Gerstenberg, Vijayan, Dittrich-Breiholz, Wang, Karsten, Gwinner, Haller, Immenschuh and Gueler.)

Mesenchymal stromal cells (MSCs) are a promising cell source for tissue engineering and regenerative medicine. In our lab, we found that MSC preparations from bone marrow of many different donors had a limited capacity of in vitro differentiation into osteogenic and chondrogenic lineages-a capacity claimed to be inherent to MSCs. The current study was designed to test the hypothesis that the amount of heparin used as anticoagulant during bone marrow harvest had an inhibitory influence on the in vitro differentiation capacity of isolated MSCs. Bone marrow was obtained from the femoral cavity of twelve donors during total hip arthroplasty in the absence or presence of heparin. No coagulation was observed in the absence of heparin. The number of mononuclear cells was independent of heparin addition. Isolated MSCs were characterized by morphology, population doubling times, expression of cell surface antigens and in vitro differentiation. Results of these analyses were independent of the amount of heparin. Transcriptome analyses of cells from three randomly chosen donors and quantitative realtime PCR (qRT-PCR) analysis from cells of all donors demonstrated no clear effect of heparin on the transcriptome of the cells. This excludes heparin as a potential source of disparate results.

In obesity, increased dietary lipids are taken up and transported by the lymphatic systems into the circulatory system. Increased fat accumulation results in impairments in the lymph fluid and lymph node (LN) atrophy. LNs filter the lymph fluid for foreign antigens to induce and control immune responses, and the alteration of this function during obesity remains underexplored. Here, the changes within the microarchitecture of mesenteric LNs (mLNs) during high levels of lipid transport were investigated, and the role of stromal cells in mice fed a high-fat diet for 10 weeks was assessed. Microarray experiments revealed that gene probes involved in lipid metabolism are expressed by mLN stromal cells. Transmission electron microscopy enabled the identification of lipid droplets in lymphatic endothelial cells, different reticulum cells, and macrophages, and the lipid droplet sizes as well as their numbers and intercellular distances increased after 10 weeks of high-fat diet feeding. The results indicate that changes in the microarchitecture and increased accumulation of lipid droplets in stromal cells and macrophages influence the immunological function of mLNs., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 THE AUTHORS. Published by Elsevier BV on behalf of Cairo University.)

How cytokine-driven changes in chromatin topology are converted into gene regulatory circuits during inflammation still remains unclear. Here, we show that interleukin (IL)-1α induces acute and widespread changes in chromatin accessibility via the TAK1 kinase and NF-κB at regions that are highly enriched for inflammatory disease-relevant SNPs. Two enhancers in the extended chemokine locus on human chromosome 4 regulate the IL-1α-inducible IL8 and CXCL1-3 genes. Both enhancers engage in dynamic spatial interactions with gene promoters in an IL-1α/TAK1-inducible manner. Microdeletions of p65-binding sites in either of the two enhancers impair NF-κB recruitment, suppress activation and biallelic transcription of the IL8/CXCL2 genes, and reshuffle higher-order chromatin interactions as judged by i4C interactome profiles. Notably, these findings support a dominant role of the IL8 "master" enhancer in the regulation of sustained IL-1α signaling, as well as for IL-8 and IL-6 secretion. CRISPR-guided transactivation of the IL8 locus or cross-TAD regulation by TNFα-responsive enhancers in a different model locus supports the existence of complex enhancer hierarchies in response to cytokine stimulation that prime and orchestrate proinflammatory chromatin responses downstream of NF-κB., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Cerebral cavernous malformations (CCMs) are low-flow vascular malformations in the brain associated with recurrent hemorrhage and seizures. The current treatment of CCMs relies solely on surgical intervention. Henceforth, alternative non-invasive therapies are urgently needed to help prevent subsequent hemorrhagic episodes. Long non-coding RNAs (lncRNAs) belong to the class of non-coding RNAs and are known to regulate gene transcription and involved in chromatin remodeling via various mechanism. Despite accumulating evidence demonstrating the role of lncRNAs in cerebrovascular disorders, their identification in CCMs pathology remains unknown. The objective of the current study was to identify lncRNAs associated with CCMs pathogenesis using patient cohorts having 10 CCM patients and 4 controls from brain. Executing next generation sequencing, we performed whole transcriptome sequencing (RNA-seq) analysis and identified 1,967 lncRNAs and 4,928 protein coding genes (PCGs) to be differentially expressed in CCMs patients. Among these, we selected top 6 differentially expressed lncRNAs each having significant correlative expression with more than 100 differentially expressed PCGs. The differential expression status of the top lncRNAs, SMIM25 and LBX2-AS1 in CCMs was further confirmed by qRT-PCR analysis. Additionally, gene set enrichment analysis of correlated PCGs revealed critical pathways related to vascular signaling and important biological processes relevant to CCMs pathophysiology. Here, by transcriptome-wide approach we demonstrate that lncRNAs are prevalent in CCMs disease and are likely to play critical roles in regulating important signaling pathways involved in the disease progression. We believe, that detailed future investigations on this set of identified lncRNAs can provide useful insights into the biology and, ultimately, contribute in preventing this debilitating disease.

