• Combined high-throughput library screening and next generation RNA sequencing uncover microRNAs controlling human cardiac fibroblast biology

      Schimmel, Katharina; Stojanović, Stevan D.; Huang, Cheng Kai; Jung, Mira; Meyer, Martin H.; Xiao, Ke; Grote-Levi, Lea; Bär, Christian; Pfanne, Angelika; Mitzka, Saskia; et al. (Elsevier, 2021-01-01)
      Background: Myocardial fibrosis is a hallmark of the failing heart, contributing to the most common causes of deaths worldwide. Several microRNAs (miRNAs, miRs) controlling cardiac fibrosis were identified in recent years; however, a more global approach to identify miRNAs involved in fibrosis is missing. Methods and results: Functional miRNA mimic library screens were applied in human cardiac fibroblasts (HCFs) to identify annotated miRNAs inducing proliferation. In parallel, miRNA deep sequencing was performed after subjecting HCFs to proliferating and resting stimuli, additionally enabling discovery of novel miRNAs. In-depth in vitro analysis confirmed the pro-fibrotic nature of selected, highly conserved miRNAs miR-20a-5p and miR-132-3p. To determine downstream cellular pathways and their role in the fibrotic response, targets of the annotated miRNA candidates were modulated by synthetic siRNA. We here provide evidence that repression of autophagy and detoxification of reactive oxygen species by miR-20a-5p and miR-132-3p explain some of their pro-fibrotic nature on a mechanistic level. Conclusion: We here identified both miR-20a-5p and miR-132-3p as crucial regulators of fibrotic pathways in an in vitro model of human cardiac fibroblast biology.
    • Identification of miRNAs associated with dendritic cell dysfunction during Acute and Chronic Hepatitis B virus infection.

      Singh, Avishek Kumar; Rooge, Sheetalnath Babasaheb; Varshney, Aditi; Vasudevan, Madavan; Kumar, Manoj; Geffers, Robert; Kumar, Vijay; Sarin, Shiv Kumar; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley, 2020-10-27)
      The uptake or expression of hepatitis B virus proteins by Dendritic cells (DCs) is considered important for disease outcome. Differential expression of microRNA may have a role in viral persistence and hepatocellular injury. The miRNA expression was investigated by microarray in DCs from different stages of HBV infection and liver disease viz., immune active (IA; n=20); low replicative (LR; n=20); HBeAg negative (n=20); acute viral hepatitis (AVH, n=20) and healthy controls (n=20). miRNA levels were analyzed by unsupervised hierarchical clustering and principal component analyses and validated by qPCR. The miRNA-mRNA regulatory networks identified 19 miRNAs and 12 target gene interactions in MHC and other immune pathways. miR-2278, miR-615-3p and miR-3681-3p were down-regulated in IA group compared to healthy control, miR-152-3p and miR-3613-3p in LR group compared to IA group and miR-152-3p and miR-503-3p in HBe negative compared to LR group. However, miR-7-1-1-3p, miR-192-5p, miR-195-5p and miR-32-5p in LR, miR-342-3p and miR-940 in HBe negative, and miR-34a-5p, miR-130b-3p, miR-221-3p, miR-320a, miR-324-5p and miR-484 in AVH were up-regulated. Further, qPCR confirmed changes in miRNA levels and their target genes associated with antigen processing and presentation. Thus, a deregulated network of miRNAs-mRNAs in DCs seems responsible for impaired immune response during HBV pathogenesis. This article is protected by copyright. All rights reserved.
    • Determining the effects of trastuzumab, cetuximab and afatinib by phosphoprotein, gene expression and phenotypic analysis in gastric cancer cell lines.

