Publications by authors named "Manfred Kopf"

140 Publications

Differential sensitivity of inflammatory macrophages and alternatively activated macrophages to ferroptosis.

Eur J Immunol 2021 Jul 17. Epub 2021 Jul 17.

Institute of Molecular Health Sciences, Department of Biology, ETH Zurich, Zurich, Switzerland.

Acumulation of oxidized membrane lipids ultimately results in ferroptotic cell death, which can be prevented by the selenoenzyme glutathione peroxidase 4 (Gpx4). In vivo conditions promoting ferroptosis and susceptible cell types are still poorly defined. In this study, we analyzed the conditional deletion of Gpx4 in mice specifically in the myeloid cell lineages. Surprisingly, development and maintenance of LysM macrophages and neutrophils, as well as CD11c monocyte-derived macrophages and dendritic cells were unaffected in the absence of Gpx4. Gpx4-deficient macrophages mounted an unaltered proinflammatory cytokine response including IL-1β production following stimulation with TLR ligands and activation of several inflammasomes. Accordingly, Gpx4 LysM-cre mice were protected from bacterial and protozoan infections. Despite having the capacity to differentiate to alternatively activated macrophages (AAM), these cells lacking Gpx4 triggered ferroptosis both in vitro and in vivo following IL-4 overexpression and nematode infection. Exposure to nitric oxide restored viability of Gpx4-deficient AAM, while inhibition of iNOS in proinflammatory macrophages had no effect. These data together suggest that activation cues of tissue macrophages determine sensitivity to lipid peroxidation and ferroptotic cell death.
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http://dx.doi.org/10.1002/eji.202049114DOI Listing
July 2021

Cytomegalovirus subverts macrophage identity.

Cell 2021 Jul 10;184(14):3774-3793.e25. Epub 2021 Jun 10.

Institute for Immunodeficiency, Center for Chronic Immunodeficiency (CCI), University Medical Center, Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany; Center for Pediatrics and Adolescent Medicine, University Medical Center, 79106 Freiburg, Germany. Electronic address:

Cytomegaloviruses (CMVs) have co-evolved with their mammalian hosts for millions of years, leading to remarkable host specificity and high infection prevalence. Macrophages, which already populate barrier tissues in the embryo, are the predominant immune cells at potential CMV entry sites. Here we show that, upon CMV infection, macrophages undergo a morphological, immunophenotypic, and metabolic transformation process with features of stemness, altered migration, enhanced invasiveness, and provision of the cell cycle machinery for viral proliferation. This complex process depends on Wnt signaling and the transcription factor ZEB1. In pulmonary infection, mouse CMV primarily targets and reprograms alveolar macrophages, which alters lung physiology and facilitates primary CMV and secondary bacterial infection by attenuating the inflammatory response. Thus, CMV profoundly perturbs macrophage identity beyond established limits of plasticity and rewires specific differentiation processes, allowing viral spread and impairing innate tissue immunity.
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http://dx.doi.org/10.1016/j.cell.2021.05.009DOI Listing
July 2021

Tissue-resident macrophages: guardians of organ homeostasis.

Trends Immunol 2021 06 7;42(6):495-507. Epub 2021 May 7.

Institute of Molecular Health Sciences, Department of Biology, ETH Zurich, 8093 Zurich, Switzerland. Electronic address:

Tissue-resident macrophages (M) have recently emerged as a key rheostat capable of regulating the balance between organ health and disease. In most organs, ontogenetically and functionally distinct macrophage subsets fulfill a plethora of functions specific to their tissue environment. In this review, we summarize recent findings regarding the ontogeny and functions of macrophage populations in different mammalian tissues, describing how these cells regulate tissue homeostasis and how they can contribute to inflammation. Furthermore, we highlight new developments concerning certain general principles of tissue macrophage biology, including the importance of metabolism for understanding macrophage activation states and the influence of intrinsic and extrinsic factors on macrophage metabolic control. We also shed light on certain open questions in the field and how answering these might pave the way for tissue-specific therapeutic approaches.
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http://dx.doi.org/10.1016/j.it.2021.04.007DOI Listing
June 2021

PPARɣ drives IL-33-dependent ILC2 pro-tumoral functions.

Nat Commun 2021 05 5;12(1):2538. Epub 2021 May 5.

Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland.

Group 2 innate lymphoid cells (ILC2s) play a critical role in protection against helminths and in diverse inflammatory diseases by responding to soluble factors such as the alarmin IL-33, that is often overexpressed in cancer. Nonetheless, regulatory factors that dictate ILC2 functions remain poorly studied. Here, we show that peroxisome proliferator-activated receptor gamma (PPARγ) is selectively expressed in ILC2s in humans and in mice, acting as a central functional regulator. Pharmacologic inhibition or genetic deletion of PPARγ in ILC2s significantly impair IL-33-induced Type-2 cytokine production and mitochondrial fitness. Further, PPARγ blockade in ILC2s disrupts their pro-tumoral effect induced by IL-33-secreting cancer cells. Lastly, genetic ablation of PPARγ in ILC2s significantly suppresses tumor growth in vivo. Our findings highlight a crucial role for PPARγ in supporting the IL-33 dependent pro-tumorigenic role of ILC2s and suggest that PPARγ can be considered as a druggable pathway in ILC2s to inhibit their effector functions. Hence, PPARγ targeting might be exploited in cancer immunotherapy and in other ILC2-driven mediated disorders, such as asthma and allergy.
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http://dx.doi.org/10.1038/s41467-021-22764-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8100153PMC
May 2021

PPARγ is essential for the development of bone marrow erythroblastic island macrophages and splenic red pulp macrophages.

