Benedikt S Saller, Svenja Wöhrle , Larissa Fischer, Clara Dufossez, Isabella L Ingerl, Susanne Kessler, Maria Mateo-Tortola, Oliver Gorka, Felix Lange, Yurong Cheng, Emilia Neuwirt, Adinarayana Marada, Christoph Koentges, Chiara Urban, Philipp Aktories, Peter Reuther, Sebastian Giese, Susanne Kirschnek, Carolin Mayer, Johannes Pilic, Hugo Falquez-Medina, Aline Oelgeklaus, Veerasikku Gopal Deepagan, Farzaneh Shojaee, Julia A Zimmermann, Damian Weber, Yi-Heng Tai, Anna Crois, Kevin Ciminski, Remi Peyronnet, Katharina S Brandenburg, Gang Wu, Ralf Baumeister, Thomas Heimbucher, Marta Rizzi, Dietmar Riedel, Martin Helmstädter, Joerg Buescher, Konstantin Neumann, Thomas Misgeld, Martin Kerschensteiner, Peter Walentek, Clemens Kreutz, Ulrich Maurer, Angelika S Rambold, James E Vince, Frank Edlich, Roland Malli, Georg Häcker, Katrin Kierdorf, Chris Meisinger, Anna Köttgen, Stefan Jakobs, Alexander N R Weber, Martin Schwemmle, Christina J Groß, Olaf Groß Immunity 2024 How mitochondria reconcile roles in functionally divergent cell death pathways of apoptosis and NLRP3 inflammasome-mediated pyroptosis remains elusive, as is their precise role in NLRP3 activation and the evolutionarily conserved physiological function of NLRP3. Here, we have shown that when cells were challenged simultaneously, apoptosis was inhibited and NLRP3 activation prevailed. Apoptosis inhibition by structurally diverse NLRP3 activators, including nigericin, imiquimod, extracellular ATP, particles, and viruses, was not a consequence of inflammasome activation but rather of their effects on mitochondria. NLRP3 activators turned out as oxidative phosphorylation (OXPHOS) inhibitors, which we found to disrupt mitochondrial cristae architecture, leading to trapping of cytochrome c. Although this effect was alone not sufficient for NLRP3 activation, OXPHOS inhibitors became triggers of NLRP3 when combined with resiquimod or Yoda-1, suggesting that NLRP3 activation requires two simultaneous cellular signals, one of mitochondrial origin. Therefore, OXPHOS and apoptosis inhibition by NLRP3 activators provide stringency in cell death decisions. Source

Christopher Berlin, Bernhard Mauerer, Pierre Cauchy, Jost Luenstedt, Roman Sankowski, Lisa Marx, Reinhild Feuerstein, Luisa Schaefer, Florian R Greten, Marina Pesic, Olaf Groß, Marco Prinz, Naomi Ruehl, Laura Miketiuk, Dominik Jauch, Claudia Laessle, Andreas Jud, Esther A Biesel, Hannes Neeff, Stefan Fichtner-Feigl, Philipp A. Holzner, Rebecca Kesselring The Journal of Clinical Investigation 2024 Metastasized colorectal cancer (CRC) is associated with a poor prognosis and rapid disease progression. Besides hepatic metastasis, peritoneal carcinomatosis is the major cause of death in Union for International Cancer Control (UICC) stage IV CRC patients. Insights into differential site-specific reconstitution of tumor cells and the corresponding tumor microenvironment are still missing. Here, we analyzed the transcriptome of single cells derived from murine multivisceral CRC and delineated the intermetastatic cellular heterogeneity regarding tumor epithelium, stroma, and immune cells. Interestingly, we found an intercellular site-specific network of cancer-associated fibroblasts and tumor epithelium during peritoneal metastasis as well as an autologous feed-forward loop in cancer stem cells. We furthermore deciphered a metastatic dysfunctional adaptive immunity by a loss of B cell–dependent antigen presentation and consecutive effector T cell exhaustion. Furthermore, we demonstrated major similarities of this murine metastatic CRC model with human disease and — based on the results of our analysis — provided an auspicious site-specific immunomodulatory treatment approach for stage IV CRC by intraperitoneal checkpoint inhibition. Source