      Ebert, Karolin; Zwingenberger, Gwen; Barbaria, Elena; Keller, Simone; Heck, Corinna; Arnold, Rouven; Hollerieth, Vanessa; Mattes, Julian; Geffers, Robert; Raimúndez, Elba; et al. (BMC, 2020-10-28)
      Background: Gastric cancer is the fifth most frequently diagnosed cancer and the third leading cause of cancer death worldwide. The molecular mechanisms of action for anti-HER-family drugs in gastric cancer cells are incompletely understood. We compared the molecular effects of trastuzumab and the other HER-family targeting drugs cetuximab and afatinib on phosphoprotein and gene expression level to gain insights into the regulated pathways. Moreover, we intended to identify genes involved in phenotypic effects of anti-HER therapies. Methods: A time-resolved analysis of downstream intracellular kinases following EGF, cetuximab, trastuzumab and afatinib treatment was performed by Luminex analysis in the gastric cancer cell lines Hs746T, MKN1, MKN7 and NCI-N87. The changes in gene expression after treatment of the gastric cancer cell lines with EGF, cetuximab, trastuzumab or afatinib for 4 or 24 h were analyzed by RNA sequencing. Significantly enriched pathways and gene ontology terms were identified by functional enrichment analysis. Furthermore, effects of trastuzumab and afatinib on cell motility and apoptosis were analyzed by time-lapse microscopy and western blot for cleaved caspase 3. Results: The Luminex analysis of kinase activity revealed no effects of trastuzumab, while alterations of AKT1, MAPK3, MEK1 and p70S6K1 activations were observed under cetuximab and afatinib treatment. On gene expression level, cetuximab mainly affected the signaling pathways, whereas afatinib had an effect on both signaling and cell cycle pathways. In contrast, trastuzumab had little effects on gene expression. Afatinib reduced average speed in MKN1 and MKN7 cells and induced apoptosis in NCI-N87 cells. Following treatment with afatinib, a list of 14 genes that might be involved in the decrease of cell motility and a list of 44 genes that might have a potential role in induction of apoptosis was suggested. The importance of one of these genes (HBEGF) as regulator of motility was confirmed by knockdown experiments. Conclusions: Taken together, we described the different molecular effects of trastuzumab, cetuximab and afatinib on kinase activity and gene expression. The phenotypic changes following afatinib treatment were reflected by altered biological functions indicated by overrepresentation of gene ontology terms. The importance of identified genes for cell motility was validated in case of HBEGF.
    • IDH1/2 mutations in acute myeloid leukemia patients and risk of coronary artery disease and cardiac dysfunction—a retrospective propensity score analysis

      Kattih, Badder; Shirvani, Amir; Klement, Piroska; Garrido, Abel Martin; Gabdoulline, Razif; Liebich, Alessandro; Brandes, Maximilian; Chaturvedi, Anuhar; Seeger, Timon; Thol, Felicitas; et al. (Springer Nature, 2020-01-01)
      Clonal hematopoiesis of indeterminate potential (CHIP) is linked to leukemia gene mutations and associates with an increased risk for coronary artery disease and poor prognosis in ischemic cardiomyopathy. Two recurrently mutated genes in CHIP and adult acute myeloid leukemia (AML) encode for isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2). Global expression of mutant IDH2 in transgenic mice-induced dilated cardiomyopathy and muscular dystrophy. In this retrospective observational study, we investigated whether mutant IDH1/2 predisposes to cardiovascular disease in AML patients. Among 363 AML patients, IDH1 and IDH2 mutations were detected in 26 (7.2%) and 39 patients (10.7%), respectively. Mutant IDH1 patients exhibited a significantly higher prevalence of coronary artery disease (26.1% vs. 6.4%, p = 0.002). Applying inverse probability-weighting analysis, patients with IDH1/2 mutations had a higher risk for a declining cardiac function during AML treatment compared to IDH1/2 wild type patients [left ventricular ejection fraction pretreatment compared to 10 months after diagnosis: 59.2% to 41.9% (p < 0.001) vs 58.5% to 55.4% (p = 0.27), respectively]. Mechanistically, RNA sequencing and immunostaining in hiPS-derived cardiomyocytes indicated that the oncometabolite R-2HG exacerbated doxorubicin mediated cardiotoxicity. Evaluation of IDH1/2 mutation status may therefore help identifying AML patients at risk for cardiovascular complications during cytotoxic treatment. Similar articles
    • Severe COVID-19 Is Marked by a Dysregulated Myeloid Cell Compartment.

      Schulte-Schrepping, Jonas; Reusch, Nico; Paclik, Daniela; Baßler, Kevin; Schlickeiser, Stephan; Zhang, Bowen; Krämer, Benjamin; Krammer, Tobias; Brumhard, Sophia; Bonaguro, Lorenzo; et al. (Elsevier /Cell Press), 2020-08-05)
      Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DRhiCD11chi inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DRlo monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19.
    • Rare heterozygous GDF6 variants in patients with renal anomalies.