Background: Mesenchymal stromal cells (MSCs) are used in over 800 clinical trials mainly due to their immune inhibitory activity. Umbilical cord (UC), the second leading source of clinically used MSCs, is usually cut in small tissue pieces. Subsequent cultivation leads to a continuous outgrowth of MSC explant monolayers (MSC-EMs) for months. Currently, the first MSC-EM culture takes approximately 2 weeks to grow out, which is then expanded and applied to patients. The initiating tissue pieces are then discarded. However, when UC pieces are transferred to new culture dishes, MSC-EMs continue to grow out. In case the functional integrity of these cells is maintained, later induced cultures could also be expanded and used for cell therapy. This would drastically increase the number of available cells for each patient. To test the functionality of MSC-EMs from early and late induction time points, we compared the first cultures to those initiated after 2 months by investigating their clonality and immunomodulatory capacity., Methods: We analyzed the clonal composition of MSC-EM cultures by umbilical cord piece transduction using integrating lentiviral vectors harboring genetic barcodes assessed by high-throughput sequencing. We investigated the transcriptome of these cultures by microarrays. Finally, the secretome was analyzed by multiplexed ELISAs, in vitro assays, and in vivo in mice., Results: DNA barcode analysis showed polyclonal MSC-EMs even after months of induction cycles. A transcriptome and secretome analyses of early and late MSC cultures showed only minor changes over time. However, upon activation with TNF-α and IFN-γ, cells from both induction time points produced a multitude of immunomodulatory cytokines. Interestingly, the later induced MSC-EMs produced higher amounts of cytokines. To test whether the different cytokine levels were in a therapeutically relevant range, we used conditioned medium (CM) in an in vitro MLR and an in vivo killing assay. CM from late induced MSC-EMs was at least as immune inhibitory as CM from early induced MSC-EMs., Conclusion: Human umbilical cord maintains a microenvironment for the long-term induction of polyclonal and immune inhibitory active MSCs for months. Thus, our results would offer the possibility to drastically increase the number of therapeutically applicable MSCs for a substantial amount of patients.

Autoreactive T cells that infiltrate into the central nervous system (CNS) are believed to have a significant role in mediating the pathology of neuroinflammatory diseases like multiple sclerosis. Their interaction with microglia and astrocytes in the CNS is crucial for the regulation of neuroinflammatory processes. Our previous work demonstrated that effectors secreted by Th1 and Th17 cells have different capacities to influence the phenotype and function of glial cells. We have shown that Th1-derived effectors altered the phenotype and function of both microglia and astrocytes whereas Th17-derived effectors induced direct effects only on astrocytes but not on microglia. Here we investigated if effector molecules associated with IFN-γ producing Th1 cells induced different gene expression profiles in microglia and astrocytes. We performed a microarray analysis of RNA isolated from microglia and astrocytes treated with medium and Th-derived culture supernatants and compared the gene expression data. By using the criteria of 2-fold change and a false discovery rate of 0.01 (corrected p < 0.01), we demonstrated that a total of 2,106 and 1,594 genes were differentially regulated in microglia and astrocytes, respectively, in response to Th1-derived factors. We observed that Th1-derived effectors induce distinct transcriptional changes in microglia and astrocytes in addition to commonly regulated transcripts. These distinct transcriptional changes regulate peculiar physiological functions, and this knowledge can help to better understand T cell mediated neuropathologies.