J Exp Med 2021 May;218(5)

Institute of Molecular Health Sciences, Swiss Federal Institute of Technology, Zurich, Switzerland.

Tissue-resident macrophages play a crucial role in maintaining homeostasis. Macrophage progenitors migrate to tissues perinatally, where environmental cues shape their identity and unique functions. Here, we show that the absence of PPARγ affects neonatal development and VCAM-1 expression of splenic iron-recycling red pulp macrophages (RPMs) and bone marrow erythroblastic island macrophages (EIMs). Transcriptome analysis of the few remaining Pparg-deficient RPM-like and EIM-like cells suggests that PPARγ is required for RPM and EIM identity, cell cycling, migration, and localization, but not function in mature RPMs. Notably, Spi-C, another transcription factor implicated in RPM development, was not essential for neonatal expansion of RPMs, even though the transcriptome of Spic-deficient RPMs was strongly affected and indicated a loss of identity. Similarities shared by Pparg- and Spic-deficient RPM-like cells allowed us to identify pathways that rely on both factors. PPARγ and Spi-C collaborate in inducing transcriptional changes, including VCAM-1 and integrin αD expression, which could be required for progenitor retention in the tissue, allowing access to niche-related signals that finalize differentiation.
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http://dx.doi.org/10.1084/jem.20191314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8006858PMC
May 2021

Comparative analysis of the role of mast cells in murine asthma models using Kit-sufficient mast cell-deficient animals.

Allergy 2021 Jul 9;76(7):2030-2043. Epub 2021 Mar 9.

Institute of Molecular Health Sciences, ETH Zurich, Zurich, Switzerland.

Background: Asthma is a frequent chronic disease that can potentially severely affect the respiratory capacity and well-being of patients. Mast cells (MCs) are regarded as major players in human asthma due to their capacity to release crucial inflammatory mediators following allergen exposure. However, unambiguous characterization of their role in animal models has long been hindered by the unavailability of specific MC-deficient models lacking confounding MC-unrelated effects. This study aims to examine the role of MCs in Kit-sufficient MC-deficient Cpa3 mice.

Methods: We used a variety of models of acute and chronic asthma employing distinct routes and regimes of sensitization. These sensitizations were done via the peritoneal cavity, the skin, or the lung. Additionally, different allergens, i.e. ovalbumin and house dust mite extract, were used.

Results: Our results show that the absence of MCs had no impact on the severity of allergic airway inflammation in any of the tested mouse models, as measured by leukocyte infiltration in the airways, cytokine expression, antibody production, airway hyper-responsiveness and mucus production.

Conclusion: This indicates that MCs do not play a major role in murine allergic airway inflammation.
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http://dx.doi.org/10.1111/all.14765DOI Listing
July 2021

GM-CSF instigates a dendritic cell-T-cell inflammatory circuit that drives chronic asthma development.

J Allergy Clin Immunol 2021 Jun 10;147(6):2118-2133.e3. Epub 2021 Jan 10.

Molecular Biomedicine, Institute of Molecular Health Sciences, Department of Biology, ETH Zurich, Zurich, Switzerland. Electronic address:

Background: Steroid-resistant asthma is often characterized by high levels of neutrophils and mixed T2/T17 immune profiles. Indeed, neutrophils are key drivers of chronic lung inflammation in multiple respiratory diseases. Their numbers correlate strongly with disease severity, and their presence is often associated with exacerbation of chronic lung inflammation.

Objective: What factors drive development of neutrophil-mediated chronic lung disease remains largely unknown, and we sought to study the role of GM-CSF as a potential regulator in chronic asthma.

Methods: Different experimental animal models of chronic asthma were used in combination with alveolar macrophage-reconstitution of global GM-CSF receptor knockout mice as well as cell-type-specific knockout animals to elucidate the role of GM-CSF signaling in chronic airway inflammation.

Results: We identify GM-CSF signaling as a critical factor regulating pulmonary accumulation of neutrophils. We show that although being not required for intrinsically regulating neutrophil migration, GM-CSF controls lung dendritic cell function, which in turn promotes T-cell-dependent recruitment of neutrophils to the airways. We demonstrate that GM-CSF regulates lung dendritic cell antigen uptake, transport, and T2/T17 cell priming in an intrinsic fashion, which in turn drives pulmonary granulocyte recruitment and contributes to development of airway hyperresponsiveness in chronic disease.

Conclusions: We identify GM-CSF as a potentially novel therapeutic target in chronic lung inflammation, describing a GM-CSF-dependent lung conventional dendritic cell-T-cell-neutrophil axis that drives chronic lung disease.
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http://dx.doi.org/10.1016/j.jaci.2020.12.638DOI Listing
June 2021

Redox regulation of immunometabolism.

Nat Rev Immunol 2021 06 18;21(6):363-381. Epub 2020 Dec 18.

Institute of Molecular Health Sciences, Department of Biology, ETH Zürich, Zürich, Switzerland.