Benedikt S. Saller, Emilia Neuwirt, Olaf Groß Methods in Molecular Biology 2023 The inflammasome-nucleating cytoplasmic sensor protein NLRP3 (NACHT-, LRR, and PYD domains-containing protein 3, also known as NOD-like receptor pyrin domain-containing 3, NALP3, or cryopyrin) is triggered by a broad spectrum of sterile endogenous danger signals and environmental irritants. Upon activation, NLRP3 engages the adapter protein ASC that in turn recruits the third inflammasome component, the protease caspase-1. Subsequent caspase-1 activation leads to its auto-processing and maturation of the leaderless IL-1 family cytokines IL-1β and IL-18 as well as cleavage of the pore-forming protein Gasdermin D (GSDMD). GSDMD plasma membrane pores, formed by its N-terminus, facilitate IL-1 release and, typically, subsequent cell lysis (pyroptosis). This protocol explains standard methods, which are routinely used in our laboratory to study NLRP3 inflammasome biology in vitro. It includes experimental approaches using primary murine bone marrow-derived macrophages (BMDMs) and bone marrow-derived dendritic cells (BMDCs), human peripheral blood mononuclear cells (PBMCs), as well as inflammasome-competent cell lines (HoxB8 and THP-1 cells). The protocol covers the use of a broad spectrum of established NLRP3 activators and outlines the use of common inhibitors blocking NLRP3 itself or its upstream triggering events. We also provide guidelines for experimental set-up and crucial experimental controls to investigate NLRP3 inflammasome signaling or study new activators and inhibitors. Source

Emilia Neuwirt, Giovanni Magnani, Tamara Ćiković, Svenja Wöhrle, Larissa Fischer, Anna Kostina, Stephan Flemming, Nora J Fischenich, Benedikt S. Saller, Oliver Gorka, Steffen Renner, Claudia Agarinis, Christian N Parker, Andreas Boettcher, Christopher J Farady, Rebecca Kesselring, Christopher Berlin, Rolf Backofen, Marta Rodriguez-Franco, Clemens Kreutz, Marco Prinz, Martina Tholen, Thomas Reinheckel, Thomas Ott, Christina J Groß, Philipp J Jost, Olaf Groß Science Signaling 2023 Inflammasomes are intracellular protein complexes that promote an inflammatory host defense in response to pathogens and damaged or neoplastic tissues and are implicated in inflammatory disorders and therapeutic- induced toxicity. We investigated the mechanisms of activation for inflammasomes nucleated by NOD-like re- ceptor (NLR) protiens. A screen of a small-molecule library revealed that several tyrosine kinase inhibitors (TKIs)—including those that are clinically approved (such as imatinib and crizotinib) or are in clinical trials (such as masitinib)—activated the NLRP3 inflammasome. Furthermore, imatinib and masitinib caused lysosom- al swelling and damage independently of their kinase target, leading to cathepsin-mediated destabilization of myeloid cell membranes and, ultimately, cell lysis that was accompanied by potassium (K+) efflux, which acti- vated NLRP3. This effect was specific to primary myeloid cells (such as peripheral blood mononuclear cells and mouse bone marrow–derived dendritic cells) and did not occur in other primary cell types or various cell lines. TKI-induced lytic cell death and NLRP3 activation, but not lysosomal damage, were prevented by stabilizing cell membranes. Our findings reveal a potential immunological off-target of some TKIs that may contribute to their clinical efficacy or to their adverse effects. Source