Rationale: Although the transplantation of induced pluripotent stem cell (iPSC)-derived cells harbors enormous potential for the treatment of pulmonary diseases, in vivo data demonstrating clear therapeutic benefits of human iPSC-derived cells in lung disease models are missing., Objectives: We have tested the therapeutic potential of iPSC-derived macrophages in a humanized disease model of hereditary pulmonary alveolar proteinosis (PAP). Hereditary PAP is caused by a genetic defect of the GM-CSF (granulocyte-macrophage colony-stimulating factor) receptor, which leads to disturbed macrophage differentiation and protein/surfactant degradation in the lungs, subsequently resulting in severe respiratory insufficiency., Methods: Macrophages derived from human iPSCs underwent intrapulmonary transplantation into humanized PAP mice, and engraftment, in vivo differentiation, and therapeutic efficacy of the transplanted cells were analyzed., Measurements and Main Results: On intratracheal application, iPSC-derived macrophages engrafted in the lungs of humanized PAP mice. After 2 months, transplanted cells displayed the typical morphology, surface markers, functionality, and transcription profile of primary human alveolar macrophages. Alveolar proteinosis was significantly reduced as demonstrated by diminished protein content and surfactant protein D levels, decreased turbidity of the BAL fluid, and reduced surfactant deposition in the lungs of transplanted mice., Conclusions: We here demonstrate for the first time that pulmonary transplantation of human iPSC-derived macrophages leads to pulmonary engraftment, their in situ differentiation to an alveolar macrophage phenotype, and a reduction of alveolar proteinosis in a humanized PAP model. To our knowledge, this finding presents the first proof-of-concept for the therapeutic potential of human iPSC-derived cells in a pulmonary disease and may have profound implications beyond the rare disease of PAP.

Fundamental research and drug development for personalized medicine necessitates cell cultures from defined genetic backgrounds. However, providing sufficient numbers of authentic cells from individuals poses a challenge. Here, we present a new strategy for rapid cell expansion that overcomes current limitations. Using a small gene library, we expanded primary cells from different tissues, donors, and species. Cell-type-specific regimens that allow the reproducible creation of cell lines were identified. In depth characterization of a series of endothelial and hepatocytic cell lines confirmed phenotypic stability and functionality. Applying this technology enables rapid, efficient, and reliable production of unlimited numbers of personalized cells. As such, these cell systems support mechanistic studies, epidemiological research, and tailored drug development.

