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1. Interleukin-22 protects intestinal stem cells against genotoxic stress

Author

Caroline Tizian, Fabian Schumacher, Konrad Gronke, Michael Kofoed-Branzk, Lukas Amann, Antigoni Triantafyllopoulou, Fabian Guendel, Mario Witkowski, Christoph S.N. Klose, Pedro P. Hernandez, Jakob Zimmermann, Hansruedi Glatt, and Andreas Diefenbach

Subjects
0301 basic medicine, Colon, DNA repair, DNA damage, T-Lymphocytes, medicine.medical_treatment, Glucosinolates, Medizin, Apoptosis, Genotoxic Stress, Biology, Ligands, Article, Interleukin 22, Mice, 03 medical and health sciences, 0302 clinical medicine, medicine, Homeostasis, Animals, ddc:610, Intestinal Mucosa, Multidisciplinary, Interleukins, Stem Cells, Innate lymphoid cell, Receptors, Interleukin, Aryl hydrocarbon receptor, Immunity, Innate, Diet, 3. Good health, Cell biology, Intestines, Cell Transformation, Neoplastic, 030104 developmental biology, Cytokine, Receptors, Aryl Hydrocarbon, 030220 oncology & carcinogenesis, Colonic Neoplasms, Mutation, biology.protein, Institut für Ernährungswissenschaft, Stem cell, DNA Damage, Mutagens
Abstract

Environmental genotoxic factors pose a challenge to the genomic integrity of epithelial cells at barrier surfaces that separate host organisms from the environment. They can induce mutations that, if they occur in epithelial stem cells, contribute to malignant transformation and cancer development1,2,3. Genome integrity in epithelial stem cells is maintained by an evolutionarily conserved cellular response pathway, the DNA damage response (DDR). The DDR culminates in either transient cell-cycle arrest and DNA repair or elimination of damaged cells by apoptosis4,5. Here we show that the cytokine interleukin-22 (IL-22), produced by group 3 innate lymphoid cells (ILC3) and γδ T cells, is an important regulator of the DDR machinery in intestinal epithelial stem cells. Using a new mouse model that enables sporadic inactivation of the IL-22 receptor in colon epithelial stem cells, we demonstrate that IL-22 is required for effective initiation of the DDR following DNA damage. Stem cells deprived of IL-22 signals and exposed to carcinogens escaped DDR-controlled apoptosis, contained more mutations and were more likely to give rise to colon cancer. We identified metabolites of glucosinolates, a group of phytochemicals contained in cruciferous vegetables, to be a widespread source of genotoxic stress in intestinal epithelial cells. These metabolites are ligands of the aryl hydrocarbon receptor (AhR)6, and AhR-mediated signalling in ILC3 and γδ T cells controlled their production of IL-22. Mice fed with diets depleted of glucosinolates produced only very low levels of IL-22 and, consequently, the DDR in epithelial cells of mice on a glucosinolate-free diet was impaired. This work identifies a homeostatic network protecting stem cells against challenge to their genome integrity by AhR-mediated ‘sensing’ of genotoxic compounds from the diet. AhR signalling, in turn, ensures on-demand production of IL-22 by innate lymphocytes directly regulating components of the DDR in epithelial stem cells.

Published
2019
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2. Murine T-Cell Transfer Colitis as a Model for Inflammatory Bowel Disease

Author

Patrick, Maschmeyer, Jakob, Zimmermann, and Anja Andrea, Kühl

Subjects
Homeodomain Proteins, Mice, Inbred C57BL, Mice, Knockout, Disease Models, Animal, Colon, Research Design, Animals, Cell Separation, T-Lymphocytes, Helper-Inducer, Colitis, Flow Cytometry, Adoptive Transfer, Workflow
Abstract

Inflammatory bowel disease (IBD) is a group of severe chronic inflammatory conditions of the human gastrointestinal tract. Murine models of colitis have been invaluable tools to improve the understanding of IBD development and pathogenesis. While the disease etiology of IBD is complex and multifactorial, CD4

Published
2021

3. Evaluation of Cytokine Production at the Single Cell Level by Flow Cytometry Upon Polyclonal Stimulation

Author

Jakob, Zimmermann

Subjects
Research Design, Ionomycin, Animals, Cytokines, Humans, Tetradecanoylphorbol Acetate, T-Lymphocytes, Helper-Inducer, Single-Cell Analysis, Flow Cytometry, Cells, Cultured, Workflow
Abstract

An important hallmark for the characterisation of Th cells is their capacity for cytokine expression. In this chapter, we describe how Th cells can be restimulated polyclonally to reveal their cytokine-producing potential that can then be analysed by intracellular staining and flow cytometry.

Published
2021

4. Long-term evolution and short-term adaptation of microbiota strains and sub-strains in mice

Author

Lars Bosshard, Bärbel Stecher, Jakob Zimmermann, Mercedes Gomez de Agüero, Bahtiyar Yilmaz, Tobias Fuhrer, Stephanie C. Ganal-Vonarburg, Wolf-Dietrich Hardt, Irene Keller, Heidi Tschanz-Lischer, Andrew J. Macpherson, Laurent Excoffier, Nerea Fernandez Trigo, Kathleen McCoy, Hai Li, Julien P. Limenitakis, Uwe Sauer, and Catherine Mooser

Subjects
Male, Silent mutation, Ralstonia, Biology, Microbiology, Mice, 03 medical and health sciences, 0302 clinical medicine, Nutrient, Virology, Animals, Metabolomics, Symbiosis, 610 Medicine & health, Gene, 030304 developmental biology, Mutualism (biology), 0303 health sciences, Bacteria, Ecotype, Microbiota, Immunity, Niche differentiation, Genomics, Adaptation, Physiological, Gastrointestinal Microbiome, Intestines, Mice, Inbred C57BL, Taxon, Evolutionary biology, 570 Life sciences, biology, Female, Parasitology, Adaptation, 030217 neurology & neurosurgery
Abstract

Isobiotic mice, with an identical stable microbiota composition, potentially allow models of host-microbial mutualism to be studied over time and between different laboratories. To understand microbiota evolution in these models, we carried out a 6-year experiment in mice colonized with 12 representative taxa. Increased non-synonymous to synonymous mutation rates indicate positive selection in multiple taxa, particularly for genes annotated for nutrient acquisition or replication. Microbial sub-strains that evolved within a single taxon can stably coexist, consistent with niche partitioning of ecotypes in the complex intestinal environment. Dietary shifts trigger rapid transcriptional adaptation to macronutrient and micronutrient changes in individual taxa and alterations in taxa biomass. The proportions of different sub-strains are also rapidly altered after dietary shift. This indicates that microbial taxa within a mouse colony adapt to changes in the intestinal environment by long-term genomic positive selection and short-term effects of transcriptional reprogramming and adjustments in sub-strain proportions. ISSN:1931-3128 ISSN:1934-6069

Published
2021
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5. T-bet and RORα control lymph node formation by regulating embryonic innate lymphoid cell differentiation