      Martens, Helge; Hennies, Imke; Getwan, Maike; Christians, Anne; Weiss, Anna-Carina; Brand, Frank; Gjerstad, Ann Christin; Christians, Arne; Gucev, Zoran; Geffers, Robert; et al. (Springer Nature, 2020-07-31)
      Although over 50 genes are known to cause renal malformation if mutated, the underlying genetic basis, most easily identified in syndromic cases, remains unsolved in most patients. In search of novel causative genes, whole-exome sequencing in a patient with renal, i.e., crossed fused renal ectopia, and extrarenal, i.e., skeletal, eye, and ear, malformations yielded a rare heterozygous variant in the GDF6 gene encoding growth differentiation factor 6, a member of the BMP family of ligands. Previously, GDF6 variants were reported to cause pleiotropic defects including skeletal, e.g., vertebral, carpal, tarsal fusions, and ocular, e.g., microphthalmia and coloboma, phenotypes. To assess the role of GDF6 in the pathogenesis of renal malformation, we performed targeted sequencing in 193 further patients identifying rare GDF6 variants in two cases with kidney hypodysplasia and extrarenal manifestations. During development, gdf6 was expressed in the pronephric tubule of Xenopus laevis, and Gdf6 expression was observed in the ureteric tree of the murine kidney by RNA in situ hybridization. CRISPR/Cas9-derived knockout of Gdf6 attenuated migration of murine IMCD3 cells, an effect rescued by expression of wild-type but not mutant GDF6, indicating affected variant function regarding a fundamental developmental process. Knockdown of gdf6 in Xenopus laevis resulted in impaired pronephros development. Altogether, we identified rare heterozygous GDF6 variants in 1.6% of all renal anomaly patients and 5.4% of renal anomaly patients additionally manifesting skeletal, ocular, or auricular abnormalities, adding renal hypodysplasia and fusion to the phenotype spectrum of GDF6 variant carriers and suggesting an involvement of GDF6 in nephrogenesis.
    • YB-1 Interferes with TNFα-TNFR Binding and Modulates Progranulin-Mediated Inhibition of TNFα Signaling.

      Hessman, Christopher L; Hildebrandt, Josephine; Shah, Aneri; Brandt, Sabine; Bock, Antonia; Frye, Björn C; Raffetseder, Ute; Geffers, Robert; Brunner-Weinzierl, Monika C; Isermann, Berend; et al. (MDPI, 2020-09-25)
      Inflammation and an influx of macrophages are common elements in many diseases. Among pro-inflammatory cytokines, tumor necrosis factor α (TNFα) plays a central role by amplifying the cytokine network. Progranulin (PGRN) is a growth factor that binds to TNF receptors and interferes with TNFα-mediated signaling. Extracellular PGRN is processed into granulins by proteases released from immune cells. PGRN exerts anti-inflammatory effects, whereas granulins are pro-inflammatory. The factors coordinating these ambivalent functions remain unclear. In our study, we identify Y-box binding protein-1 (YB-1) as a candidate for this immune-modulating activity. Using a yeast-2-hybrid assay with YB-1 protein as bait, clones encoding for progranulin were selected using stringent criteria for strong interaction. We demonstrate that at physiological concentrations, YB-1 interferes with the binding of TNFα to its receptors in a dose-dependent manner using a flow cytometry-based binding assay. We show that YB-1 in combination with progranulin interferes with TNFα-mediated signaling, supporting the functionality with an NF-κB luciferase reporter assay. Together, we show that YB-1 displays immunomodulating functions by affecting the binding of TNFα to its receptors and influencing TNFα-mediated signaling via its interaction with progranulin.
    • Complete Genome Sequence and Manual Reannotation of Mycobacterium avium subsp. Strain DSM 44135.

      Goethe, Ralph; Basler, Tina; Meissner, Thorsten; Goethe, Elke; Spröer, Cathrin; Swiderski, Jolantha; Gerlach, Gerald-F; Weiss, Siegfried; Jarek, Michael; Bunk, Boyke; et al. (ASM, 2020-08-13)
      Here, we report the complete genome sequence of the Mycobacterium avium subsp. paratuberculosis reference strain DSM 44135, amended with a manual genome reannotation. The strain was originally described as M. paratuberculosis strain 6783. It was isolated from feces from a dairy cow in northern Germany.
    • Low-load pathogen spillover predicts shifts in skin microbiome and survival of a terrestrial-breeding amphibian.