Metabolic pathways and redox reactions are at the core of life. In the past decade(s), numerous discoveries have shed light on how metabolic pathways determine the cellular fate and function of lymphoid and myeloid cells, giving rise to an area of research referred to as immunometabolism. Upon activation, however, immune cells not only engage specific metabolic pathways but also rearrange their oxidation-reduction (redox) system, which in turn supports metabolic reprogramming. In fact, studies addressing the redox metabolism of immune cells are an emerging field in immunology. Here, we summarize recent insights revealing the role of reactive oxygen species (ROS) and the differential requirement of the main cellular antioxidant pathways, including the components of the thioredoxin (TRX) and glutathione (GSH) pathways, as well as their transcriptional regulator NF-E2-related factor 2 (NRF2), for proliferation, survival and function of T cells, B cells and macrophages.
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http://dx.doi.org/10.1038/s41577-020-00478-8DOI Listing
June 2021

The GM-CSF-IRF5 signaling axis in eosinophils promotes antitumor immunity through activation of type 1 T cell responses.

J Exp Med 2020 12;217(12)

Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland.

The depletion of eosinophils represents an efficient strategy to alleviate allergic asthma, but the consequences of prolonged eosinophil deficiency for human health remain poorly understood. We show here that the ablation of eosinophils severely compromises antitumor immunity in syngeneic and genetic models of colorectal cancer (CRC), which can be attributed to defective Th1 and CD8+ T cell responses. The specific loss of GM-CSF signaling or IRF5 expression in the eosinophil compartment phenocopies the loss of the entire lineage. GM-CSF activates IRF5 in vitro and in vivo and can be administered recombinantly to improve tumor immunity. IL-10 counterregulates IRF5 activation by GM-CSF. CRC patients whose tumors are infiltrated by large numbers of eosinophils also exhibit robust CD8 T cell infiltrates and have a better prognosis than patients with eosinophillow tumors. The combined results demonstrate a critical role of eosinophils in tumor control in CRC and introduce the GM-CSF-IRF5 axis as a critical driver of the antitumor activities of this versatile cell type.
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http://dx.doi.org/10.1084/jem.20190706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7953737PMC
December 2020

The thioredoxin-1 inhibitor Txnip restrains effector T-cell and germinal center B-cell expansion.

Eur J Immunol 2021 01 28;51(1):115-124. Epub 2020 Sep 28.

Institute of Molecular Health Sciences, ETH Zürich, Zürich, Switzerland.

Thioredoxin-1 (Trx1) is a vital component for cellular redox homeostasis. In T cells, Trx1 donates electrons for the de novo synthesis of deoxyribonucleotides to allow rapid cell proliferation. The Trx-interacting protein (Txnip) binds to the reduced Trx1 and inhibits its activity. However, the role of Txnip in adaptive immunity in vivo is unknown. Here, we show that absence of Txnip increased proliferation of effector T cells and GC B-cell responses in response to lymphocytic choriomeningitis virus and Qβ virus-like particles, respectively, but did not affect development and homeostasis of T and B cells. While downregulation of Txnip and concomitant upregulation of Trx1 is critical for rapid T-cell expansion upon viral infection, re-expression of Txnip and consequently inhibition of Trx1 is important to restrain late T-cell expansion. Importantly, we demonstrated that T-cell receptor (TCR) engagement but not CD28 costimulation is critically required for Txnip downregulation. Thus, this study further uncovers positive and negative control of lymphocyte proliferation by the Trx1 system.
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http://dx.doi.org/10.1002/eji.202048851DOI Listing
January 2021

Electrophilic Nrf2 activators and itaconate inhibit inflammation at low dose and promote IL-1β production and inflammatory apoptosis at high dose.

Redox Biol 2020 09 21;36:101647. Epub 2020 Jul 21.

Institute of Molecular Health Sciences, ETH Zürich, 8093, Zürich, Switzerland. Electronic address:

Controlling inflammation is critical for preventing many diseases including cancer, autoimmune disorders and hypersensitivity reactions. NF-E2-related factor 2 (Nrf2) is a key transcription factor that controls the cellular antioxidant and cytoprotective response. Moreover, Nrf2 has been implicated in the regulation of inflammatory processes, although the ultimate mechanism by which this is achieved is unknown. Here, we investigated mechanisms of inflammation and cell death pathways induced by a variety of Nrf2 activators including dimethyl fumarate (DMF) and the endogenous metabolite itaconate. We found that exposure of bone marrow-derived dendritic cells (BMDCs) to low concentrations of a variety of electrophilic Nrf2 activators including itaconate prior to Toll-like receptor (TLR) stimulation inhibits transcription of pro-inflammatory cytokines (such as interleukin [IL]-12 and IL-1β) by activation of Nrf2. By contrast, high doses of these electrophilic compounds after TLR activation promote inflammatory apoptosis and caspase-8-dependent IL-1β processing and release independently of Nrf2. Interestingly, tert-butylhydroquinone (tBHQ), a non-electrophilic Nrf2-activator, failed to induce IL-1β production. These results have important implications for clinical application of electrophilic compounds.
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http://dx.doi.org/10.1016/j.redox.2020.101647DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7387846PMC
September 2020

High-Dimensional T Helper Cell Profiling Reveals a Broad Diversity of Stably Committed Effector States and Uncovers Interlineage Relationships.

Immunity 2020 09 30;53(3):597-613.e6. Epub 2020 Jul 30.