Following the acute phase of an inflammatory reaction, a strictly controlled resolution of inflammation is necessary. A dysregulation of this process leads to hyperinflammation, chronic inflammatory disease, or immune paralysis. Different mechanisms participate in the coordinated termination of the inflammatory process, e.g. the expression of antiinflammatory molecules and different forms of tolerance. To better understand the processes which mediate resolution of TNF-dependent inflammation and induce tolerance, it is necessary to characterize the signal transduction quality during TNF long-term (pre)incubation. Within a time frame from 12 to 48h, designated as phase III of the TNF response, we measured an ongoing, constitutive activation of TNFR1/NF-κB-dependent pathways in monocytic cells. Phase III signalling which was also named "constitutive signaling in TNF tolerant cells" induces the expression of low- and high-sensitive target genes including A20 which is differentially regulated by transcriptional and proteolytic events. A20 strictly controls TNF long-term constitutive signalling in an IκB kinase complex- and partially RIP-dependent manner supported by adjuvant ABIN1. In addition, CYLD proteins participate in the regulation of this late-phase signal transduction, whereas downstream molecules such as Bcl3 and p50 are not involved. A20 and CYLD are expressed with different mRNA kinetics resulting in a strong or only a modest increase in protein levels, respectively. The identification of mechanisms which contribute to the termination of inflammation will provide additional diagnostic and therapeutic aspects to specifically diagnose certain aspects of inflammation and specifically modulate them., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Formation of motile cilia in vertebrate embryos is essential for proper development and tissue function. Key regulators of motile ciliogenesis are the transcription factors FOXJ1 and NOTO, which are conserved throughout vertebrates. Downstream target genes of FOXJ1 have been identified in a variety of species, organs and cultured cell lines; in murine embryonic and foetal tissues, however, FOXJ1 and NOTO effectors have not been comprehensively analysed and our knowledge of the downstream genetic programme driving motile ciliogenesis in the mammalian lung and ventral node is fragmentary. We compared genome-wide expression profiles of undifferentiated E14.5 vs. abundantly ciliated E18.5 micro-dissected airway epithelia as well as Foxj1 + vs. Foxj1-deficient foetal (E16.5) lungs of the mouse using microarray hybridisation. 326 genes deregulated in both screens are candidates for FOXJ1-dependent, ciliogenesis-associated factors at the endogenous onset of motile ciliogenesis in the lung, including 123 genes that have not been linked to ciliogenesis before; 46% of these novel factors lack known homologues outside mammals. Microarray screening of Noto + vs. Noto null early headfold embryos (E7.75) identified 59 of the lung candidates as NOTO/FOXJ1-dependent factors in the embryonic left-right organiser that carries a different subtype of motile cilia. For several uncharacterised factors from this small overlap - including 1700012B09Rik, 1700026L06Rik and Fam183b - we provide extended experimental evidence for a ciliary function., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Interleukin (IL)-36α - one of the novel members of the IL-1 family of cytokines - is a potent regulator of dendritic and T cells and plays an important role in inflammatory processes like experimental skin inflammation in mice and in mouse models for human psoriasis. Here, we demonstrate that C/EBPβ, a transcription factor required for the selective expression of inflammatory genes, is a key activator of the Il36A gene in murine macrophages. RNAi-mediated suppression of C/EBPβ expression in macrophages (C/EBPβ(low) cells) significantly impaired Il36A gene induction following challenge with LPS. Despite the presence of five predicted C/EBP binding sites, luciferase reporter assays demonstrated that C/EBPβ confers responsiveness to LPS primarily through a half-CRE•C/EBP element in the proximal Il36A promoter. Electrophoretic mobility shift assays showed that C/EBPβ but not CREB proteins interact with this critical half-CRE•C/EBP element. In addition, overexpression of C/EBPβ in C/EBPβ(low) cells enhanced the expression of Il36A whereas CREB-1 had no effect. Finally, chromatin immunoprecipitation confirmed that C/EBPβ but neither CREB-1, ATF-2 nor ATF4 is directly recruited to the proximal promoter region of the Il36A gene. Together, these findings demonstrate an essential role of C/EBPβ in the regulation of the Il36A gene via the proximal half-CRE•C/EBP element in response to inflammatory stimuli., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The adult small intestine contains more than half of the body's lymphocytes in order to maintain homeostasis with the commensal microbiota. Birth marks a transition of the intestine from a sterile to an increasingly colonized environment. The data described in this article are incremented into the work published by Torow et al. titled "Active suppression of intestinal CD4(+) TCRαβ(+) T lymphocyte maturation during the postnatal period" [1]. While most of the CD4 T cells found in the adult small intestine have an activated phenotype marked by expression of helper lineage specific genes neonatal lymphocytes exhibit a naïve phenotype. Further, direct comparison of neonatal CD4 T cells from the small intestine and the gut draining mesenteric lymph node (mLN) reveals a global transcriptional 'inactivity' of the small intestinal CD4 T cells. Here, we describe in more detail the experimental design, sample preparation and analysis that were performed to obtain and interpret the microarray data. The data set is publicly available through the Gene Expression Omnibus (GEO) database with accession number GSE60515, and the analysis and interpretation of these data are included in Torow et al. [1].