Author

Christina, Stehle, Timo, Rückert, Rémi, Fiancette, Dominika W, Gajdasik, Claire, Willis, Carolin, Ulbricht, Pawel, Durek, Mir-Farzin, Mashreghi, Daniela, Finke, Anja Erika, Hauser, David R, Withers, Hyun-Dong, Chang, Jakob, Zimmermann, and Chiara, Romagnani

Subjects
Mice, Lymphoid Tissue, Animals, Cell Differentiation, Cell Lineage, Female, Nuclear Receptor Subfamily 1, Group F, Member 1, Lymph Nodes, Lymphocytes, T-Lymphocytes, Helper-Inducer, Nuclear Receptor Subfamily 1, Group F, Member 3, T-Box Domain Proteins, Immunity, Innate
Abstract

The generation of lymphoid tissues during embryogenesis relies on group 3 innate lymphoid cells (ILC3) displaying lymphoid tissue inducer (LTi) activity and expressing the master transcription factor RORγt. Accordingly, RORγt-deficient mice lack ILC3 and lymphoid structures, including lymph nodes (LN). Whereas T-bet affects differentiation and functions of ILC3 postnatally, the role of T-bet in regulating fetal ILC3 and LN formation remains completely unknown. Using multiple mouse models and single-cell analyses of fetal ILCs and ILC progenitors (ILCP), here we identify a key role for T-bet during embryogenesis and show that its deficiency rescues LN formation in RORγt-deficient mice. Mechanistically, T-bet deletion skews the differentiation fate of fetal ILCs and promotes the accumulation of PLZF

Published
2020

6. Resident macrophages acquire innate immune memory in staphylococcal skin infection

Author

Florens Lohrmann, Neftali Jose Ramirez, Julia Kolter, Mercedes Gomez de Agüero, Jakob Zimmermann, Reinhild Feuerstein, Philipp Henneke, and Aaron James Forde

Subjects
Male, 0301 basic medicine, Mouse, Skin infection, medicine.disease_cause, immune memory, Monocytes, Mice, Immunology and Inflammation, 0302 clinical medicine, Biology (General), Mice, Inbred BALB C, integumentary system, General Neuroscience, General Medicine, macrophages, dermis, 3. Good health, medicine.anatomical_structure, Staphylococcus aureus, Medicine, Female, Staphylococcal Skin Infections, medicine.symptom, Research Article, QH301-705.5, Science, Secondary infection, Inflammation, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Dermis, medicine, Animals, Innate immune system, General Immunology and Microbiology, business.industry, Monocyte, medicine.disease, Immunity, Innate, Mice, Inbred C57BL, Disease Models, Animal, 030104 developmental biology, Immunology, Bone marrow, business, Immunologic Memory, 030215 immunology
Abstract

Staphylococcus aureus (S. aureus) is a common colonizer of healthy skin and mucous membranes. At the same time, S. aureus is the most frequent cause of skin and soft tissue infections. Dermal macrophages (Mφ) are critical for the coordinated defense against invading S. aureus, yet they have a limited life span with replacement by bone marrow derived monocytes. It is currently poorly understood whether localized S. aureus skin infections persistently alter the resident Mφ subset composition and resistance to a subsequent infection. In a strictly dermal infection model we found that mice, which were previously infected with S. aureus, showed faster monocyte recruitment, increased bacterial killing and improved healing upon a secondary infection. However, skin infection decreased Mφ half-life, thereby limiting the duration of memory. In summary, resident dermal Mφ are programmed locally, independently of bone marrow-derived monocytes during staphylococcal skin infection leading to transiently increased resistance against a second infection.

Published
2020
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7. Discrete populations of isotype-switched memory B lymphocytes are maintained in murine spleen and bone marrow

Author

Stefanie Hahne, Katrin Lehmann, Claudia Haftmann, Jannis Kummer, Melanie Weber, Silvia Kühnel, Daniel Schulz, Stefan Kröger, Ralf Köhler, Richard K. Addo, Jakob Zimmermann, Ulrike Menzel, Frederik Heinrich, Andreas Radbruch, Patrick Maschmeyer, Rebecca Cornelis, Anja E. Hauser, Sai T. Reddy, Francesco Siracusa, Gitta Anne Heinz, Ulrik Stervbo, Özen Sercan-Alp, Mir-Farzin Mashreghi, Jonathan Stefanowski, Pawel Durek, René Riedel, Hyun-Dong Chang, Victor Greiff, Marta Ferreira-Gomes, Cora Klaeden, Mairi McGrath, Kerstin Westendorf, and Sandra Naundorf

Subjects
0301 basic medicine, General Physics and Astronomy, Immunological memory, Transcriptome, 0302 clinical medicine, 610 Medicine & health, lcsh:Science, education.field_of_study, B-Lymphocytes, Multidisciplinary, Cell Cycle, RNA sequencing, Isotype, medicine.anatomical_structure, Antibody, Stromal cell, Science, B-cell receptor, Population, Receptors, Antigen, B-Cell, Vascular Cell Adhesion Molecule-1, Spleen, Animals, Wild, Bone Marrow Cells, Mice, Transgenic, Biology, General Biochemistry, Genetics and Molecular Biology, Article, Antibodies, 03 medical and health sciences, Antigen, Adjuvants, Immunologic, medicine, Animals, ddc:610, education, B cell, Cell Proliferation, B cells, General Chemistry, Immunoglobulin Class Switching, B-1 cell, Mice, Inbred C57BL, 030104 developmental biology, Gene Expression Regulation, Immunology, biology.protein, lcsh:Q, Bone marrow, Stromal Cells, 610 Medizin und Gesundheit, Immunologic Memory, 030215 immunology
Abstract

At present, it is not clear how memory B lymphocytes are maintained over time, and whether only as circulating cells or also residing in particular tissues. Here we describe distinct populations of isotype-switched memory B lymphocytes (Bsm) of murine spleen and bone marrow, identified according to individual transcriptional signature and B cell receptor repertoire. A population of marginal zone-like cells is located exclusively in the spleen, while a population of quiescent Bsm is found only in the bone marrow. Three further resident populations, present in spleen and bone marrow, represent transitional and follicular B cells and B1 cells, respectively. A population representing 10-20% of spleen and bone marrow memory B cells is the only one qualifying as circulating. In the bone marrow, all cells individually dock onto VCAM1+ stromal cells and, reminiscent of resident memory T and plasma cells, are void of activation, proliferation and mobility., Nature Communications, 11 (1), ISSN:2041-1723

Published
2020
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8. Specific microbiota enhances intestinal IgA levels by inducing TGF‐β in T follicular helper cells of Peyer's patches in mice

Author

Justus Ninnemann, René Maier, Katharina Werner, Kevin Heiking, Gitta Anne Heinz, Ute Hoffmann, Jakob Zimmermann, Alexander Beller, Britta Siegmund, Andreas Radbruch, Victoria von Goetze, René Riedel, Andrey Kruglov, Hyun-Dong Chang, Katrin Lehmann, Pawel Durek, and Mir-Farzin Mashreghi