Institute of Molecular Health Sciences, ETH Zurich, 8093 Zurich, Switzerland. Electronic address:

CD4 T helper (Th) cells are fundamental players in immunity. Based on the expression of signature cytokines and transcription factors, several Th subsets have been defined. Th cells are thought to be far more heterogeneous and multifunctional than originally believed, but characterization of the full diversity has been hindered by technical limitations. Here, we employ mass cytometry to analyze the diversity of Th cell responses generated in vitro and in animal disease models, revealing a vast heterogeneity of effector states with distinct cytokine footprints. The diversities of cytokine responses established during primary antigen encounters in Th1- and Th2-cell-polarizing conditions are largely maintained after secondary challenge, regardless of the new inflammatory environment, highlighting many of the identified states as stable Th cell sublineages. We also find that Th17 cells tend to upregulate Th2-cell-associated cytokines upon challenge, indicating a closer developmental connection between Th17 and Th2 cells than previously anticipated.
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http://dx.doi.org/10.1016/j.immuni.2020.07.001DOI Listing
September 2020

Endothelial Lactate Controls Muscle Regeneration from Ischemia by Inducing M2-like Macrophage Polarization.

Cell Metab 2020 06;31(6):1136-1153.e7

Laboratory of Exercise and Health, Department Health Sciences and Technology, Swiss Federal Institute of Technology (ETH) Zurich, 8603 Zurich, Switzerland. Electronic address:

Endothelial cell (EC)-derived signals contribute to organ regeneration, but angiocrine metabolic communication is not described. We found that EC-specific loss of the glycolytic regulator pfkfb3 reduced ischemic hindlimb revascularization and impaired muscle regeneration. This was caused by the reduced ability of macrophages to adopt a proangiogenic and proregenerative M2-like phenotype. Mechanistically, loss of pfkfb3 reduced lactate secretion by ECs and lowered lactate levels in the ischemic muscle. Addition of lactate to pfkfb3-deficient ECs restored M2-like polarization in an MCT1-dependent fashion. Lactate shuttling by ECs enabled macrophages to promote proliferation and fusion of muscle progenitors. Moreover, VEGF production by lactate-polarized macrophages was increased, resulting in a positive feedback loop that further stimulated angiogenesis. Finally, increasing lactate levels during ischemia rescued macrophage polarization and improved muscle reperfusion and regeneration, whereas macrophage-specific mct1 deletion prevented M2-like polarization. In summary, ECs exploit glycolysis for angiocrine lactate shuttling to steer muscle regeneration from ischemia.
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http://dx.doi.org/10.1016/j.cmet.2020.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7267778PMC
June 2020

Comprehensive characterization of myeloid cells during wound healing in healthy and healing-impaired diabetic mice.

Eur J Immunol 2020 09 13;50(9):1335-1349. Epub 2020 May 13.

Institute of Molecular Health Sciences, Department of Biology, ETH Zurich, Zurich, Switzerland.

Wound healing involves the concerted action of various lymphoid and in particular myeloid cell populations. To characterize and quantitate different types of myeloid cells and to obtain information on their kinetics during wound healing, we performed multiparametric flow cytometry analysis. In healthy mice, neutrophil numbers increased early after injury and returned to near basal levels after completion of healing. Macrophages, monocyte-derived dendritic cells (DCs), and eosinophils were abundant throughout the healing phase, in particular in early wounds, and Langerhans cells increased after wounding and remained elevated after epithelial closure. Major differences in healing-impaired diabetic mice were a much higher percentage of immune cells in late wounds, mainly as a result of neutrophil, macrophage, and monocyte persistence; reduced numbers and percentages of macrophages and monocyte-derived DCs in early wounds; and of Langerhans cells, conventional DCs, and eosinophils throughout the healing process. Finally, unbiased cluster analysis (PhenoGraph) identified a large number of different clusters of myeloid cells in skin wounds. These results provide insight into myeloid cell diversity and dynamics during wound repair and highlight the abnormal inflammatory response associated with impaired healing.
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http://dx.doi.org/10.1002/eji.201948438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496577PMC
September 2020

Cyclopentenone Prostaglandins and Structurally Related Oxidized Lipid Species Instigate and Share Distinct Pro- and Anti-inflammatory Pathways.

Cell Rep 2020 03;30(13):4399-4417.e7

Institute of Molecular Health Sciences, ETH Zürich, 8093 Zürich, Switzerland. Electronic address:

Oxidized lipids play a critical role in a variety of diseases with two faces: pro- and anti-inflammatory. The molecular mechanisms of this Janus-faced activity remain largely unknown. Here, we have identified that cyclopentenone-containing prostaglandins such as 15d-PGJ2 and structurally related oxidized phospholipid species possess a dual and opposing bioactivity in inflammation, depending on their concentration. Exposure of dendritic cells (DCs)/macrophages to low concentrations of such lipids before Toll-like receptor (TLR) stimulation instigates an anti-inflammatory response mediated by nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent inhibition of nuclear factor κB (NF-κB) activation and downstream targets. By contrast, high concentrations of such lipids upon TLR activation of DCs/macrophages result in inflammatory apoptosis characterized by mitochondrial depolarization and caspase-8-mediated interleukin (IL)-1β maturation independently of Nrf2 and the classical inflammasome pathway. These results uncover unexpected pro- and anti-inflammatory activities of physiologically relevant lipid species generated by enzymatic and non-enzymatic oxidation dependent on their concentration, a phenomenon known as hormesis.
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http://dx.doi.org/10.1016/j.celrep.2020.03.019DOI Listing
March 2020

Mice carrying an analogous heterozygous dynamin 2 K562E mutation that causes neuropathy in humans develop predominant characteristics of a primary myopathy.