Lymph node stromal cells are known to be immunorelevant during inflammation and tolerance. Differences between peripheral lymph nodes and mesenteric lymph nodes are important for an efficient and effective immune defense. Stromal cells were considered to be perfectly adapted to their draining area and not changeable concerning their expression pattern. Here we show that stromal cells can change their profile after isolation and transplantation into a different draining area. Subsequently, these newly organized lymph nodes are able to induce not only a region-specific but also an antigen-specific immune response. Thus, stromal cells are trend-setters for immune cells in producing a microenvironment that allows an optimized immune defense.

The inflammatory gene response requires activation of the protein kinase TAK1, but it is currently unknown how TAK1-derived signals coordinate transcriptional programs in the genome. We determined the genome-wide binding of the TAK1-controlled NF-κB subunit p65 in relation to active enhancers and promoters of transcribed genes by chromatin immunoprecipitation sequencing (ChIP-seq) experiments. Out of 35,000 active enhancer regions, 410 H3K4me1-positive enhancers show interleukin 1 (IL-1)-induced H3K27ac and p65 binding. Inhibition of TAK1 or IKK2 or depletion of p65 blocked inducible enhancer activation and gene expression. As exemplified by the CXC chemokine cluster located on chromosome 4, the TAK1-p65 pathway also regulates the recruitment kinetics of the histone acetyltransferase CBP, of NF-κB p50, and of AP-1 transcription factors to both promoters and enhancers. This study provides a high-resolution view of epigenetic changes occurring during the IL-1 response and allows the genome-wide identification of a distinct class of inducible p65 NF-κB-dependent enhancers in epithelial cells., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Natural killer (NK) cells have been detected in the lesional skin of patients with inflammatory skin diseases, where high levels of histamine are also present. Therefore, we investigated the effect of histamine, in particular via the histamine H4 receptor (H4R), on gene expression levels in human NK cells., Methods: Comprehensive microarray-based mRNA expression profiling was performed to assess the gene expression levels in human NK cells in response to H4R stimulation in an unbiased approach. The expression of selected cytokines and chemokines was quantified by real-time PCR and enzyme-linked immunosorbent assay., Results: The microarray analysis identified only few genes which were differentially regulated upon H4R stimulation. In follow-up studies, a significant upregulation of CCL3 and CCL4 at the mRNA level and in addition for CCL3 also at the protein level via the H4R was observed., Conclusion: The elevated expression levels of chemokines in response to H4R stimulation might foster the inflammation in allergic skin diseases and characterize the H4R as a promising therapeutic target., (© 2015 S. Karger AG, Basel.)

Transcription of immediate early genes (IEGs) in response to extrinsic and intrinsic signals is tightly regulated at multiple stages. It is known that untranslated regions of the RNA can play a role in these processes. Here we show that THOC5, a member of the TREX (transcription/export) complex, plays a role in expression of only a subset of constitutively active genes, however transcriptome analysis reveals that more than 90% of IEG were not induced by serum in THOC5 depleted cells. Furthermore, THOC5 depletion does not influence the expression of the most rapidly induced IEGs, e.g. Fos and Jun. One group of THOC5 target genes, including Id1, Id3 and Wnt11 transcripts, were not released from chromatin in THOC5 depleted cells. Genes in another group, including Myc and Smad7 transcripts, were released with shortening of 3'UTR by alternative cleavage, and were spliced but export was impaired in THOC5 depleted cells. By interactome analysis using THOC5 as bait, we show that upon stimulation with serum THOC5 forms a complex with polyadenylation-specific factor 100 (CPSF100). THOC5 is required for recruitment of CPSF100 to 3'UTR of THOC5 target genes. These data suggest the presence of a novel mechanism for the control of IEG response by THOC5 via 3'end-processing., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The molecular basis of TNF tolerance is poorly understood. In human monocytes we detected two forms of TNF refractoriness, as follows: absolute tolerance was selective, dose dependently affecting a small group of powerful effector molecules; induction tolerance represented a more general phenomenon. Preincubation with a high TNF dose induces both absolute and induction tolerance, whereas low-dose preincubation predominantly mediates absolute tolerance. In cells preincubated with the high TNF dose, we observed blockade of IκBα phosphorylation/proteolysis and nuclear p65 translocation. More prominent in cells preincubated with the high dose, reduced basal IκBα levels were found, accompanied by increased IκBα degradation, suggesting an increased IκBα turnover. In addition, a nuclear elevation of p50 was detected in tolerant cells, which was more visible following high-dose preincubation. TNF-induced phosphorylation of p65-Ser(536), p38, and c-jun was inhibited, and basal inhibitory p65-Ser(468) phosphorylation was increased in tolerant cells. TNF tolerance induced by the low preincubation dose is mediated by glycogen synthesis kinase-3, whereas high-dose preincubation-mediated tolerance is regulated by A20/glycogen synthesis kinase-3 and protein phosphatase 1-dependent mechanisms. To our knowledge, we present the first genome-wide analysis of TNF tolerance in monocytic cells, which differentially inhibits NF-κB/AP-1-associated signaling and shifts the kinase/phosphatase balance. These forms of refractoriness may provide a cellular paradigm for resolution of inflammation and may be involved in immune paralysis.

