Publications by authors named "Rudi Beyaert"

202 Publications

Reprogramming of glucocorticoid receptor function by hypoxia.

EMBO Rep 2021 Oct 26:e53083. Epub 2021 Oct 26.

VIB Center for Inflammation Research, Ghent, Belgium.

Here, we investigate the impact of hypoxia on the hepatic response of glucocorticoid receptor (GR) to dexamethasone (DEX) in mice via RNA-sequencing. Hypoxia causes three types of reprogramming of GR: (i) much weaker induction of classical GR-responsive genes by DEX in hypoxia, (ii) a number of genes is induced by DEX specifically in hypoxia, and (iii) hypoxia induces a group of genes via activation of the hypothalamic-pituitary-adrenal (HPA) axis. Transcriptional profiles are reflected by changed GR DNA-binding as measured by ChIP sequencing. The HPA axis is induced by hypothalamic HIF1α and HIF2α activation and leads to GR-dependent lipolysis and ketogenesis. Acute inflammation, induced by lipopolysaccharide, is prevented by DEX in normoxia but not during hypoxia, and this is attributed to HPA axis activation by hypoxia. We unfold new physiological pathways that have consequences for patients suffering from GC resistance.
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http://dx.doi.org/10.15252/embr.202153083DOI Listing
October 2021

Polo-like kinase 1 (PLK1) signaling in cancer and beyond.

Biochem Pharmacol 2021 Aug 26;193:114747. Epub 2021 Aug 26.

Center for Inflammation Research, Unit of Molecular Signal Transduction in Inflammation, VIB, B-9052 Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, B-9052 Ghent, Belgium.

PLK1 is an evolutionary conserved Ser/Thr kinase that is best known for its role in cell cycle regulation and is expressed predominantly during the G2/S and M phase of the cell cycle. PLK1-mediated phosphorylation of specific substrates controls cell entry into mitosis, centrosome maturation, spindle assembly, sister chromatid cohesion and cytokinesis. In addition, a growing body of evidence describes additional roles of PLK1 beyond the cell cycle, more specifically in the DNA damage response, autophagy, apoptosis and cytokine signaling. PLK1 has an indisputable role in cancer as it controls several key transcription factors and promotes cell proliferation, transformation and epithelial-to-mesenchymal transition. Furthermore, deregulation of PLK1 results in chromosome instability and aneuploidy. PLK1 is overexpressed in many cancers, which is associated with poor prognosis, making PLK1 an attractive target for cancer treatment. Additionally, PLK1 is involved in immune and neurological disorders including Graft versus Host Disease, Huntington's disease and Alzheimer's disease. Unfortunately, newly developed small compound PLK1 inhibitors have only had limited success so far, due to low therapeutic response rates and toxicity. In this review we will highlight the current knowledge about the established roles of PLK1 in mitosis regulation and beyond. In addition, we will discuss its tumor promoting but also tumor suppressing capacities, as well as the available PLK1 inhibitors, elaborating on their efficacy and limitations.
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http://dx.doi.org/10.1016/j.bcp.2021.114747DOI Listing
August 2021

Cyclin D2 overexpression drives B1a-derived MCL-like lymphoma in mice.

J Exp Med 2021 Oct 18;218(10). Epub 2021 Aug 18.

Department of Biomolecular Medicine, Ghent University, Ghent, Belgium.

Mantle cell lymphoma (MCL) is an aggressive B cell lymphoma with poor long-term overall survival. Currently, MCL research and development of potential cures is hampered by the lack of good in vivo models. MCL is characterized by recurrent translocations of CCND1 or CCND2, resulting in overexpression of the cell cycle regulators cyclin D1 or D2, respectively. Here, we show, for the first time, that hematopoiesis-specific activation of cyclin D2 is sufficient to drive murine MCL-like lymphoma development. Furthermore, we demonstrate that cyclin D2 overexpression can synergize with loss of p53 to form aggressive and transplantable MCL-like lymphomas. Strikingly, cyclin D2-driven lymphomas display transcriptional, immunophenotypic, and functional similarities with B1a B cells. These MCL-like lymphomas have B1a-specific B cell receptors (BCRs), show elevated BCR and NF-κB pathway activation, and display increased MALT1 protease activity. Finally, we provide preclinical evidence that inhibition of MALT1 protease activity, which is essential for the development of early life-derived B1a cells, can be an effective therapeutic strategy to treat MCL.
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http://dx.doi.org/10.1084/jem.20202280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8377631PMC
October 2021

ZBTB32 performs crosstalk with the glucocorticoid receptor and is crucial in glucocorticoid responses to starvation.

iScience 2021 Jul 28;24(7):102790. Epub 2021 Jun 28.