Hum Mol Genet 2020 05;29(8):1253-1273

Department of Biology, Institute of Molecular Health Sciences, Swiss Federal Institute of Technology, ETH Zurich, 8093 Zurich, Switzerland.

Some mutations affecting dynamin 2 (DNM2) can cause dominantly inherited Charcot-Marie-Tooth (CMT) neuropathy. Here, we describe the analysis of mice carrying the DNM2 K562E mutation which has been associated with dominant-intermediate CMT type B (CMTDIB). Contrary to our expectations, heterozygous DNM2 K562E mutant mice did not develop definitive signs of an axonal or demyelinating neuropathy. Rather, we found a primary myopathy-like phenotype in these mice. A likely interpretation of these results is that the lack of a neuropathy in this mouse model has allowed the unmasking of a primary myopathy due to the DNM2 K562E mutation which might be overshadowed by the neuropathy in humans. Consequently, we hypothesize that a primary myopathy may also contribute to the disease mechanism in some CMTDIB patients. We propose that these findings should be considered in the evaluation of patients, the determination of the underlying disease processes and the development of tailored potential treatment strategies.
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http://dx.doi.org/10.1093/hmg/ddaa034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7254847PMC
May 2020

Thioredoxin-1 distinctly promotes NF-κB target DNA binding and NLRP3 inflammasome activation independently of Txnip.

Elife 2020 02 25;9. Epub 2020 Feb 25.

Institute of Molecular Health Sciences, ETH Zürich, Zürich, Switzerland.

Antioxidant systems, such as the thioredoxin-1 (Trx1) pathway, ensure cellular redox homeostasis. However, how such systems regulate development and function of myeloid cells is barely understood. Here we show that in contrast to its critical role in T cells, the murine Trx1 system is dispensable for steady-state myeloid-cell hematopoiesis due to their capacity to tap the glutathione/glutaredoxin pathway for DNA biosynthesis. However, the Trx1 pathway instrumentally enables nuclear NF-κB DNA-binding and thereby pro-inflammatory responses in monocytes and dendritic cells. Moreover, independent of this activity, Trx1 is critical for NLRP3 inflammasome activation and IL-1β production in macrophages by detoxifying excessive ROS levels. Notably, we exclude the involvement of the Trx1 inhibitor Txnip as a redox-sensitive ligand of NLRP3 as previously proposed. Together, this study suggests that targeting Trx1 may be exploited to treat inflammatory diseases.
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http://dx.doi.org/10.7554/eLife.53627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7062472PMC
February 2020

Fetal monocytes possess increased metabolic capacity and replace primitive macrophages in tissue macrophage development.

EMBO J 2020 02 2;39(3):e103205. Epub 2020 Jan 2.

Department of Biology, Institute of Molecular Health Sciences, ETH Zürich, Zürich, Switzerland.

Tissue-resident macrophages (MΦ ) originate from at least two distinct waves of erythro-myeloid progenitors (EMP) arising in the yolk sac (YS) at E7.5 and E8.5 with the latter going through a liver monocyte intermediate. The relative potential of these precursors in determining development and functional capacity of MΦ remains unclear. Here, we studied development of alveolar macrophages (AM) after single and competitive transplantation of different precursors from YS, fetal liver, and fetal lung into neonatal Csf2ra mice, which lack endogenous AM. Fetal monocytes, promoted by Myb, outcompeted primitive MΦ (pMΦ) in empty AM niches and preferentially developed to mature AM, which is associated with enhanced mitochondrial respiratory and glycolytic capacity and repression of the transcription factors c-Maf and MafB. Interestingly, AM derived from pMΦ failed to efficiently clear alveolar proteinosis and protect from fatal lung failure following influenza virus infection. Thus, our data demonstrate superior developmental and functional capacity of fetal monocytes over pMΦ in AM development and underlying mechanisms explaining replacement of pMΦ in fetal tissues.
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http://dx.doi.org/10.15252/embj.2019103205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996567PMC
February 2020

Apolipoprotein C3 induces inflammation and organ damage by alternative inflammasome activation.

Nat Immunol 2020 01 9;21(1):30-41. Epub 2019 Dec 9.

Institute of Molecular Health Science, ETH, Zurich, Switzerland.

NLRP3-inflammasome-driven inflammation is involved in the pathogenesis of a variety of diseases. Identification of endogenous inflammasome activators is essential for the development of new anti-inflammatory treatment strategies. Here, we identified that apolipoprotein C3 (ApoC3) activates the NLRP3 inflammasome in human monocytes by inducing an alternative NLRP3 inflammasome via caspase-8 and dimerization of Toll-like receptors 2 and 4. Alternative inflammasome activation in human monocytes is mediated by the Toll-like receptor adapter protein SCIMP. This triggers Lyn/Syk-dependent calcium entry and the production of reactive oxygen species, leading to activation of caspase-8. In humanized mouse models, ApoC3 activated human monocytes in vivo to impede endothelial regeneration and promote kidney injury in an NLRP3- and caspase-8-dependent manner. These data provide new insights into the regulation of the NLRP3 inflammasome and the pathophysiological role of triglyceride-rich lipoproteins containing ApoC3. Targeting ApoC3 might prevent organ damage and provide an anti-inflammatory treatment for vascular and kidney diseases.
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http://dx.doi.org/10.1038/s41590-019-0548-1DOI Listing
January 2020

B1 and Marginal Zone B Cells but Not Follicular B2 Cells Require Gpx4 to Prevent Lipid Peroxidation and Ferroptosis.