      Becker, C Guilherme; Bletz, Molly C; Greenspan, Sasha E; Rodriguez, David; Lambertini, Carolina; Jenkinson, Thomas S; Guimarães, Paulo R; Assis, Ana Paula A; Geffers, Robert; Jarek, Michael; et al. (Royal Society of London, 2019-08-14)
      Wildlife disease dynamics are strongly influenced by the structure of host communities and their symbiotic microbiota. Conspicuous amphibian declines associated with the waterborne fungal pathogen Batrachochytrium dendrobatidis (Bd) have been observed in aquatic-breeding frogs globally. However, less attention has been given to cryptic terrestrial-breeding amphibians that have also been declining in tropical regions. By experimentally manipulating multiple tropical amphibian assemblages harbouring natural microbial communities, we tested whether Bd spillover from naturally infected aquatic-breeding frogs could lead to Bd amplification and mortality in our focal terrestrial-breeding host: the pumpkin toadlet Brachycephalus pitanga. We also tested whether the strength of spillover could vary depending on skin bacterial transmission within host assemblages. Terrestrial-breeding toadlets acquired lethal spillover infections from neighbouring aquatic hosts and experienced dramatic but generally non-protective shifts in skin bacterial composition primarily attributable to their Bd infections. By contrast, aquatic-breeding amphibians maintained mild Bd infections and higher survival, with shifts in bacterial microbiomes that were unrelated to Bd infections. Our results indicate that Bd spillover from even mildly infected aquatic-breeding hosts may lead to dysbiosis and mortality in terrestrial-breeding species, underscoring the need to further investigate recent population declines of terrestrial-breeding amphibians in the tropics.
    • Integrative Bioinformatic Analyses of Global Transcriptome Data Decipher Novel Molecular Insights into Cardiac Anti-Fibrotic Therapies.

      Fuchs, Maximilian; Kreutzer, Fabian Philipp; Kapsner, Lorenz A; Mitzka, Saskia; Just, Annette; Perbellini, Filippo; Terracciano, Cesare M; Xiao, Ke; Geffers, Robert; Bogdan, Christian; et al. (MDPI, 2020-07-02)
      Integrative bioinformatics is an emerging field in the big data era, offering a steadily increasing number of algorithms and analysis tools. However, for researchers in experimental life sciences it is often difficult to follow and properly apply the bioinformatical methods in order to unravel the complexity and systemic effects of omics data. Here, we present an integrative bioinformatics pipeline to decipher crucial biological insights from global transcriptome profiling data to validate innovative therapeutics. It is available as a web application for an interactive and simplified analysis without the need for programming skills or deep bioinformatics background. The approach was applied to an ex vivo cardiac model treated with natural anti-fibrotic compounds and we obtained new mechanistic insights into their anti-fibrotic action and molecular interplay with miRNAs in cardiac fibrosis. Several gene pathways associated with proliferation, extracellular matrix processes and wound healing were altered, and we could identify micro (mi) RNA-21-5p and miRNA-223-3p as key molecular components related to the anti-fibrotic treatment. Importantly, our pipeline is not restricted to a specific cell type or disease and can be broadly applied to better understand the unprecedented level of complexity in big data research.
    • Worlds Apart - Transcriptome Profiles of Key Oral Microbes in the Periodontal Pocket Compared to Single Laboratory Culture Reflect Synergistic Interactions.

      Deng, Zhi-Luo; Sztajer, Helena; Jarek, Michael; Bhuju, Sabin; Wagner-Döbler, Irene; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2018-02-06)
      Periodontitis is a worldwide prevalent oral disease which results from dysbiosis of the periodontal microbiome. Some of the most active microbial players, e.g., Porphyromonas gingivalis, Treponema denticola, and Fusobacterium nucleatum, have extensively been studied in the laboratory, but it is unclear to which extend these findings can be transferred to in vivo conditions. Here we show that the transcriptional profiles of P. gingivalis, T. denticola, and F. nucleatum in the periodontal niche are distinct from those in single laboratory culture and exhibit functional similarities. GO (gene ontology) term enrichment analysis showed up-regulation of transporters, pathogenicity related traits and hemin/heme uptake mechanisms for all three species in vivo. Differential gene expression analysis revealed that cysteine proteases, transporters and hemin/heme-binding proteins were highly up-regulated in the periodontal niche, while genes involved in DNA modification were down-regulated. The data suggest strong interactions between those three species regarding protein degradation, iron up-take, and mobility in vivo, explaining their enhanced synergistic pathogenicity. We discovered a strikingly high frequency of Single Nucleotide Polymorphisms (SNPs) in vivo. For F. nucleatum we discovered a total of 127,729 SNPs in periodontal niche transcripts, which were found in similar frequency in health and disease and covered the entire genome, suggesting continuous evolution in the host. We conclude that metabolic interactions shape gene expression in vivo. Great caution is required when inferring pathogenicity of microbes from laboratory data, and microdiversity is an important adaptive trait of natural communities.
    • Long-Term Neuroinflammation Induced by Influenza A Virus Infection and the Impact on Hippocampal Neuron Morphology and Function.