Center for Inflammation Research, VIB Center for Inflammation Research, 9000 Ghent, Belgium.

The hypothalamic-pituitary-adrenal (HPA) axis forms a complex neuroendocrine system that regulates the body's response to stress such as starvation. In contrast with the glucocorticoid receptor (GR), Zinc finger and BTB domain containing 32 (ZBTB32) is a transcription factor with poorly described functional relevance in physiology. This study shows that ZBTB32 is essential for the production of glucocorticoids (GCs) in response to starvation, since ZBTB32 mice fail to increase their GC production in the absence of nutrients. In terms of mechanism, GR-mediated upregulation of adrenal gene expression was absent in ZBTB32 mice, implicating defective cholesterol import as the cause of the poor GC synthesis. These lower GC levels are further associated with aberrations in the metabolic adaptation to starvation, which could explain the progressive weight gain of ZBTB32 mice. In conclusion, ZBTB32 performs a crosstalk with the GR in the metabolic adaptation to starvation via regulation of adrenal GC production.
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http://dx.doi.org/10.1016/j.isci.2021.102790DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8324811PMC
July 2021

Bidirectional Crosstalk Between Hypoxia Inducible Factors and Glucocorticoid Signalling in Health and Disease.

Front Immunol 2021 4;12:684085. Epub 2021 Jun 4.

Centre for Inflammation Research, Flanders Institute for Biotechnology (VIB), Ghent, Belgium.

Glucocorticoid-induced (GC) and hypoxia-induced transcriptional responses play an important role in tissue homeostasis and in the regulation of cellular responses to stress and inflammation. Evidence exists that there is an important crosstalk between both GC and hypoxia effects. Hypoxia is a pathophysiological condition to which cells respond quickly in order to prevent metabolic shutdown and death. The hypoxia inducible factors (HIFs) are the master regulators of oxygen homeostasis and are responsible for the ability of cells to cope with low oxygen levels. Maladaptive responses of HIFs contribute to a variety of pathological conditions including acute mountain sickness (AMS), inflammation and neonatal hypoxia-induced brain injury. Synthetic GCs which are analogous to the naturally occurring steroid hormones (cortisol in humans, corticosterone in rodents), have been used for decades as anti-inflammatory drugs for treating pathological conditions which are linked to hypoxia (i.e. asthma, ischemic injury). In this review, we investigate the crosstalk between the glucocorticoid receptor (GR), and HIFs. We discuss possible mechanisms by which GR and HIF influence one another, and , and the therapeutic effects of GCs on HIF-mediated diseases.
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http://dx.doi.org/10.3389/fimmu.2021.684085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8211996PMC
June 2021

IL-33trap-mediated IL-33 neutralization does not exacerbate choroidal neovascularization, but fails to protect against retinal degeneration in a dry age-related macular degeneration model.

Exp Eye Res 2021 06 27;207:108608. Epub 2021 Apr 27.

Oxurion NV, Gaston Geenslaan 1, 3001, Heverlee, Belgium.

The progressive and sight-threatening disease, age-related macular degeneration (AMD), is a growing public health concern due to ageing demographics, with the highest unmet medical need for the advanced stage of dry AMD, geographic atrophy. The pathogenesis underlying AMD is driven by a complex interplay of genetic and environmental factors. There is ample evidence that inflammation is strongly involved in AMD development. Interleukin-33 (IL-33) has been proposed to be critically involved in retinal degeneration, but a protective role in eye pathophysiology was also demonstrated. The current study investigated the therapeutic potential of IL-33trap, a novel IL-33-neutralizing biologic, in dry AMD/geographic atrophy and, based on controversial data regarding the protective versus detrimental functions of IL-33 in neovascularization, evaluated the risk of progression to wet AMD by IL-33 neutralization. Repeated intravitreal (IVT) injections of IL-33trap in the mouse laser-induced choroidal neovascularization model did not exacerbate neovascularization or leakage, while it significantly inhibited inflammatory cell infiltration in the retinal pigment epithelium and choroid. On the contrary, IVT treatment with IL-33trap significantly induced retinal inflammation and could not prevent retinopathy induction in the mouse sodium iodate (NaIO) model. Overall, these data suggest a complex and dichotomous role of IL-33 in eye pathology and indicate that IL-33 neutralization is not able to prevent onset and progression of dry AMD pathogenesis.
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http://dx.doi.org/10.1016/j.exer.2021.108608DOI Listing
June 2021

Tailored Modulation of Cellular Pro-inflammatory Responses With Disaccharide Lipid A Mimetics.

Front Immunol 2021 18;12:631797. Epub 2021 Mar 18.

Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria.