Cell Rep 2019 Nov;29(9):2731-2744.e4

Institute of Molecular Health Sciences, ETH Zurich, 8093 Zürich, Switzerland. Electronic address:

Aerobic organisms need to maintain cellular redox homeostasis. Glutathione peroxidase-4 (Gpx4) has the unique ability to protect cells against lipid peroxidation. Here, we show that Gpx4 is absolutely required to prevent ferroptosis during development, maintenance, and responses of innate-like B cells, namely, the B1 and marginal zone (MZ) B cells. In contrast, Gpx4 is dispensable for the development, germinal center reactions, and antibody responses of follicular B2 cells. Mechanistically, we show increased lipid metabolism and sensitivity to lipid peroxidation and ferroptosis in B1 and MZ B cells compared to follicular B2 cells, consistent with the requirement of Gpx4 in innate-like B cells. This high sensitivity to ferroptosis of innate-like B cells may be used to therapeutically target Gpx4 in certain forms of B cell malignancies involving B1 cells.
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http://dx.doi.org/10.1016/j.celrep.2019.10.070DOI Listing
November 2019

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition).

Eur J Immunol 2019 Oct;49(10):1457-1973

Flow Cytometry Laboratory, Institute of Molecular Toxicology and Pharmacology, Helmholtz Zentrum München, German Research Center for Environmental Health, München, Germany.

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.
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http://dx.doi.org/10.1002/eji.201970107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7350392PMC
October 2019

Interleukin-36 cytokines alter the intestinal microbiome and can protect against obesity and metabolic dysfunction.

Nat Commun 2019 09 5;10(1):4003. Epub 2019 Sep 5.

Department of Clinical Medicine, School of Medicine,Trinity Translational Medicine Institute, Trinity College Dublin, Dublin 8, Ireland.

Members of the interleukin-1 (IL-1) family are important mediators of obesity and metabolic disease and have been described to often play opposing roles. Here we report that the interleukin-36 (IL-36) subfamily can play a protective role against the development of disease. Elevated IL-36 cytokine expression is found in the serum of obese patients and negatively correlates with blood glucose levels among those presenting with type 2 diabetes. Mice lacking IL-36Ra, an IL-36 family signalling antagonist, develop less diet-induced weight gain, hyperglycemia and insulin resistance. These protective effects correlate with increased abundance of the metabolically protective bacteria Akkermansia muciniphila in the intestinal microbiome. IL-36 cytokines promote its outgrowth as well as increased colonic mucus secretion. These findings identify a protective role for IL-36 cytokines in obesity and metabolic disease, adding to the current understanding of the role the broader IL-1 family plays in regulating disease pathogenesis.
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http://dx.doi.org/10.1038/s41467-019-11944-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6728358PMC
September 2019

Glutathione peroxidase 4 and vitamin E control reticulocyte maturation, stress erythropoiesis and iron homeostasis.

Haematologica 2020 04 27;105(4):937-950. Epub 2019 Jun 27.

Institute of Clinical Molecular Biology and Tumor Genetics, Helmholtz Zentrum München, Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH), München, Germany

Glutathione peroxidase 4 (GPX4) is unique as it is the only enzyme that can prevent detrimental lipid peroxidation by reducing lipid peroxides to the respective alcohols thereby stabilizing oxidation products of unsaturated fatty acids. During reticulocyte maturation, lipid peroxidation mediated by 15-lipoxygenase in humans and rabbits and by 12/15-lipoxygenase (ALOX15) in mice was considered the initiating event for the elimination of mitochondria but is now known to occur through mitophagy. Yet, genetic ablation of the gene in mice failed to provide evidence for this hypothesis. We designed a different genetic approach to tackle this open conundrum. Since either other lipoxygenases or non-enzymatic autooxidative mechanisms may compensate for the loss of , we asked whether ablation of in the hematopoietic system would result in the perturbation of reticulocyte maturation. Quantitative assessment of erythropoiesis indices in the blood, bone marrow (BM) and spleen of chimeric mice with ablated in hematopoietic cells revealed anemia with an increase in the fraction of erythroid precursor cells and reticulocytes. Additional dietary vitamin E depletion strongly aggravated the anemic phenotype. Despite strong extramedullary erythropoiesis reticulocytes failed to mature and accumulated large autophagosomes with engulfed mitochondria. -deficiency in hematopoietic cells led to systemic hepatic iron overload and simultaneous severe iron demand in the erythroid system. Despite extremely high erythropoietin and erythroferrone levels in the plasma, hepcidin expression remained unchanged. Conclusively, perturbed reticulocyte maturation in response to loss in hematopoietic cells thus causes ineffective erythropoiesis, a phenotype partially masked by dietary vitamin E supplementation.
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http://dx.doi.org/10.3324/haematol.2018.212977DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7109755PMC
April 2020

A spontaneous leptin receptor point mutation causes obesity and differentially affects leptin signaling in hypothalamic nuclei resulting in metabolic dysfunctions distinct from db/db mice.