      Hosseini, Shirin; Wilk, Esther; Michaelsen-Preusse, Kristin; Gerhauser, Ingo; Baumgärtner, Wolfgang; Geffers, Robert; Schughart, Klaus; Korte, Martin; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Society for Neuroscience, 2018-02-27)
      Acute influenza infection has been reported to be associated with neurological symptoms. However, the long-term consequences of an infection with neurotropic and non-neurotropic influenza A virus (IAV) variants for the CNS remain elusive. We can show that spine loss in the hippocampus after infection with neurotropic H7N7 (rSC35M) and non-neurotropic H3N2 (maHK68) in female C57BL/6 mice persists well beyond the acute phase of the disease. Although spine number was significantly reduced at 30 d postinfection (dpi) with H7N7 or H3N2, full recovery could only be observed much later at 120 dpi. Infection with H1N1 virus, which was shown previously to affect spine number and hippocampus-dependent learning acutely, had no significant long-term effects. Spine loss was associated with an increase in the number of activated microglia, reduced long-term potentiation in the hippocampus, and impairment in spatial memory formation, indicating that IAV-associated inflammation induced functional and structural alterations in hippocampal networks. Transcriptome analyses revealed regulation of many inflammatory and neuron- and glia-specific genes in H3N2- and H7N7-infected mice at day 18 and in H7N7-infected mice at day 30 pi that related to the structural and functional alterations. Our data provide evidence that neuroinflammation induced by neurotropic H7N7 and infection of the lung with a non-neurotropic H3N2 IAV result in long-term impairments in the CNS. IAV infection in humans may therefore not only lead to short-term responses in infected organs, but may also trigger neuroinflammation and associated chronic alterations in the CNS.SIGNIFICANCE STATEMENT In the acute phase of influenza infection, neuroinflammation can lead to alterations in hippocampal neuronal morphology and cognitive deficits. The results of this study now also provide evidence that neuroinflammation induced by influenza A virus (IAV) infection can induce longer-lasting, virus-specific alterations in neuronal connectivity that are still detectable 1 month after infection and are associated with impairments in spatial memory formation. IAV infection in humans may therefore not only lead to short-term responses in infected organs, but may also trigger neuroinflammation and associated chronic alterations in the CNS.
    • Synergistic activity of IDH1 inhibitor BAY1436032 with azacitidine in IDH1 mutant acute myeloid leukemia.

      Chaturvedi, Anuhar; Gupta, Charu; Gabdoulline, Razif; Borchert, Nora M; Goparaju, Ramya; Kaulfuss, Stefan; Görlich, Kerstin; Schottmann, Renate; Othman, Basem; Welzenbach, Julia; et al. (Ferrraata Storti Foundation, 2020-04-02)
      Mutant IDH1 (mIDH1) inhibitors have shown single-agent activity in relapsed/refractory AML, though most patients eventually relapse. We evaluated the efficacy and molecular mechanism of the combination treatment with azacitidine, which is currently the standard of care in older AML patients, and mIDH1 inhibitor BAY1436032. Both compounds were evaluated in vivo as single agents and in combination with sequential (azacitidine, followed by BAY1436032) or simultaneous application in two human IDH1 mutated AML xenograft models. Combination treatment significantly prolonged survival compared to single agent or control treatment (P<.005). The sequential combination treatment depleted leukemia stem cells (LSC) by 470-fold. Interestingly, the simultaneous combination treatment depleted LSCs by 33,150-fold compared to control mice. This strong synergy is mediated through inhibition of MAPK/ERK and RB/E2F signaling. Our data strongly argues for the concurrent application of mIDH1 inhibitors and azacitidine and predicts improved outcome of this regimen in IDH1 mutated AML patients.
    • Donor-derived IL-17A and IL-17F deficiency triggers Th1 allo-responses and increases gut leakage during acute GVHD.