Subjects
0301 basic medicine, medicine.medical_treatment, Immunology, Plasma cell, 03 medical and health sciences, Mice, Peyer's Patches, 0302 clinical medicine, Follicular phase, medicine, Immunology and Allergy, Animals, Intestinal Mucosa, Gene, Immunity, Mucosal, Mice, Knockout, Mice, Inbred BALB C, biology, T-Lymphocytes, Helper-Inducer, biology.organism_classification, 3. Good health, Gastrointestinal Microbiome, Immunoglobulin A, 030104 developmental biology, Cytokine, medicine.anatomical_structure, Immunoglobulin class switching, biology.protein, 570 Life sciences, Antibody, Bacteria, Tenericutes, 030215 immunology, Transforming growth factor
Abstract

In humans and mice, mucosal immune responses are dominated by IgA antibodies and the cytokine TGF-β, suppressing unwanted immune reactions but also targeting Ig class switching to IgA. It had been suggested that eosinophils promote the genera- tion and maintenance of mucosal IgA-expressing plasma cells. Here, we demonstrate that not eosinophils, but specific bacteria determine mucosal IgA production. Co-housing of eosinophil-deficient mice with mice having high intestinal IgA levels, as well as the intentional microbiota transfer induces TGF-β expression in intestinal T follicular helper cells, thereby promoting IgA class switching in Peyer’s patches, enhancing IgA+ plasma cell numbers in the small intestinal lamina propria and levels of mucosal IgA. We show that bacteria highly enriched for the genus Anaeroplasma are sufficient to induce these changes and enhance IgA levels when adoptively transferred. Thus, specific members of the intestinal microbiota and not the microbiota as such regulate gut homeostasis, by promoting the expression of immune-regulatory TGF-β and of mucosal IgA.

Published
2020
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9. The intestinal microbiota determines the colitis-inducing potential of T-bet-deficient Th cells in mice

Author

Florian Schattenberg, Patrick Maschmeyer, Pawel Durek, Katrin Lehmann, René Riedel, Anja A. Kühl, Susann Müller, Andreas Radbruch, Jakob Zimmermann, Francesco Siracusa, Melanie Weber, Kerstin Westendorf, and Hyun-Dong Chang

Subjects
0301 basic medicine, Adoptive cell transfer, Short Communication, Cellular differentiation, Immunology, 610 Medicine & health, chemical and pharmacologic phenomena, Inflammation, Biology, Lymphocyte Activation, Inflammatory bowel disease, T‐bet, Pathogenesis, Mice, T helper cells, 03 medical and health sciences, 0302 clinical medicine, Allergy and inflammation, T-Lymphocyte Subsets, medicine, Animals, Immunology and Allergy, Microbiome, Basic, Colitis, Homeodomain Proteins, Mice, Knockout, T cell transfer colitis, Microbiota, Cell Differentiation, hemic and immune systems, T-Lymphocytes, Helper-Inducer, medicine.disease, Adoptive Transfer, Gastrointestinal Microbiome, Mice, Inbred C57BL, Short Communication|Basic, Disease Models, Animal, 030104 developmental biology, 030220 oncology & carcinogenesis, T cell differentiation, medicine.symptom, T-Box Domain Proteins
Abstract

Conflicting evidence has been provided as to whether induction of intestinal inflammation by adoptive transfer of naïve T cells into Rag-/- mice requires expression of the transcription factor T-bet by the T cells. Here, we formally show that the intestinal microbiota composition of the Rag-/- recipient determines whether or not T-bet-deficient Th cells can induce colitis and we have resolved the differences of the two microbiomes, permissive or non-permissive to T-bet-independent colitis. Our data highlight the dominance of the microbiota over particular T cell differentiation programs in the pathogenesis of chronic intestinal inflammation. © 2017 WILEY-VCH Verlag GmbH Co. KGaA, Weinheim.

Published
2017
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10. High‐resolution microbiota flow cytometry reveals dynamic colitis‐associated changes in fecal bacterial composition

Author

Marie Friedrich, Joachim Schumann, René Riedel, Susann Müller, Hyun-Dong Chang, Britta Siegmund, Jakob Zimmermann, Thomas Hübschmann, Rainer Glauben, Andreas Radbruch, Pawel Durek, and Florian Schattenberg

Subjects
0301 basic medicine, Immunology, IBD, Biology, Technical Comment, Microbiology, Flow cytometry, 03 medical and health sciences, chemistry.chemical_compound, Feces, 0302 clinical medicine, fluids and secretions, Single-cell analysis, RNA, Ribosomal, 16S, medicine, Immunology and Allergy, Animals, Humans, DAPI, Colitis, Single‐cell analysis, Phylogeny, medicine.diagnostic_test, Bacteria, Microbiota, 16S ribosomal RNA, medicine.disease, biology.organism_classification, Flow Cytometry, Inflammatory Bowel Diseases, Staining, Gastrointestinal Microbiome, 030104 developmental biology, chemistry, 030220 oncology & carcinogenesis, T‐cell transfer colitis
Abstract

Using high-resolution flow cytometry of bacterial shape (forward scatter) and DNA content (DAPI staining), we detected dramatic differences in the fecal microbiota composition during murine colitis that were validated using 16S rDNA sequencing. This innovative method provides a fast and inexpensive tool to interrogate the microbiota on the single-cell level.

Published
2016

11. Selective targeting of pro-inflammatory Th1 cells by microRNA-148a-specific antagomirs in vivo

Author

Katrin Lehmann, Mir-Farzin Mashreghi, Georg Petkau, Franziska Zügel, Nikolaus Rajewsky, Markus Bardua, Melanie Weber, Falk Hiepe, Gitta Anne Heinz, Sebastian Herzog, Cam Loan Tran, Sarah Schimmelpfennig, Jakob Zimmermann, Claudia Haftmann, Fritz Melchers, René Riedel, Anja A. Kühl, Francesco Siracusa, Hyun-Dong Chang, Jürgen Wittmann, Andreas Radbruch, Patrick Maschmeyer, Bimba F. Hoyer, Petkau, Georg [0000-0002-6687-2667], and Apollo - University of Cambridge Repository

Subjects
0301 basic medicine, Oligonucleotide therapy, Colon, Immunology, Inflammation, Article, Inflammatory bowel disease, Mice, 03 medical and health sciences, In vivo, microRNA, medicine, Animals, Humans, Immunology and Allergy, Colitis, Cells, Cultured, Oligonucleotide, Chemistry, Effector, Twist-Related Protein 1, Antibody titer, Antagomirs, Nuclear Proteins, Pro-inflammatory Th1 cells, Cell Differentiation, Gamma globulin, Chronic inflammation, Th1 Cells, medicine.disease, miRNA-148a, 3. Good health, Mice, Inbred C57BL, Disease Models, Animal, MicroRNAs, 030104 developmental biology, Cancer research, medicine.symptom, T-Box Domain Proteins, Pre-clinical study
Abstract