Jan Hettwer, Julia Hinterdobler, Benedikt Miritsch, Marcus-André Deutsch, Xinghai Li, Carina Mauersberger, Aldo Moggio, Quinte Braster, Hermann Gram, Avril AB Robertson, Matthew A Cooper, Olaf Groß, Markus Krane, Christian Weber, Wolfgang Koenig, Oliver Soehnlein, Nicholas H Adamstein, Paul Ridker, Heribert Schunkert, Peter Libby, Thorsten Kessler, Hendrik B Sager Cardiovascular Research 2022 Aims: Targeting vascular inflammation represents a novel therapeutic approach to reduce complications of atherosclerosis. Neutralizing the pro-inflammatory cytokine interleukin-1β (IL-1β) using canakinumab, a monoclonal antibody, reduces the incidence of cardiovascular events in patients after myocardial infarction (MI). The biological basis for these beneficial effects remains incompletely understood. We sought to explore the mechanisms of IL-1β-targeted therapies. Methods and results: In mice with early atherosclerosis (ApoE-/- mice on a high-cholesterol diet for 6 weeks), we found that 3 weeks of NACHT, LRR, and PYD domains-containing protein 3 (NLRP3)-inflammasome inhibition or anti-IL-1β treatment (using either MCC950, an NLRP3-inflammasome inhibitor which blocks production and release of active IL-1β, or a murine analogue of canakinumab) dampened accumulation of leucocytes in atherosclerotic aortas, which consequently resulted in slower progression of atherosclerosis. Causally, we found that endothelial cells from atherosclerotic aortas lowered expression of leucocyte chemoattractants and adhesion molecules upon NLRP3-inflammasome inhibition, indicating that NLRP3-inflammasome- and IL-1β-targeted therapies reduced blood leucocyte recruitment to atherosclerotic aortas. In accord, adoptive transfer experiments revealed that anti-IL-1β treatment mitigated blood myeloid cell uptake to atherosclerotic aortas. We further report that anti-IL-1β treatment and NLRP3-inflammasome inhibition reduced inflammatory leucocyte supply by decreasing proliferation of bone marrow haematopoietic stem and progenitor cells, demonstrating that suppression of IL-1β and the NLRP3-inflammasome lowered production of disease-propagating leucocytes. Using bone marrow reconstitution experiments, we observed that haematopoietic cell-specific NLRP3-inflammasome activity contributed to both enhanced recruitment and increased supply of blood inflammatory leucocytes. Further experiments that queried whether anti-IL-1β treatment reduced vascular inflammation also in post-MI accelerated atherosclerosis documented the operation of convergent mechanisms (reduced supply and uptake of inflammatory leucocytes). In line with our pre-clinical findings, post-MI patients on canakinumab treatment showed reduced blood monocyte numbers. Conclusions: Our murine and human data reveal that anti-IL-1β treatment and NLRP3-inflammasome inhibition dampened vascular inflammation and progression of atherosclerosis through reduced blood inflammatory leucocyte (i) supply and (ii) uptake into atherosclerotic aortas providing additional mechanistic insights into links between haematopoiesis and atherogenesis, and into the beneficial effects of NLRP3-inflammasome- and IL-1β-targeted therapies. Source

Emilia Neuwirt, Oliver Gorka, Benedikt S Saller, Christina J Groß, Tobias Madl, Olaf Groß Current Opinion in Biotechnology 2021 The NLRP3 inflammasome is an important player in innate immunity and pathogenic inflammation. Numerous studies have implicated it in sensing endogenous danger signals, yet the precise mechanisms remain unknown. Here, we review the current knowledge on the organismal and cellular metabolic triggers engaging NLRP3, and the mechanisms involved in integrating the diverse signals. Source

Shaima’a Hamarsheh, Olaf Groß, Tilman Brummer, Robert Zeiser Nature Communications 2020 Oncogenic KRAS mutations are the most frequent mutations in human cancer, but most difficult to target. While sustained proliferation caused by oncogenic KRAS-downstream signalling is a main driver of carcinogenesis, there is increasing evidence that it also mediates autocrine effects and crosstalk with the tumour microenvironment (TME). Here, we discuss recent reports connecting KRAS mutations with tumour-promoting inflammation and immune modulation caused by KRAS that leads to immune escape in the TME. We discuss the preclinical work on KRAS-induced inflammation and immune modulation in the context of currently ongoing clinical trials targeting cancer entities that carry KRAS mutations and strategies to overcome the oncogene-induced effects on the immune system. Source