      Odak, Ivan; Depkat-Jakob, Alina; Beck, Maleen; Jarek, Michael; Yu, Yan; Seidler, Ursula; David, Sascha; Ganser, Arnold; Förster, Reinhold; Prinz, Immo; et al. (PLOS, 2020-04-06)
      s Metrics Comments Media Coverage Abstract Introduction Material and methods Results Discussion Supporting information Acknowledgments References Reader Comments (0) Media Coverage (0) Figures Abstract IL-17A and IL-17F cytokines are important regulators of acute graft-versus-host-disease (GVHD). However, contrary effects of these cytokines in inflammatory diseases have been reported. To investigate the effects of donor-derived IL-17A and IL-17F on GVHD, we made use of single (Il17a-/- or Il17f-/-) and double deficient (Il17af-/-) allogeneic donor CD4+ T cells. We could demonstrate that transplantation of Il17af-/- CD4+ donor T cells led to aggravated GVHD. However, this phenotype was not observed after transplantation of single, Il17a-/- or Il17f-/-, deficient CD4+ T cells, suggesting redundant effects of IL-17A and IL-17F. Moreover, Il17af-/- cell recipients showed an increase of systemic IFNγ, indicating a heightened pro-inflammatory state, as well as infiltration of IFNγ-secreting CD4+ T cells in the recipients’ intestinal tract. These recipients exhibited significant gut leakage, and markedly macrophage infiltration in the gastrointestinal epithelial layer. Moreover, we saw evidence of impaired recovery of gut epithelial cells in recipients of Il17af-/- CD4+ T cells. In this study, we show that IL-17A/F double deficiency of donor CD4+ T cells leads to accelerated GVHD and therefore highlight the importance of these cytokines. Together, IL-17 cytokines might serve as a brake to an intensified Th1 response, leading to the exacerbated gut damage in acute GVHD.
    • Natural Compound Library Screening Identifies New Molecules for the Treatment of Cardiac Fibrosis and Diastolic Dysfunction.

      Schimmel, Katharina; Jung, Mira; Foinquinos, Ariana; José, Gorka San; Beaumont, Javier; Bock, Katharina; Grote-Levi, Lea; Xiao, Ke; Bär, Christian; Pfanne, Angelika; et al. (Lippincott, Williams & Wilkins, 2020-01-17)
      High-throughput natural compound library screening identified 15 substances with antiproliferative effects in human cardiac fibroblasts. Using multiple in vitro fibrosis assays and stringent selection algorithms, we identified the steroid bufalin (from Chinese toad venom) and the alkaloid lycorine (from Amaryllidaceae species) to be effective antifibrotic molecules both in vitro and in vivo, leading to improvement in diastolic function in 2 hypertension-dependent rodent models of cardiac fibrosis. Administration at effective doses did not change plasma damage markers or the morphology of kidney and liver, providing the first toxicological safety data. Using next-generation sequencing, we identified the conserved microRNA 671-5p and downstream the antifibrotic selenoprotein P1 as common effectors of the antifibrotic compounds.
    • Chimeric antigen receptor-induced BCL11B suppression propagates NK-like cell development.

      Maluski, Marcel; Ghosh, Arnab; Herbst, Jessica; Scholl, Vanessa; Baumann, Rolf; Huehn, Jochen; Geffers, Robert; Meyer, Johann; Maul, Holger; Eiz-Vesper, Britta; et al. (American Society for Clinical Investigation, 2019-12-02)
      The transcription factor B cell CLL/lymphoma 11B (BCL11B) is indispensable for T lineage development of lymphoid progenitors. Here, we show that chimeric antigen receptor (CAR) expression during early phases of ex vivo generation of lymphoid progenitors suppressed BCL11B, leading to suppression of T cell-associated gene expression and acquisition of NK cell-like properties. Upon adoptive transfer into hematopoietic stem cell transplant recipients, CAR-expressing lymphoid progenitors differentiated into CAR-induced killer (CARiK) cells that mediated potent antigen-directed antileukemic activity even across MHC barriers. CD28 and active immunoreceptor tyrosine-based activation motifs were critical for a functional CARiK phenotype. These results give important insights into differentiation of murine and human lymphoid progenitors driven by synthetic CAR transgene expression and encourage further evaluation of ex vivo-generated CARiK cells for targeted immunotherapy.
    • Identification of Ppar-modulated miRNA hubs that target the fibrotic tumor microenvironment.