In T lymphocytes, expression of miR-148a is induced by T-bet and Twist1, and is specific for pro-inflammatory Th1 cells. In these cells, miR-148a inhibits the expression of the pro-apoptotic protein Bim and promotes their survival. Here we use sequence-specific cholesterol-modified oligonucleotides against miR-148a (antagomir-148a) for the selective elimination of pro-inflammatory Th1 cells in vivo. In the murine model of transfer colitis, antagomir-148a treatment reduced the number of pro-inflammatory Th1 cells in the colon of colitic mice by 50% and inhibited miR-148a expression by 71% in the remaining Th1 cells. Expression of Bim protein in colonic Th1 cells was increased. Antagomir-148a-mediated reduction of Th1 cells resulted in a significant amelioration of colitis. The effect of antagomir-148a was selective for chronic inflammation. Antigen-specific memory Th cells that were generated by an acute immune reaction to nitrophenylacetyl-coupled chicken gamma globulin (NP-CGG) were not affected by treatment with antagomir-148a, both during the effector and the memory phase. In addition, antibody titers to NP-CGG were not altered. Thus, antagomir-148a might qualify as an effective drug to selectively deplete pro-inflammatory Th1 cells of chronic inflammation without affecting the protective immunological memory., Highlights • Th1 cells expressing miR-148a mediate colitis in a murine model of IBD. • Antagomir-148a inhibits colitis by selectively depleting Th1 cells from the colon. • Antagomir-148a does not affect the protective immunological memory.

Published
2017

12. T-bet expression by Th cells promotes type 1 inflammation but is dispensable for colitis

Author

Julia Löffler, Patrick Maschmeyer, Claudia Haftmann, Jakob Zimmermann, Max Löhning, René Riedel, H.-D. Chang, Anja A. Kühl, Katrin Lehmann, Joachim R. Grün, Michael Weber, Mir-Farzin Mashreghi, Matthias Mack, Andreas Radbruch, and Kerstin Westendorf

Subjects
0301 basic medicine, Chemokine, Cellular differentiation, Immunology, Gene Expression, chemical and pharmacologic phenomena, Inflammation, Biology, Monocytes, 03 medical and health sciences, Chemokine receptor, Interferon-gamma, Mice, medicine, Immunology and Allergy, Animals, Interferon gamma, Lymphocyte Count, Colitis, Mice, Knockout, Macrophages, Interleukin-17, hemic and immune systems, Cell Differentiation, T-Lymphocytes, Helper-Inducer, Th1 Cells, biology.organism_classification, medicine.disease, Disease Models, Animal, 030104 developmental biology, biology.protein, Th17 Cells, Interleukin 17, Helicobacter hepaticus, medicine.symptom, T-Box Domain Proteins, medicine.drug
Abstract

The transcription factor T-bet is highly expressed by Th cells isolated from the inflamed intestine of Crohn's disease patients, and has been regarded a critical driver of murine T cell-induced colitis. However, we show here that T-bet expression by Th cells is not required for the manifestation of T-cell-induced colitis in the presence of segmented filamentous bacteria and Helicobacter hepaticus. T-bet expression by Th cells controls their survival and localization, their repertoire of chemokine and chemokine receptor expression, the accumulation of monocytes and macrophages in the inflamed colon, and their differentiation to the M1 type, i.e., type 1 inflammation. Nevertheless, T-bet-deficient Th cells efficiently induce colitis, as reflected by weight loss, diarrhea, and colon histopathology. T-bet-deficient Th cells differentiate into Th1/17 cells, able to express IFN-γ and IL-17A upon restimulation. While neutralization of IL-17A exacerbated colitis induced by wild-type or T-bet-deficient Th cells, neutralization of IFN-γ completely abolished colitis.

Published
2015

13. MiR-148a is upregulated by Twist1 and T-bet and promotes Th1-cell survival by regulating the proapoptotic gene Bim

Author

Gitta Anne Heinz, Nikolaus Rajewsky, Markus Bardua, Hans-Martin Jäck, Marina Backhaus, Anna-Barbara Stittrich, Wei-Wei Chen, Max Löhning, Claudia Haftmann, Jakob Zimmermann, Mareen Matz, Joachim Sieper, Christian Neumann, Joachim R. Grün, Uta Lauer, Kristyna Hradilkova, Alexander Scheffold, Julia Siede, René Riedel, Jürgen Wittmann, Hyun-Dong Chang, Andreas Radbruch, Esther E. Weinberger, Mir-Farzin Mashreghi, Ute Hoffmann, Martina Porstner, Katrin Lehmann, Thomas Häupl, Ria Baumgrass, David Zimmel, Kerstin Westendorf, and Zhuo Fang

Subjects
Apoptosis, Arthritis, Rheumatoid, Th1, Twist transcription factor, Mice, 0302 clinical medicine, RNA interference, hemic and lymphatic diseases, Immunology and Allergy, RNA, Small Interfering, Cells, Cultured, Regulation of gene expression, Mice, Knockout, 0303 health sciences, Gene knockdown, Mice, Inbred BALB C, Bcl-2-Like Protein 11, Nuclear Proteins, hemic and immune systems, 3. Good health, Cell biology, miR-148a, Bim, Mir-148a, T-bet, Twist1, RNA Interference, medicine.symptom, Technology Platforms, biological phenomena, cell phenomena, and immunity, Molecular Immunology, Cell Survival, Immunology, Inflammation, chemical and pharmacologic phenomena, Biology, 03 medical and health sciences, Downregulation and upregulation, Proto-Oncogene Proteins, microRNA, medicine, Animals, Humans, Transcription factor, 030304 developmental biology, Twist-Related Protein 1, Membrane Proteins, Th1 Cells, Molecular biology, Mice, Inbred C57BL, MicroRNAs, Gene Expression Regulation, Cardiovascular and Metabolic Diseases, Apoptosis Regulatory Proteins, T-Box Domain Proteins, 030215 immunology
Abstract

Repeatedly activated T helper 1 (Th1) cells present during chronic inflammation can efficiently adapt to the inflammatory milieu, for example, by expressing the transcription factor Twist1, which limits the immunopathology caused by Th1 cells. Here, we show that in repeatedly activated murine Th1 cells, Twist1 and T-bet induce expression of microRNA-148a (miR-148a). miR-148a regulates expression of the proapoptotic gene Bim, resulting in a decreased Bim/Bcl2 ratio. Inhibition of miR-148a by antagomirs in repeatedly activated Th1 cells increases the expression of Bim, leading to enhanced apoptosis. Knockdown of Bim expression by siRNA in miR-148a antagomir-treated cells restores viability of the Th1 cells, demonstrating that miR-148a controls survival by regulating Bim expression. Thus, Twist1 and T-bet not only control the differentiation and function of Th1 cells, but also their persistence in chronic inflammation.