Given the intimate link between inflammation and dysregulated cell proliferation in cancer, we investigated cytokine-triggered gene expression in different cell cycle stages. Transcriptome analysis revealed that G1 release through cyclin-dependent kinase 6 (CDK6) and CDK4 primes and cooperates with the cytokine-driven gene response. CDK6 physically and functionally interacts with the NF-κB subunit p65 in the nucleus and is found at promoters of many transcriptionally active NF-κB target genes. CDK6 recruitment to distinct chromatin regions of inflammatory genes was essential for proper loading of p65 to its cognate binding sites and for the function of p65 coactivators, such as TRIP6. Furthermore, cytokine-inducible nuclear translocation and chromatin association of CDK6 depends on the kinase activity of TAK1 and p38. These results have widespread biological implications, as aberrant CDK6 expression or activation that is frequently observed in human tumors modulates NF-κB to shape the cytokine and chemokine repertoires in chronic inflammation and cancer., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Hematopoiesis and commitment to a restricted lineage are guided by a timely expressed set of cytokine receptors and their downstream transcription factors. A member of the mRNA export complex, THOC5 (suppressors of the transcriptional defects of hpr1 delta by overexpression complex 5) is a substrate for several tyrosine kinases such as macrophage colony-stimulating factor (M-CSF) receptor and various leukemogenic tyrosine kinases, such as Bcr-Abl, or NPM-ALK. THOC5 tyrosine phosphorylation is elevated in stem cells from patients with chronic myeloid leukemia, suggesting that THOC5 may be involved in leukemia development. THOC5 is also an essential element in the maintenance of hematopoiesis in adult mice. In this report, we show that THOC5 is located in the nuclear speckles, and that it is translocated from the nucleus to cytoplasm during M-CSF-induced bone marrow-derived macrophage differentiation. Furthermore, we have identified THOC5 target genes by trancriptome analysis, using tamoxifen-inducible THOC5 knockout macrophages. Although only 99 genes were downregulated in THOC5-depleted macrophages, half of the genes are involved in differentiation and/or migration. These include well-known regulators of myeloid differentiation inhibitor of DNA binding (Id)1, Id3, Smad family member 6 (Smad6) and Homeobox (Hox)A1. In addition, a subset of M-CSF-inducible genes, such as Ets family mRNAs are THOC5 target mRNAs. Upon depletion of THOC5, unspliced v-ets erythroblastosis virus E26 oncogene homolog (Ets1) mRNA was accumulated in the nucleus. Furthermore, THOC5 was recruited to chromatin where Ets1 was transcribed and bound to unspliced and spliced Ets1 transcripts, indicating that THOC5 has a role in processing/export of M-CSF-inducible genes. In conclusion, regulation of immediate-early gene response by THOC5, a member of mRNA export complex contributes to the M-CSF-induced macrophage differentiation.