Shaima’a Hamarsheh, Lena Osswald, Benedikt S Saller, Susanne Unger, Donatella De Feo, Janaki Manoja Vinnakota, Martina Konantz, Franziska M Uhl, Heiko Becker, Michael Lübbert, Khalid Shoumariyeh, Christoph Schürch, Geoffroy Andrieux, Nils Venhoff, Annette Schmitt-Graeff, Sandra Duquesne, Dietmar Pfeifer, Matthew A Cooper, Claudia Lengerke, Melanie Boerries, Justus Duyster, Charlotte M Niemeyer, Miriam Erlacher, Bruce R Blazar, Burkard Becher, Olaf Groß, Tilman Brummer, Robert Zeiser Nature Communications 2020 Oncogenic Ras mutations occur in various leukemias. It was unclear if, besides the direct transforming effect via constant RAS/MEK/ERK signaling, an inflammation-related effect of KRAS contributes to the disease. Here, we identify a functional link between oncogenic KrasG12D and NLRP3 inflammasome activation in murine and human cells. Mice expressing active KrasG12D in the hematopoietic system developed myeloproliferation and cytopenia, which is reversed in KrasG12D mice lacking NLRP3 in the hematopoietic system. Therapeutic IL-1-receptor blockade or NLRP3-inhibition reduces myeloproliferation and improves hematopoiesis. Mechanistically, KrasG12D-RAC1 activation induces reactive oxygen species (ROS) production causing NLRP3 inflammasome-activation. In agreement with our observations in mice, patient-derived myeloid leukemia cells exhibit KRAS/RAC1/ROS/NLRP3/IL-1β axis activity. Our findings indicate that oncogenic KRAS not only act via its canonical oncogenic driver function, but also enhances the activation of the pro-inflammatory RAC1/ROS/NLRP3/IL-1β axis. This paves the way for a therapeutic approach based on immune modulation via NLRP3 blockade in KRAS-mutant myeloid malignancies. Source

Marta Joana Costa Jordão, Roman Sankowski, Stefanie M. Brendecke, Sagar, Giuseppe Locatelli, Yi-Heng Tai, Tuan Leng Tay, Eva Schramm, Stephan Armbruster, Nora Hagemeyer, Olaf Groß, Dominic Mai, Özgün Çiçek, Thorsten Falk, Martin Kerschensteiner, Dominic Grün, Marco Prinz Science 2019 The innate immune cell compartment is highly diverse in the healthy central nervous system (CNS), including parenchymal and non-parenchymal macrophages. However, this complexity is increased in inflammatory settings by the recruitment of circulating myeloid cells. It is unclear which disease-specific myeloid subsets exist and what their transcriptional profiles and dynamics during CNS pathology are. Combining deep single-cell transcriptome analysis, fate mapping, in vivo imaging, clonal analysis, and transgenic mouse lines, we comprehensively characterized unappreciated myeloid subsets in several CNS compartments during neuroinflammation. During inflammation, CNS macrophage subsets undergo self-renewal, and random proliferation shifts toward clonal expansion. Last, functional studies demonstrated that endogenous CNS tissue macrophages are redundant for antigen presentation. Our results highlight myeloid cell diversity and provide insights into the brain's innate immune system. Source