Published
2015

14. Guidelines for the use of flow cytometry and cell sorting in immunological studies

Author

Guadalupe Herrera, Jens Geginat, Daryl Grummitt, Vincenzo Barnaba, Joanne Lannigan, Beate Rückert, Elisabetta Traggiai, Christian Münz, Susanne Melzer, Ari Waisman, Pratip K. Chattopadhyay, Jonas Hahn, T. Vincent Shankey, S Schmid, Julia Tornack, David W. Hedley, Paolo Dellabona, Jürgen Wienands, Ana Cumano, Ester B. M. Remmerswaal, Christopher A. Hunter, Van Duc Dang, Anis Larbi, Timothy P. Bushnell, Mor Gross, Wenjun Ouyang, Vera S. Donnenberg, Lilly Lopez, Holden T. Maecker, Jenny Mjösberg, Christina Stehle, Yanling Liu, Alan M. Stall, Anja E. Hauser, Yousuke Takahama, Mark C. Dessing, Gergely Toldi, Klaus Warnatz, Raghav Palankar, Sussan Nourshargh, Enrico Lugli, Bimba F. Hoyer, Pleun Hombrink, Bartek Rajwa, Sarah Warth, Isabel Panse, Rachael C. Walker, Silvia Piconese, Andrew Filby, Pärt Peterson, Kilian Schober, Silvia Della Bella, Leonie Wegener, Merle Stein, Anne Cooke, Alessandro Moretta, Deborah Kienhöfer, Andrea Cossarizza, Hyun-Dong Chang, Konrad von Volkmann, Jessica P. Houston, Mübeccel Akdis, Andreas Grützkau, Tristan Holland, Jakob Zimmermann, Jonni S. Moore, Dirk Mielenz, Iain B. McInnes, Bo Huang, Paulo Vieira, Thomas Kroneis, Tobit Steinmetz, Kerstin Juelke, Sharon Sanderson, James V. Watson, Srijit Khan, Sally A. Quataert, Winfried F. Pickl, Annika Wiedemann, Sara De Biasi, Andreas Radbruch, James B. Wing, Susann Müller, Ton N. Schumacher, Katy Rezvani, Gloria Martrus, Alexander Scheffold, Toralf Kaiser, Carlo Pucillo, Lara Gibellini, Anna Rubartelli, Qingyu Cheng, Luca Battistini, David Mirrer, David W. Galbraith, Giovanna Borsellino, Ryan R. Brinkman, Tim R. Mosmann, Laura G. Rico, Anita Dreher, Désirée Kunkel, Francesco Annunziato, Pia Kvistborg, Andrea Gori, Chiara Romagnani, Anat Shemer, Toshinori Nakayama, Francisco Sala-de-Oyanguren, Attila Tárnok, Alfonso Blanco, Anna Iannone, Giuseppe Matarese, Thomas Dörner, Virginia Litwin, Michael Lohoff, Petra Bacher, Jordi Petriz, Lorenzo Moretta, Götz R. A. Ehrhardt, Qianjun Zhang, Andrea Cavani, Barry Moran, Christian Maueröder, Immanuel Andrä, Dirk H. Busch, Joe Trotter, Timothy R D J Radstake, Stipan Jonjić, Fritz Melchers, Hans-Martin Jäck, Beatriz Jávega, Gerald Willimsky, Martin Büscher, Henrik E. Mei, Christine S. Falk, Zhigang Tian, Martin Herrmann, Alice Yue, Steffen Jung, Bart Everts, Frank A. Schildberg, John Bellamy Foster, Giovanna Lombardi, Milena Nasi, John P. Nolan, Todd A. Fehniger, Francesco Dieli, Steffen Schmitt, Andreas Dolf, A. Graham Pockley, Claudia Berek, Josef Spidlen, Megan K. Levings, Werner Müller, Baerbel Keller, René A. W. van Lier, Daisy Philips, Susanne Ziegler, Christian Kurts, Malgorzata J. Podolska, Jürgen Ruland, David Voehringer, Kenneth M. Murphy, Marlous van der Braber, Maria Dolores García-Godoy, Sabine Baumgart, Yi Zhao, Antonio Cosma, Falk Hiepe, Charlotte Esser, Pablo Engel, Marcello Veldhoen, Irmgard Förster, Amy E. Lovett-Racke, Günnur Deniz, Burkhard Ludewig, Esther Schimisky, Cristiano Scottà, Marcello Pinti, Jonathan Rebhahn, Regina Stark, Mario Clerici, Liping Yu, Shimon Sakaguchi, Derek Davies, Anna Katharina Simon, Lorenzo Cosmi, Gabriele Multhoff, Kamran Ghoreschi, Quirin Hammer, Henning Ulrich, J. Paul Robinson, Yvonne Samstag, Olivier Lantz, Hannes Stockinger, Xuetao Cao, Simon Fillatreau, David L. Haviland, Natalio Garbi, C. Neudörfl, Kingston H. G. Mills, Salvador Vento-Asturias, Christian Peth, Philip E. Boulais, Diether J. Recktenwald, Burkhard Becher, Tomas Kalina, Michael D. Leipold, Christoph Goettlinger, Gemma A. Foulds, Jane L. Grogan, Axel R. Schulz, James P. Di Santo, Matthias Schiemann, Michael D. Ward, Britta Engelhardt, Birgit Sawitzki, Annette Oxenius, Carl S. Goodyear, Salomé LeibundGut-Landmann, Wolfgang Beisker, Sue Chow, Carsten Watzl, Marie Follo, Erik Lubberts, Peter Wurst, Thomas Schüler, Andreas Diefenbach, Wolfgang Bauer, Hans-Dieter Volk, Luis E. Muñoz, Elmar Endl, Genny Del Zotto, José-Enrique O'Connor, Mairi McGrath, Paul S. Frenette, Dipartimento di Scienze Biomediche, Università degli Studi di Modena e Reggio Emilia (UNIMORE), Cell Biology, Klinik für Dermatologie, Venerologie und Allergologie, Department of Internal Medicine, Università degli Studi di Firenze = University of Florence [Firenze] (UNIFI)-DENOTHE Center, Neuroimmunology Unit, Santa Lucia Foundation (IRCCS), Inorganic Chemistry II, Universität Bayreuth, Caprotec Bioanalytics GmbH, International Occultation Timing Association European Section (IOTA ES), International Occultation Timing Association European Section, Institut der Leibniz-Gemeinschaft, Berlin, Fondazione Santa Lucia (IRCCS), Terry Fox Laboratory, BC Cancer Agency (BCCRC)-British Columbia Cancer Agency Research Centre, Department of Immunology, Chinese Academy of Medical Sciences, Fondazione Don Carlo Gnocchi, Immunologie des Maladies Virales et Autoimmunes (IMVA - U1184), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Experimental Immunology Unit, Dept. of Oncology, DIBIT San Raffaele Scientific Institute, Département d'Immunologie - Department of Immunology, Institut Pasteur [Paris], Charité Hospital, Humboldt-Universität zu Berlin, Universitat de Barcelona (UB), Rheumatologie, Charité - UniversitätsMedizin = Charité - University Hospital [Berlin], Department of Internal Medicine I, University Hospital Schleswig-Holstein, Campus Kiel, Department of Histology and Embryology, University of Rijeka, Weizmann Institute of Science [Rehovot, Israël], Régulation des Infections Rétrovirales, Institutes of Molecular Medicine and Experimental Immunology, University of Bonn, Immunité et cancer (U932), Université Paris Descartes - Paris 5 (UPD5)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Singapore Immunology Network (SIgN), Biomedical Sciences Institute (BMSI), Institute of Virology [Zürich], College of Food Science and Technology [Shangai], Shanghai Ocean University, Institute for Medical Microbiology and Hygiene, University of Marburg, Centre for Transplantation, King's College London (MRC), Guy's Hospital [London], Erasmus University Medical Center [Rotterdam] (Erasmus MC), Unit of Clinical and Experimental Immunology, Humanitas Clinical and Research Institute, Heinrich Pette Institute [Hamburg], Institute of Translational Medicine, Department of Medicine Huddinge, Karolinska Institutet [Stockholm]-Karolinska University Hospital [Stockholm]-Lipid Laboratory, Università di Genova, Dipartimento di Medicina Sperimentale, Department of Environmental Microbiology, Helmholtz Zentrum für Umweltforschung = Helmholtz Centre for Environmental Research (UFZ), Experimental Immunology, Helmholtz Centre for Infection Research (HZI), Viral Immunobiology, Universität Zürich [Zürich] = University of Zurich (UZH)-Institute of Experimental Immunology [Zurich], Department of Radiation Oncology [Munich], Ludwig-Maximilians-Universität München (LMU), Department of Mathematics and Statistics, American University, William Harvey Research Institute, Barts and the London Medical School, Cytometry Laboratories and School of Veterinary Medicine, Purdue University [West Lafayette], Osaka University [Osaka], FACS and Array Core Facility, Johannes Gutenberg - Universität Mainz (JGU), Institute for Cognitive Science, University of Osnabrueck, Department of Molecular Immunology, Medizinische Universität Wien = Medical University of Vienna, Universität Leipzig [Leipzig], Institute of Immunology, School of Life Sciences-University of Science & Technology of China [Suzhou], Lymphopoïèse (Lymphopoïèse (UMR_1223 / U1223 / U-Pasteur_4)), Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institute for Immunology, Processus de Transfert et d'Echanges dans l'Environnement - EA 3819 (PROTEE), Université de Toulon (UTLN), Heinrich-Pette-Institut, Leibniz Institute for Experimental Virology, Enrico Lugli and Pratip K. Chattopadhyay were supported by grants from the Fondazione Cariplo (Grant Ricerca Biomedica 2012/0683), the Italian Ministry of Health (Bando Giovani Ricercatori GR-2011-02347324) and the European Union Marie Curie Career Integration Grant 322093 (all to E.L.). E.L. and P.K.C. are International Society for the Advancement of Cytometry (ISAC) Marylou Ingram scholars. Alice Yue and Ryan R. Brinkman were funded by Genome BC and NSERC. Klaus Warnatz received funding from the German Federal Ministry of Education and Research (BMBF 01EO1303) and the Deutsche Forschungsgemeinschaft (DECIDE, DFG WA 1597/4-1 and the TRR130). The Jung laboratory is supported by funds of the ERC and ISF. Henrik Mei is a 2017-2021 ISAC scholar. Antonio Cosma is supported by the French government program: 'Investissement d'avenir: Equipements d'Excellence' (EQUIPEX)-2010 FlowCyTech, Grant number: ANR-10-EQPX-02-01. Henrik Mei is supported by the Deutsche Forschungsgemeinschaft (DFG, grants Me3644/5-1 and TRR130/TP24)., Università degli Studi di Modena e Reggio Emilia = University of Modena and Reggio Emilia (UNIMORE), Università degli Studi di Firenze = University of Florence (UniFI)-DENOTHE Center, Institut Pasteur [Paris] (IP), Humboldt University Of Berlin, Universität