Changes in gene expression during inflammation are in part caused by post-transcriptional mechanisms. A transcriptome-wide screen for changes in ribosome occupancy indicated that the inflammatory cytokine IL-17 activates translation of a group of mRNAs that overlaps partially with those affected similarly by IL-1. Included are mRNAs of IκBζ and of MCPIP1, important regulators of the quality and course of immune and inflammatory responses. Evidence for increased ribosome association of these mRNAs was also obtained in LPS-activated RAW264.7 macrophages and human peripheral blood mononuclear cells. Like IL-1, IL-17 activated translation of IκBζ mRNA by counteracting the function of a translational silencing element in its 3'-UTR defined previously. Translational silencing of MCPIP1 mRNA in unstimulated cells resulted from the combined suppressive activities of its 5'-UTR, which contains upstream open reading frames, and of its 3'-UTR, which silences independently of the 5'-UTR. Only the silencing function of the 3'-UTR was counteracted by IL-17 as well as by IL-1. Translational silencing by the 3'-UTR was dependent on a putative stem-loop-forming region previously associated with rapid degradation of the mRNA. The results suggest that translational control exerted by IL-1 and IL-17 plays an important role in the coordination of an inflammatory reaction.

Unlabelled: Coxsackievirus B3 (CVB3) is a major cause of acute and chronic forms of myocarditis. Previously, direct viral injury and post-infectious autoimmune response were suspected as main pathogenetic mechanisms. However, induction of pro-inflammatory cytokines may be crucial for pathogenesis in spite of host protein shut off caused by CVB3 replication. We investigated the global expression profile of pro-inflammatory genes induced by acute and persistent (carrier state) CVB3 infection in human fibroblast cell cultures with DNA microarrays, quantitative RT-PCR and ELISA. Rapid induction of a typical spectrum of about 30 inflammation-related genes (e.g., PTGS2, CCL2, IL-1β, IL-6, IL-8, CSF2, MMP-1, MMP-3, and MMP-15) suggested an essential, autocrine role of IL-1. This hypothesis was confirmed by over-expression of IL-1RI, which resulted in a cytokine response upon CVB3 infection in HEK 293 cells otherwise refractory to CVB3-caused gene expression. Blocking IL-1 receptor type I (IL-1RI)-signaling during CVB3 infection with an IL-1 receptor antagonist (IL-1ra) as well as knockdown of IL-1RI using siRNA abrogated cytokine response in human fibroblasts. Both IL-1α and IL-1β are relevant for the induction of inflammation-related genes during CVB3 infection as shown by neutralization experiments. Paracrine effects of IL-1 on the subset of non-infected cells in carrier state infected fibroblast cultures enhanced induction of inflammation-related genes., Conclusions: A broad spectrum of inflammatory cytokines was induced by CVB3 replication via a pathway that requires IL-1 signaling. Our results suggest that IL-1ra may be used as a therapeutic agent to limit inflammation and tissue destruction in myocarditis.

Histone deacetylase (HDAC) 3, as a cofactor in co-repressor complexes containing silencing mediator for retinoid or thyroid-hormone receptors (SMRT) and nuclear receptor co-repressor (N-CoR), has been shown to repress gene transcription in a variety of contexts. Here, we reveal a novel role for HDAC3 as a positive regulator of IL-1-induced gene expression. Various experimental approaches involving RNAi-mediated knockdown, conditional gene deletion or small molecule inhibitors indicate a positive role of HDAC3 for transcription of the majority of IL-1-induced human or murine genes. This effect was independent from the gene regulatory effects mediated by the broad-spectrum HDAC inhibitor trichostatin A (TSA) and thus suggests IL-1-specific functions for HDAC3. The stimulatory function of HDAC3 for inflammatory gene expression involves a mechanism that uses binding to NF-κB p65 and its deacetylation at various lysines. NF-κB p65-deficient cells stably reconstituted to express acetylation mimicking forms of p65 (p65 K/Q) had largely lost their potential to stimulate IL-1-triggered gene expression, implying that the co-activating property of HDAC3 involves the removal of inhibitory NF-κB p65 acetylations at K122, 123, 314 and 315. These data describe a novel function for HDAC3 as a co-activator in inflammatory signaling pathways and help to explain the anti-inflammatory effects frequently observed for HDAC inhibitors in (pre)clinical use.