      Winkler, Ivana; Bitter, Catrin; Winkler, Sebastian; Weichenhan, Dieter; Thavamani, Abhishek; Hengstler, Jan G; Borkham-Kamphorst, Erawan; Kohlbacher, Oliver; Plass, Christoph; Geffers, Robert; et al. (National Academy of Sciences, 2020-01-07)
      Liver fibrosis interferes with normal liver function and facilitates hepatocellular carcinoma (HCC) development, representing a major threat to human health. Here, we present a comprehensive perspective of microRNA (miRNA) function on targeting the fibrotic microenvironment. Starting from a murine HCC model, we identify a miRNA network composed of 8 miRNA hubs and 54 target genes. We show that let-7, miR-30, miR-29c, miR-335, and miR-338 (collectively termed antifibrotic microRNAs [AF-miRNAs]) down-regulate key structural, signaling, and remodeling components of the extracellular matrix. During fibrogenic transition, these miRNAs are transcriptionally regulated by the transcription factor Pparγ and thus we identify a role of Pparγ as regulator of a functionally related class of AF-miRNAs. The miRNA network is active in human HCC, breast, and lung carcinomas, as well as in 2 independent mouse liver fibrosis models. Therefore, we identify a miRNA:mRNA network that contributes to formation of fibrosis in tumorous and nontumorous organs of mice and humans.
    • Antiviral potential of human IFN-α subtypes against influenza A H3N2 infection in human lung explants reveals subtype-specific activities.

      Matos, Aline da Rocha; Wunderlich, Katharina; Schloer, Sebastian; Schughart, Klaus; Geffers, Robert; Seders, Martine; Witt, Marlous de; Christersson, Anmari; Wiewrodt, Rainer; Wiebe, Karsten; et al. (Taylor & Francis Open, 2019-01-01)
      Influenza is an acute respiratory infection causing high morbidity and mortality in annual outbreaks worldwide. Antiviral drugs are limited and pose the risk of resistance development, calling for new treatment options. IFN-α subtypes are immune-stimulatory cytokines with strong antiviral activities against IAV in vitro and in vivo. However, the clinical use of IFN-α2, the only licensed subtype of this multi-gene family, could not prevent or limit IAV infections in humans. However, the other subtypes were not investigated.Therefore, this study evaluated the induction and antiviral potential of all human IFN-α subtypes during H3N2 IAV infection in human lung explants. We found that subtypes with weak antiviral activities were preferentially induced during IAV infection in human lungs. Intriguingly, non-induced subtypes α16, α5 and α4 suppressed viral replication up to 230-fold more efficiently than α2. Furthermore, our results demonstrate that subtypes with stronger antiviral activities induce higher expression of IAV-specific restriction factors and that MxA expression is a determinant of the subtype-specific antiviral activity towards H3N2 IAV. These results corroborate that IFN-α subtypes exhibit differential antiviral activities and emphasize that subtypes α16, α5 and α4 should be further investigated for the prevention and treatment of severe infections with seasonal H3N2 IAV.
    • Reprogramming of Small Noncoding RNA Populations in Peripheral Blood Reveals Host Biomarkers for Latent and Active Mycobacterium tuberculosis Infection.