Mol Metab 2019 07 25;25:131-141. Epub 2019 Apr 25.

Institute of Molecular Biomedicine, Department Biology, ETH Zürich, Switzerland. Electronic address:

Objective: Leptin (Lep) plays a crucial role in controlling food intake and energy expenditure. Defective Lep/LepRb-signaling leads to fat accumulation, massive obesity, and the development of diabetes. We serendipitously noticed spontaneous development of obesity similar to LepR-deficient (db/db) mice in offspring from a C57BL/6J breeding and transmittance of the phenotype in a Mendelian manner. Candidate gene sequencing revealed a spontaneous point mutation in the LepRb gene. We investigated leptin responsiveness, leptin receptor signaling and metabolic phenotype of this novel LepRb mutant mouse variant.

Methods: Overexpression and functional tests of the mutant LepRb in 3T3 cells. Measurement of leptin responsiveness in hypothalamic nuclei, glucose tolerance, food uptake and energy expenditure in the mutant mice.

Results: The mutation results in the exchange of a glycine for serine (G506S) and introduces an alternative splice acceptor which, when used, encodes for a protein with a 40aa deletion that is retained in the cytoplasm. LepRb signaling was abrogated in the hypothalamic ventromedial nucleus (VMN) and dorsomedial nucleus (DMN), but only partially reduced in the hypothalamic arcuate nucleus (ARC) of LepRb mice, most likely due to differential splicing in neurons located in the respective regions of the hypothalamus. Extensive metabolic characterization of these mice revealed interesting differences in the control of food intake, glucose tolerance, energy expenditure, and fat accumulation in LepRb compared with LepRb-deficient db/db mice.

Conclusions: This study provides further insight into differences of the leptin responsiveness in VMN, DMN, and ARC and its metabolic consequences.
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http://dx.doi.org/10.1016/j.molmet.2019.04.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6601129PMC
July 2019

Author Correction: Deciphering CD4 T cell specificity using novel MHC-TCR chimeric receptors.

Nat Immunol 2019 May;20(5):663

Institute of Molecular Health Sciences, ETH Zürich, Zürich, Switzerland.

In the version of this article initially published, a reference (23) was cited incorrectly and two references were not included in the second sentence of the first paragraph of the second Results subsection ('Screening for gp61 mimotopes with different functional properties'). The correct citation is as follows: "... we replaced the very stable GFP with a slow fluorescent timer (FT)." Full details on the added references can be found in the correction notice. The errors have been corrected in the print, PDF and HTML versions of the paper.
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http://dx.doi.org/10.1038/s41590-019-0383-4DOI Listing
May 2019

Deciphering CD4 T cell specificity using novel MHC-TCR chimeric receptors.

Nat Immunol 2019 05 11;20(5):652-662. Epub 2019 Mar 11.

Institute of Molecular Health Sciences, ETH Zürich, Zürich, Switzerland.

αβ T cell antigen receptors (TCRs) bind complexes of peptide and major histocompatibility complex (pMHC) with low affinity, which poses a considerable challenge for the direct identification of αβ T cell cognate peptides. Here we describe a platform for the discovery of MHC class II epitopes based on the screening of engineered reporter cells expressing novel pMHC-TCR (MCR) hybrid molecules carrying cDNA-derived peptides. This technology identifies natural epitopes of CD4 T cells in an unbiased and efficient manner and allows detailed analysis of TCR cross-reactivity that provides recognition patterns beyond discrete peptides. We determine the cognate peptides of virus- and tumor-specific T cells in mouse disease models and present a proof of concept for human T cells. Furthermore, we use MCR to identify immunogenic tumor neo-antigens and show that vaccination with a peptide naturally recognized by tumor-infiltrating lymphocytes efficiently protects mice from tumor challenge. Thus, the MCR technology holds promise for basic research and clinical applications, allowing the personalized identification of T cell-specific neo-antigens in patients.
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http://dx.doi.org/10.1038/s41590-019-0335-zDOI Listing
May 2019

The thioredoxin-1 and glutathione/glutaredoxin-1 systems redundantly fuel murine B-cell development and responses.

Eur J Immunol 2019 05 1;49(5):709-723. Epub 2019 Mar 1.

Institute of Molecular Health Sciences, ETH Zurich, Zürich, Switzerland.

Antioxidant systems maintain cellular redox homeostasis. The thioredoxin-1 (Trx1) and the glutathione (GSH)/glutaredoxin-1 (Grx1) systems are key players in preserving cytosolic redox balance. In fact, T lymphocytes critically rely on reducing equivalents from the Trx1 system for DNA biosynthesis during metabolic reprogramming upon activation. We here show that the Trx1 system is also indispensable for development and functionality of marginal zone (MZ) B cells and B1 cells in mice. In contrast, development of conventional B cells, follicular B-cell homeostasis, germinal center reactions, and antibody responses are redundantly sustained by both antioxidant pathways. Proliferating B2 cells lacking Txnrd1 have increased glutathione (GSH) levels and upregulated cytosolic Grx1, which is barely detectable in expanding thymocytes. These results suggest that the redox capacity driving proliferation is more robust and flexible in B cells than in T cells, which may have profound implications for the therapy of B and T-cell neoplasms.
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http://dx.doi.org/10.1002/eji.201848044DOI Listing
May 2019

A Macrophage-Pericyte Axis Directs Tissue Restoration via Amphiregulin-Induced Transforming Growth Factor Beta Activation.