Kaposi's sarcoma (KS) is a mesenchymal tumour, which is caused by Kaposi's sarcoma herpesvirus (KSHV) and develops under inflammatory conditions. KSHV-infected endothelial spindle cells, the neoplastic cells in KS, show increased invasiveness, attributed to the elevated expression of metalloproteinases (MMPs) and cyclooxygenase-2 (COX-2). The majority of these spindle cells harbour latent KSHV genomes, while a minority undergoes lytic reactivation with subsequent production of new virions and viral or cellular chemo- and cytokines, which may promote tumour invasion and dissemination. In order to better understand KSHV pathogenesis, we investigated cellular mechanisms underlying the lytic reactivation of KSHV. Using a combination of small molecule library screening and siRNA silencing we found a STE20 kinase family member, MAP4K4, to be involved in KSHV reactivation from latency and to contribute to the invasive phenotype of KSHV-infected endothelial cells by regulating COX-2, MMP-7, and MMP-13 expression. This kinase is also highly expressed in KS spindle cells in vivo. These findings suggest that MAP4K4, a known mediator of inflammation, is involved in KS aetiology by regulating KSHV lytic reactivation, expression of MMPs and COX-2, and, thereby modulating invasiveness of KSHV-infected endothelial cells.

Interleukin-1 (IL-1) induces the internalization of its cognate receptor from the plasma membrane. However, it has remained elusive as to how this mechanism affects the IL-1-induced signal transduction. In this study, we used small-molecule inhibitors of receptor endocytosis to analyze the effects on IL-1-induced signal transduction pathways. We demonstrate that the inhibition of endocytosis down-modulates IL-1-induced NF-κB-dependent gene expression at a level downstream of nuclear translocation and DNA binding of NF-κB. Moreover, we report that the reduced NF-κB-dependent gene expression disrupts feedback inhibition loops terminating the activation of mitogen-activated protein kinases and down-regulating the expression of IL-1-induced mRNAs. Collectively, we show that the inhibition of endocytosis causes a dysregulation of IL-1-induced signal transduction and gene expression demonstrating an important role for receptor internalization in IL-1 signaling., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Kaposi's Sarcoma (KS), caused by Kaposi's Sarcoma Herpesvirus (KSHV), is a highly vascularised angiogenic tumor of endothelial cells, characterized by latently KSHV-infected spindle cells and a pronounced inflammatory infiltrate. Several KSHV proteins, including LANA-1 (ORF73), vCyclin (ORF72), vGPCR (ORF74), vIL6 (ORF-K2), vCCL-1 (ORF-K6), vCCL-2 (ORF-K4) and K1 have been shown to exert effects that can lead to the proliferation and atypical differentiation of endothelial cells and/or the secretion of cytokines with angiogenic and inflammatory properties (VEGF, bFGF, IL6, IL8, GROα, and TNFβ). To investigate a role of the KSHV K15 protein in KSHV-mediated angiogenesis, we carried out a genome wide gene expression analysis on primary endothelial cells infected with KSHV wildtype (KSHVwt) and a KSHV K15 deletion mutant (KSHVΔK15). We found RCAN1/DSCR1 (Regulator of Calcineurin 1/Down Syndrome critical region 1), a cellular gene involved in angiogenesis, to be differentially expressed in KSHVwt- vs KSHVΔK15-infected cells. During physiological angiogenesis, expression of RCAN1 in endothelial cells is regulated by VEGF (vascular endothelial growth factor) through a pathway involving the activation of PLCγ1, Calcineurin and NFAT1. We found that K15 directly recruits PLCγ1, and thereby activates Calcineurin/NFAT1-dependent RCAN1 expression which results in the formation of angiogenic tubes. Primary endothelial cells infected with KSHVwt form angiogenic tubes upon activation of the lytic replication cycle. This effect is abrogated when K15 is deleted (KSHVΔK15) or silenced by an siRNA targeting the K15 expression. Our study establishes K15 as one of the KSHV proteins that contribute to KSHV-induced angiogenesis.