Anna Lorentzen, Paul F Becker, Jan Kosla, Massimo Saini, Kathrin Weidele, Paolo Ronchi, Corinna Klein, Monika J Wolf, Felix Geist, Bastian Seubert, Marc Ringelhan, Daniela Mihic-Probst, Knud Esser, Marko Roblek, Felix Kuehne, Gaia Bianco, Tracy O’Connor, Quentin Müller, Kathleen Schuck, Sebastian Lange, Daniel Hartmann, Saskia Spaich, Olaf Groß, Jochen Utikal, Sebastian Haferkamp, Martin R. Sprick, Amruta Damle-Vartak, Alexander Hapfelmeier, Norbert Hüser, Ulrike Protzer, Andreas Trumpp, Dieter Saur, Nachiket Vartak, Christoph A Klein, Bernhard Polzer, Lubor Borsig, Mathias Heikenwalder Nature Communications 2018 Dynamic polarisation of tumour cells is essential for metastasis. While the role of polarisation during dedifferentiation and migration is well established, polarisation of metastasising tumour cells during phases of detachment has not been investigated. Here we identify and characterise a type of polarisation maintained by single cells in liquid phase termed single-cell (sc) polarity and investigate its role during metastasis. We demonstrate that sc polarity is an inherent feature of cells from different tumour entities that is observed in circulating tumour cells in patients. Functionally, we propose that the sc pole is directly involved in early attachment, thereby affecting adhesion, transmigration and metastasis. In vivo, the metastatic capacity of cell lines correlates with the extent of sc polarisation. By manipulating sc polarity regulators and by generic depolarisation, we show that sc polarity prior to migration affects transmigration and metastasis in vitro and in vivo. Source

Christina J Groß, Ritu Mishra, Katharina S Schneider, Guillaume Médard, Jennifer Wettmarshausen, Daniela C Dittlein, Hexin Shi, Oliver Gorka, Paul-Albert Koenig, Stephan Fromm, Giovanni Magnani, Tamara Ćiković, Lara Hartjes, Joachim Smollich, Avril A B Robertson, Matthew A Cooper, Marc Schmidt-Supprian, Michael Schuster, Kate Schroder, Petr Broz, Claudia Traidl-Hoffmann, Bruce Beutler, Bernhard Kuster, Jürgen Ruland, Sabine Schneider, Fabiana Perocchi, Olaf Groß Immunity 2016 Imiquimod is a small-molecule ligand of Toll-like receptor-7 (TLR7) that is licensed for the treatment of viral infections and cancers of the skin. Imiquimod has TLR7-independent activities that are mechanistically unexplained, including NLRP3 inflammasome activation in myeloid cells and apoptosis induction in cancer cells. We investigated the mechanism of inflammasome activation by imiquimod and the related molecule CL097 and determined that K+ efflux was dispensable for NLRP3 activation by these compounds. Imiquimod and CL097 inhibited the quinone oxidoreductases NQO2 and mitochondrial Complex I. This induced a burst of reactive oxygen species (ROS) and thiol oxidation, and led to NLRP3 activation via NEK7, a recently identified component of this inflammasome. Metabolic consequences of Complex I inhibition and endolysosomal effects of imiquimod might also contribute to NLRP3 activation. Our results reveal a K+ efflux-independent mechanism for NLRP3 activation and identify targets of imiquimod that might be clinically relevant. Source

Ulrike Höckendorf, Monica Yabal, Tobias Herold, Enkhtsetseg Munkhbaatar, Stephanie Rott, Stefanie Jilg, Johanna Kauschinger, Giovanni Magnani, Florian Reisinger, Michael Heuser, Hans Kreipe, Karl Sotlar, Thomas Engleitner, Roland Rad, Wilko Weichert, Christian Peschel, Jürgen Ruland, Mathias Heikenwalder, Karsten Spiekermann, Julia Slotta-Huspenina, Olaf Groß, Philipp J Jost Cancer Cell 2016 Since acute myeloid leukemia (AML) is characterized by the blockade of hematopoietic differentiation and cell death, we interrogated RIPK3 signaling in AML development. Genetic loss of Ripk3 converted murine FLT3-ITD-driven myeloproliferation into an overt AML by enhancing the accumulation of leukemia-initiating cells (LIC). Failed inflammasome activation and cell death mediated by tumor necrosis factor receptor caused this accumulation of LIC exemplified by accelerated leukemia onset in Il1r1−/−, Pycard–/–, and Tnfr1/2−/− mice. RIPK3 signaling was partly mediated by mixed lineage kinase domain-like. This link between suppression of RIPK3, failed interleukin-1β release, and blocked cell death was supported by significantly reduced RIPK3 in primary AML patient cohorts. Our data identify RIPK3 and the inflammasome as key tumor suppressors in AML. Source