      de Araujo, Leonardo Silva; Ribeiro-Alves, Marcelo; Leal-Calvo, Thyago; Leung, Janaína; Durán, Verónica; Samir, Mohamed; Talbot, Steven; Tallam, Aravind; Mello, Fernanda Carvalho de Queiroz; Geffers, Robert; et al. (America Society of Microbiology (ASM), 2019-12-03)
      In tuberculosis (TB), as in other infectious diseases, studies of small noncoding RNAs (sncRNA) in peripheral blood have focused on microRNAs (miRNAs) but have neglected the other major sncRNA classes in spite of their potential functions in host gene regulation. Using RNA sequencing of whole blood, we have therefore determined expression of miRNA, PIWI-interacting RNA (piRNA), small nucleolar RNA (snoRNA), and small nuclear RNA (snRNA) in patients with TB (n = 8), latent TB infection (LTBI; n = 21), and treated LTBI (LTBItt; n = 6) and in uninfected exposed controls (ExC; n = 14). As expected, sncRNA reprogramming was greater in TB than in LTBI, with the greatest changes seen in miRNA populations. However, substantial dynamics were also evident in piRNA and snoRNA populations. One miRNA and 2 piRNAs were identified as moderately accurate (area under the curve [AUC] = 0.70 to 0.74) biomarkers for LTBI, as were 1 miRNA, 1 piRNA, and 2 snoRNAs (AUC = 0.79 to 0.91) for accomplished LTBI treatment. Logistic regression identified the combination of 4 sncRNA (let-7a-5p, miR-589-5p, miR-196b-5p, and SNORD104) as a highly sensitive (100%) classifier to discriminate TB from all non-TB groups. Notably, it reclassified 8 presumed LTBI cases as TB cases, 5 of which turned out to have features of Mycobacterium tuberculosis infection on chest radiographs. SNORD104 expression decreased during M. tuberculosis infection of primary human peripheral blood mononuclear cells (PBMC) and M2-like (P = 0.03) but not M1-like (P = 0.31) macrophages, suggesting that its downregulation in peripheral blood in TB is biologically relevant. Taken together, the results demonstrate that snoRNA and piRNA should be considered in addition to miRNA as biomarkers and pathogenesis factors in the various stages of TB.IMPORTANCE Tuberculosis is the infectious disease with the worldwide largest disease burden and there remains a great need for better diagnostic biomarkers to detect latent and active M. tuberculosis infection. RNA molecules hold great promise in this regard, as their levels of expression may differ considerably between infected and uninfected subjects. We have measured expression changes in the four major classes of small noncoding RNAs in blood samples from patients with different stages of TB infection. We found that, in addition to miRNAs (which are known to be highly regulated in blood cells from TB patients), expression of piRNA and snoRNA is greatly altered in both latent and active TB, yielding promising biomarkers. Even though the functions of many sncRNA other than miRNA are still poorly understood, our results strongly suggest that at least piRNA and snoRNA populations may represent hitherto underappreciated players in the different stages of TB infection.
    • Multiplex profiling of inflammation-related bioactive lipid mediators in Toxocara canis- and Toxocara cati-induced neurotoxocarosis.

      Waindok, Patrick; Janecek-Erfurth, Elisabeth; Lindenwald, Dimitri; Wilk, Esther; Schughart, Klaus; Geffers, Robert; Balas, Laurence; Durand, Thierry; Rund, Katharina Maria; Schebb, Nils Helge; et al. (PLOS, 2019-09-01)
      BACKGROUND: Somatic migration of Toxocara canis- and T. cati-larvae in humans may cause neurotoxocarosis (NT) when larvae accumulate and persist in the central nervous system (CNS). Host- or parasite-induced immunoregulatory processes contribute to the pathogenesis; however, detailed data on involvement of bioactive lipid mediators, e.g. oxylipins or eico-/docosanoids, which are involved in the complex molecular signalling network during infection and inflammation, are lacking. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate if T. canis- and T. cati-induced NT affects the homeostasis of oxylipins during the course of infection, a comprehensive lipidomic profiling in brains (cerebra and cerebella) of experimentally infected C57BL/6J mice was conducted at six different time points post infection (pi) by liquid-chromatography coupled to electrospray tandem mass spectrometry (LC-ESI-MS/MS). Only minor changes were detected regarding pro-inflammatory prostaglandins (cyclooxygenase pathway). In contrast, a significant increase of metabolites resulting from lipoxygenase pathways was observed for both infection groups and brain regions, implicating a predominantly anti-inflammatory driven immune response. This observation was supported by a significantly increased 13-hydroxyoctadecadienoic acid (HODE)/9-HODE ratio during the subacute phase of infection, indicating an anti-inflammatory response to neuroinfection. Except for the specialised pro-resolving mediator (SPM) neuroprotectin D1 (NPD1), which was detected in mice infected with both pathogens during the subacute phase of infection, no other SPMs were detected. CONCLUSIONS/SIGNIFICANCE: The obtained results demonstrate the influence of Toxocara spp. on oxylipins as part of the immune response of the paratenic hosts. Furthermore, this study shows differences in the alteration of the oxylipin composition between T. canis- and T. cati-brain infection. Results contribute to a further understanding of the largely unknown pathogenesis and mechanisms of host-parasite interactions during NT.