Bonn = University of Bonn, Università degli studi di Genova = University of Genoa (UniGe), Johannes Gutenberg - Universität Mainz = Johannes Gutenberg University (JGU), Universität Leipzig, Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), Obstetrics & Gynecology, Rheumatology, Pediatrics, Landsteiner Laboratory, Other departments, AII - Inflammatory diseases, Università di Modena e Reggio Emilia, DENOTHE Center-University of Florence, Santa Lucia Foundation ( IRCCS ), International Occultation Timing Association European Section ( IOTA ES ), Fondazione Santa Lucia ( IRCCS ), BC Cancer Agency ( BCCRC ) -British Columbia Cancer Agency Research Centre, Fondazione don Carlo Gnocchi, Fondazione IRCCS, Immunologie des Maladies Virales et Autoimmunes ( IMVA - U1184 ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), Département d'Immunologie, Humboldt Universität zu Berlin, Universitat de Barcelona ( UB ), Charité, Weizmann Institute of Science, Université de Bonn, Immunité et cancer ( U932 ), Université Paris Descartes - Paris 5 ( UPD5 ) -Institut Curie-Institut National de la Santé et de la Recherche Médicale ( INSERM ), Singapore Immunology Network ( SIgN ), Agency for Science Technology and Research, College of Food Science and Technology, Centre for Transplantation, King's College London ( MRC ), Erasmus MC University Medical Center, Helmholtz Centre for Environmental Research ( UFZ ), Helmholtz Centre for Infection Research ( HZI ), University of Zürich [Zürich] ( UZH ) -Institute of Experimental Immunology [Zurich], Ludwig-Maximilians-Universität München, Johannes Gutenberg - Universität Mainz ( JGU ), Medical University of Vienna, Lymphopoïèse, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale ( INSERM ), Johannes Gutenberg - University of Mainz ( JGU ), Processus de Transfert et d'Echanges dans l'Environnement - EA 3819 ( PROTEE ), Université de Toulon ( UTLN ), Universita degli studi di Genova, Cossarizza, Andrea, Chang, Hyun-Dong, Radbruch, Andrea, Akdis, Mübeccel, Andrä, Immanuel, Annunziato, Francesco, Bacher, Petra, Barnaba, Vincenzo, Battistini, Luca, Bauer, Wolfgang M., Baumgart, Sabine, Becher, Burkhard, Beisker, Wolfgang, Berek, Claudia, Blanco, Alfonso, Borsellino, Giovanna, Boulais, Philip E., Brinkman, Ryan R., Büscher, Martin, Busch, Dirk H., Bushnell, Timothy P., Cao, Xuetao, Cavani, Andrea, Chattopadhyay, Pratip K., Cheng, Qingyu, Chow, Sue, Clerici, Mario, Cooke, Anne, Cosma, Antonio, Cosmi, Lorenzo, Cumano, Ana, Dang, Van Duc, Davies, Derek, De Biasi, Sara, Del Zotto, Genny, Della Bella, Silvia, Dellabona, Paolo, Deniz, Günnur, Dessing, Mark, Diefenbach, Andrea, Di Santo, Jame, Dieli, Francesco, Dolf, Andrea, Donnenberg, Vera S., Dörner, Thoma, Ehrhardt, Götz R. A., Endl, Elmar, Engel, Pablo, Engelhardt, Britta, Esser, Charlotte, Everts, Bart, Dreher, Anita, Falk, Christine S., Fehniger, Todd A., Filby, Andrew, Fillatreau, Simon, Follo, Marie, Förster, Irmgard, Foster, John, Foulds, Gemma A., Frenette, Paul S., Galbraith, David, Garbi, Natalio, García-Godoy, Maria Dolore, Geginat, Jen, Ghoreschi, Kamran, Gibellini, Lara, Goettlinger, Christoph, Goodyear, Carl S., Gori, Andrea, Grogan, Jane, Gross, Mor, Grützkau, Andrea, Grummitt, Daryl, Hahn, Jona, Hammer, Quirin, Hauser, Anja E., Haviland, David L., Hedley, David, Herrera, Guadalupe, Herrmann, Martin, Hiepe, Falk, Holland, Tristan, Hombrink, Pleun, Houston, Jessica P., Hoyer, Bimba F., Huang, Bo, Hunter, Christopher A., Iannone, Anna, Jäck, Hans-Martin, Jávega, Beatriz, Jonjic, Stipan, Juelke, Kerstin, Jung, Steffen, Kaiser, Toralf, Kalina, Toma, Keller, Baerbel, Khan, Srijit, Kienhöfer, Deborah, Kroneis, Thoma, Kunkel, Désirée, Kurts, Christian, Kvistborg, Pia, Lannigan, Joanne, Lantz, Olivier, Larbi, Ani, LeibundGut-Landmann, Salome, Leipold, Michael D., Levings, Megan K., Litwin, Virginia, Liu, Yanling, Lohoff, Michael, Lombardi, Giovanna, Lopez, Lilly, Lovett-Racke, Amy, Lubberts, Erik, Ludewig, Burkhard, Lugli, Enrico, Maecker, Holden T., Martrus, Glòria, Matarese, Giuseppe, Maueröder, Christian, Mcgrath, Mairi, Mcinnes, Iain, Mei, Henrik E., Melchers, Fritz, Melzer, Susanne, Mielenz, Dirk, Mills, Kingston, Mirrer, David, Mjösberg, Jenny, Moore, Jonni, Moran, Barry, Moretta, Alessandro, Moretta, Lorenzo, Mosmann, Tim R., Müller, Susann, Müller, Werner, Münz, Christian, Multhoff, Gabriele, Munoz, Luis Enrique, Murphy, Kenneth M., Nakayama, Toshinori, Nasi, Milena, Neudörfl, Christine, Nolan, John, Nourshargh, Sussan, O'Connor, José-Enrique, Ouyang, Wenjun, Oxenius, Annette, Palankar, Raghav, Panse, Isabel, Peterson, Pärt, Peth, Christian, Petriz, Jordi, Philips, Daisy, Pickl, Winfried, Piconese, Silvia, Pinti, Marcello, Pockley, A. Graham, Podolska, Malgorzata Justyna, Pucillo, Carlo, Quataert, Sally A., Radstake, Timothy R. D. J., Rajwa, Bartek, Rebhahn, Jonathan A., Recktenwald, Diether, Remmerswaal, Ester B. M., Rezvani, Katy, Rico, Laura G., Robinson, J. Paul, Romagnani, Chiara, Rubartelli, Anna, Ruckert, Beate, Ruland, Jürgen, Sakaguchi, Shimon, Sala-de-Oyanguren, Francisco, Samstag, Yvonne, Sanderson, Sharon, Sawitzki, Birgit, Scheffold, Alexander, Schiemann, Matthia, Schildberg, Frank, Schimisky, Esther, Schmid, Stephan A., Schmitt, Steffen, Schober, Kilian, Schüler, Thoma, Schulz, Axel Ronald, Schumacher, Ton, Scotta, Cristiano, Shankey, T. Vincent, Shemer, Anat, Simon, Anna-Katharina, Spidlen, Josef, Stall, Alan M., Stark, Regina, Stehle, Christina, Stein, Merle, Steinmetz, Tobit, Stockinger, Hanne, Takahama, Yousuke, Tarnok, Attila, Tian, Zhigang, Toldi, Gergely, Tornack, Julia, Traggiai, Elisabetta, Trotter, Joe, Ulrich, Henning, van der Braber, Marlou, van Lier, René A. W., Veldhoen, Marcello, Vento-Asturias, Salvador, Vieira, Paulo, Voehringer, David, Volk, Hans-Dieter, von Volkmann, Konrad, Waisman, Ari, Walker, Rachael, Ward, Michael D., Warnatz, Klau, Warth, Sarah, Watson, James V., Watzl, Carsten, Wegener, Leonie, Wiedemann, Annika, Wienands, Jürgen, Willimsky, Gerald, Wing, Jame, Wurst, Peter, Yu, Liping, Yue, Alice, Zhang, Qianjun, Zhao, Yi, Ziegler, Susanne, Zimmermann, Jakob, Cossarizza, A., Chang, H., Radbruch, A., Akdis, M., Andrã¤, I., Annunziato, F., Bacher, P., Barnaba, V., Battistini, L., Bauer, W., Baumgart, S., Becher, B., Beisker, W., Berek, C., Blanco, A., Borsellino, G., Boulais, P., Brinkman, R., Bã¼scher, M., Busch, D., Bushnell, T., Cao, X., Cavani, A., Chattopadhyay, P., Cheng, Q., Chow, S., Clerici, M., Cooke, A., Cosma, A., Cosmi, L., Cumano, A., Dang, V., Davies, D., De Biasi, S., Del Zotto, G., Della Bella, S., Dellabona, P., Deniz, G., Dessing, M., Diefenbach, A., Di Santo, J., Dieli, F., Dolf, A., Donnenberg, V., Dã¶rner, T., Ehrhardt, G., Endl, E., Engel, P., Engelhardt, B., Esser, C., Everts, B., Dreher, A., Falk, C., Fehniger, T., Filby, A., Fillatreau, S., Follo, M., Fã¶rster, I., Foster, J., Foulds, G., Frenette, P., Galbraith, D., Garbi, N., García-Godoy, M., Geginat, J., Ghoreschi, K., Gibellini, L., Goettlinger, C., Goodyear, C., Gori, A., Grogan, J., Gross, M., Grã¼tzkau, A., Grummitt, D., Hahn, J., Hammer, Q., Hauser, A., Haviland, D., Hedley, D., Herrera, G., Herrmann, M., Hiepe, F., Holland, T., Hombrink, P., Houston, J., Hoyer, B., Huang, B., Hunter, C., Iannone, A., Jã¤ck, H., Jã¡vega, B., Jonjic, S., Juelke, K., Jung, S., Kaiser, T., Kalina, T., Keller, B., Khan, S., Kienhã¶fer, D., Kroneis, T., Kunkel, D., Kurts, C., Kvistborg, P., Lannigan, J., Lantz, O., Larbi, A., LeibundGut-Landmann, S., Leipold, M., Levings, M., Litwin, V., Liu, Y., Lohoff, M., Lombardi, G., Lopez, L., Lovett-Racke, A., Lubberts, E., Ludewig, B., Lugli, E., Maecker, H., Martrus, G., Matarese, G., Mauerã¶der, C., Mcgrath, M., Mcinnes, I., Mei, H., Melchers, F., Melzer, S., Mielenz, D., Mills, K., Mirrer, D., Mjã¶sberg, J., Moore, J., Moran, B., Moretta, A., Moretta, L., Mosmann, T., Mã¼ller, S., Mã¼ller, W., Mã¼nz, C., Multhoff, G., Munoz, L., Murphy, K., Nakayama, T., Nasi, M., Neudã¶rfl, C., Nolan, J., Nourshargh, S., O'Connor, J., Ouyang, W., Oxenius, A., Palankar, R., Panse, I., Peterson, P., Peth, C., Petriz, J., Philips, D., Pickl, W., Piconese, S., Pinti, M., Pockley, A., Podolska, M., Pucillo, C., Quataert, S., Radstake, T., Rajwa, B., Rebhahn, J., Recktenwald, D., Remmerswaal, E., Rezvani, K., Rico, L., Robinson, J., Romagnani, C., Rubartelli, A., Ruckert, B., Ruland, J., Sakaguchi, S., Sala-de-Oyanguren, F., Samstag, Y., Sanderson, S., Sawitzki, B., Scheffold, A., Schiemann, M., Schildberg, F., Schimisky, E., Schmid, S., Schmitt, S., Schober, K., Schã¼ler, T., Schulz, A., Schumacher, T., Scotta, C., Shankey, T., Shemer, A., Simon, A., Spidlen, J., Stall, A., Stark, R., Stehle, C., Stein, M., Steinmetz, T., Stockinger, H., Takahama, Y., Tarnok, A., Tian, Z., Toldi, G., Tornack, J., Traggiai, E., Trotter, J., Ulrich, H., van der Braber, M., van Lier, R., Veldhoen, M., Vento-Asturias, S., Vieira, P., Voehringer, D., Volk, H., von Volkmann, K., Waisman, A., Walker, R., Ward, M., Warnatz, K., Warth, S., Watson, J., Watzl, C., Wegener, L., Wiedemann, A., Wienands, J., Willimsky, G., Wing, J., Wurst, P., Liping, Y., Yue, A., Zhang, Q., Zhao, Y., Ziegler, S., and Zimmermann, J.