Immunity 2019 03 12;50(3):645-654.e6. Epub 2019 Feb 12.

Institute of Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3FL, UK. Electronic address:

The epidermal growth factor receptor ligand Amphiregulin has a well-documented role in the restoration of tissue homeostasis after injury; however, the mechanism by which Amphiregulin contributes to wound repair remains unknown. Here we show that Amphiregulin functioned by releasing bioactive transforming growth factor beta (TGF-β) from latent complexes via integrin-α activation. Using acute injury models in two different tissues, we found that by inducing TGF-β activation on mesenchymal stromal cells (pericytes), Amphiregulin induced their differentiation into myofibroblasts, thereby selectively contributing to the restoration of vascular barrier function within injured tissue. Furthermore, we identified macrophages as a critical source of Amphiregulin, revealing a direct effector mechanism by which these cells contribute to tissue restoration after acute injury. Combined, these observations expose a so far under-appreciated mechanism of how cells of the immune system selectively control the differentiation of tissue progenitor cells during tissue repair and inflammation.
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http://dx.doi.org/10.1016/j.immuni.2019.01.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6436929PMC
March 2019

IL-21 promotes allergic airway inflammation by driving apoptosis of FoxP3 regulatory T cells.

J Allergy Clin Immunol 2019 06 14;143(6):2178-2189.e5. Epub 2019 Jan 14.

Institute of Molecular Health Sciences, Department of Biology, ETH Zurich, Zurich, Switzerland. Electronic address:

Background: IL-21 is a key player of adaptive immunity, with well-established roles in B-cell and cytotoxic T-cell responses. IL-21 has been implicated in promotion of effector CD4 T cells and inhibition of forkhead box P3-positive regulatory T (Treg) cells, but the mechanism and functional relevance of these findings remain controversial.

Objective: We sought to understand the mechanisms by which IL-21 controls effector CD4 cell responses and Treg cell homeostasis.

Methods: We used IL-21 receptor-deficient mice to study the effect of IL-21 on T-cell responses in models of asthma and colitis. We used mixed bone marrow chimeras and adoptive transfer of naive CD4 T cells and Treg cells into lymphopenic mice to assess the cell-intrinsic effects of IL-21. Using various in vitro T-cell assays, we characterized the mechanism of IL-21-mediated inhibition of Treg cells.

Results: We show that IL-21 production by T2 and follicular helper T/ex-follicular helper T cells promotes asthma by inhibiting Treg cells. Il21r mice displayed reduced generation of T2 cells and increased generation of Treg cells. In mixed chimeras we demonstrate that IL-21 promotes T2 responses indirectly through inhibition of Treg cells. Depleting Treg cells in Il21r mice restored T2 generation and eosinophilia. Furthermore, IL-21 inhibited Treg cell generation in mice with colitis. Using competitive transfer of Il21r and Il21r CD4 cells, we show that IL-21 directly inhibited expansion of differentiated Treg cells but was dispensable for T1/T17 effectors. We show that IL-21 sensitizes Treg cells to apoptosis by interfering with the expression of Bcl-2 family genes.

Conclusion: IL-21 directly promotes apoptosis of Treg cells and therefore indirectly sustains generation of inflammatory T cells and related effector responses.
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http://dx.doi.org/10.1016/j.jaci.2018.11.047DOI Listing
June 2019

GM-CSF intrinsically controls eosinophil accumulation in the setting of allergic airway inflammation.

J Allergy Clin Immunol 2019 04 20;143(4):1513-1524.e2. Epub 2018 Sep 20.

Molecular Biomedicine, Institute of Molecular Health Sciences, Department of Biology, ETH Zurich, Zurich, Switzerland. Electronic address:

Background: Eosinophils are a therapeutic target in asthmatic patients, and GM-CSF has been suggested to control various aspects of eosinophil biology, including development, function, and survival. However, to date, the role of GM-CSF signaling in eosinophils in vivo is largely unclear.

Objective: We sought to elucidate the role of GM-CSF signaling in asthmatic inflammation.

Methods: Wild-type and GM-CSF receptor α (Csf2ra)-deficient mice reconstituted with Csf2ra-proficient alveolar macrophages were subjected to different models of airway inflammation to evaluate the effect of GM-CSF signaling deficiency on asthmatic inflammation in general and on eosinophils in particular.

Results: We demonstrate that GM-CSF signaling, although being largely dispensable for eosinophil development at steady state, intrinsically promotes accumulation of eosinophils in the lung during allergic airway inflammation. In contrast, chitin-induced eosinophil accumulation in the peritoneal cavity occurs independent of GM-CSF, indicating organ specificity. We show that GM-CSF induces chemokinesis and promotes eosinophil survival in vitro, which likely contribute to eosinophil accumulation in the airways in vivo.

Conclusion: GM-CSF intrinsically promotes eosinophil accumulation in the setting of pulmonary allergic inflammation.
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http://dx.doi.org/10.1016/j.jaci.2018.08.044DOI Listing
April 2019
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