Subjects
0301 basic medicine, T-Lymphocytes, Cell Separation, T cell precursors, 0302 clinical medicine, Immunophenotyping, Human lymphopoiesis, [ SDV.IMM ] Life Sciences [q-bio]/Immunology, Immunology and Allergy, Non-U.S. Gov't, Immunologic Technique, medicine.diagnostic_test, Research Support, Non-U.S. Gov't, virus diseases, hemic and immune systems, False Positive Reaction, Cell sorting, Flow Cytometry, natural killer and innate lymphoid cells differentiation, 3. Good health, Research Design, [SDV.IMM]Life Sciences [q-bio]/Immunology, Human, Quality Control, medicine.drug_class, Immunology, Animals, Cell Proliferation, DNA, False Positive Reactions, Humans, RNA, Software, Guidelines as Topic, Immunologic Techniques, chemical and pharmacologic phenomena, Computational biology, Biology, Monoclonal antibody, Research Support, Article, Flow cytometry, N.I.H, 03 medical and health sciences, Immune system, Research Support, N.I.H., Extramural, medicine, early lymphoid progenitors, Journal Article, Mass cytometry, IMUNOLOGIA, Animal, Extramural, B cell ontogeny, 030104 developmental biology, T-Lymphocyte, Cytometry, 030215 immunology
Abstract

The marriage between immunology and cytometry is one of the most stable and productive in the recent history of science. A rapid search in PubMed shows that, as of July 2017, using “flow cytometry immunology” as a search term yields more than 68 000 articles, the first of which, interestingly, is not about lymphocytes. It might be stated that, after a short engagement, the exchange of the wedding rings between immunology and cytometry officially occurred when the idea to link fluorochromes to monoclonal antibodies came about. After this, recognizing different types of cells became relatively easy and feasible not only by using a simple fluorescence microscope, but also by a complex and sometimes esoteric instrument, the flow cytometer that is able to count hundreds of cells in a single second, and can provide repetitive results in a tireless manner. Given this, the possibility to analyse immune phenotypes in a variety of clinical conditions has changed the use of the flow cytometer, which was incidentally invented in the late 1960s to measure cellular DNA by using intercalating dyes, such as ethidium bromide. The epidemics of HIV/AIDS in the 1980s then gave a dramatic impulse to the technology of counting specific cells, since it became clear that the quantification of the number of peripheral blood CD4+ T cells was crucial to follow the course of the infection, and eventually for monitoring the therapy. As a consequence, the development of flow cytometers that had to be easy-to-use in all clinical laboratories helped to widely disseminate this technology. Nowadays, it is rare to find an immunological paper or read a conference abstract in which the authors did not use flow cytometry as the main tool to dissect the immune system and identify its fine and complex functions. Of note, recent developments have created the sophisticated technology of mass cytometry, which is able to simultaneously identify dozens of molecules at the single cell level and allows us to better understand the complexity and beauty of the immune system.

Published
2017
Full Text
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15. A Ca(2+) concentration of 1.5 mM, as present in IMDM but not in RPMI, is critical for maximal response of Th cells to PMA/ionomycin

Author

Hyun-Dong Chang, Jakob Zimmermann, and Andreas Radbruch

Subjects
Immunology, Cell Culture Techniques, chemistry.chemical_element, Biology, Calcium, Lymphocyte Activation, Technical Comment, Flow cytometry, Cellular activation, chemistry.chemical_compound, Mice, medicine, Immunology and Allergy, Animals, Humans, Cytokine genes, Mice, Knockout, medicine.diagnostic_test, Ionomycin, T-Lymphocytes, Helper-Inducer, Colitis, Molecular biology, CD4+ T cells, Culture Media, Calcineurin, Mice, Inbred C57BL, PMA/ionomycin, chemistry, Cell culture, Lymphocyte activation, Cytokines
Abstract

Restimulation of memory–effector T helper lymphocytes with PMA/ionomycin is an established technique to determine their imprinting for the expression of cytokine genes, that is their functional potential. Here, we show that for the maximal reexpression of cytokine genes, the Ca2+ concentration of the medium has to be greater than 1.5 mM. For RPMI (Roswell Park Memorial Institute 1640), the most commonly used medium, the Ca2+ concentration has to be increased from the regular 0.42 to 1.5 mM.

Published
2014

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