Pro-inflammatory signaling mediated by Toll-like receptor 4 (TLR4)/myeloid differentiation-2 (MD-2) complex plays a crucial role in the instantaneous protection against infectious challenge and largely contributes to recovery from Gram-negative infection. Activation of TLR4 also boosts the adaptive immunity which is implemented in the development of vaccine adjuvants by application of minimally toxic TLR4 activating ligands. The modulation of pro-inflammatory responses via the TLR4 signaling pathway was found beneficial for management of acute and chronic inflammatory disorders including asthma, allergy, arthritis, Alzheimer disease pathology, sepsis, and cancer. The TLR4/MD-2 complex can recognize the terminal motif of Gram-negative bacterial lipopolysaccharide (LPS)-a glycophospholipid lipid A. Although immense progress in understanding the molecular basis of LPS-induced TLR4-mediated signaling has been achieved, gradual, and predictable TLR4 activation by structurally defined ligands has not yet been attained. We report on controllable modulation of cellular pro-inflammatory responses by application of novel synthetic glycolipids-disaccharide-based lipid A mimetics (DLAMs) having picomolar affinity for TLR4/MD-2. Using crystal structure inspired design we have developed endotoxin mimetics where the inherently flexible β(1 → 6)-linked diglucosamine backbone of lipid A is replaced by a conformationally restricted α,α-(1↔1)-linked disaccharide scaffold. The tertiary structure of the disaccharide skeleton of DLAMs mirrors the 3-dimensional shape of TLR4/MD-2 bound lipid A. Due to exceptional conformational rigidity of the sugar scaffold, the specific 3D organization of DLAM must be preserved upon interaction with proteins. These structural factors along with specific acylation and phosphorylation pattern can ensure picomolar affinity for TLR4 and permit efficient dimerization of TLR4/MD-2/DLAM complexes. Since the binding pose of lipid A in the binding pocket of MD-2 (±180°) is crucial for the expression of biological activity, the chemical structure of DLAMs was designed to permit a predefined binding orientation in the binding groove of MD-2, which ensured tailored and species-independent (human and mice) TLR4 activation. Manipulating phosphorylation and acylation pattern at the sugar moiety facing the secondary dimerization interface allowed for adjustable modulation of the TLR4-mediated signaling. Tailored modulation of cellular pro-inflammatory responses by distinct modifications of the molecular structure of DLAMs was attained in primary human and mouse immune cells, lung epithelial cells and TLR4 transfected HEK293 cells.
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http://dx.doi.org/10.3389/fimmu.2021.631797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8012497PMC
September 2021

Analysis of T cells in mouse lymphoid tissue and blood with flow cytometry.

STAR Protoc 2021 Mar 20;2(1):100351. Epub 2021 Feb 20.

Center for Inflammation Research, VIB, Technologiepark-Zwijnaarde 71, 9052 Ghent, Belgium.

T cells play a key role in adaptive immunity. Defects in specific T cell receptors or signaling proteins can alter their frequency and activation status, which may be associated with immune disease or cancer. Monitoring of T cell frequency and function in genetically modified mice or murine models of disease is therefore of high interest. Here, we provide a detailed protocol to analyze regulatory T cells, T cell activation, and cytokine production in thymus, spleen, or blood via flow cytometry. For complete details on the use and execution of this protocol, please refer to Demeyer et al. (2020).
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http://dx.doi.org/10.1016/j.xpro.2021.100351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7907922PMC
March 2021

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

Inflammatory cell-derived CXCL3 promotes pancreatic cancer metastasis through a novel myofibroblast-hijacked cancer escape mechanism.

Gut 2021 Feb 10. Epub 2021 Feb 10.

Department of Microbiology, Tumor and Cell Biology, Biomedicum, Karolinska Institutet, Stockholm, Sweden

Objective: Pancreatic ductal adenocarcinoma (PDAC) is the most lethal malignancy and lacks effective treatment. We aimed to understand molecular mechanisms of the intertwined interactions between tumour stromal components in metastasis and to provide a new paradigm for PDAC therapy.

Design: Two unselected cohorts of 154 and 20 patients with PDAC were subjected to correlation between interleukin (IL)-33 and CXCL3 levels and survivals. Unbiased expression profiling, and genetic and pharmacological gain-of-function and loss-of-function approaches were employed to identify molecular signalling in tumour-associated macrophages (TAMs) and myofibroblastic cancer-associated fibroblasts (myoCAFs). The role of the IL-33-ST2-CXCL3-CXCR2 axis in PDAC metastasis was evaluated in three clinically relevant mouse PDAC models.

Results: IL-33 was specifically elevated in human PDACs and positively correlated with tumour inflammation in human patients with PDAC. CXCL3 was highly upregulated in IL-33-stimulated macrophages that were the primary source of CXCL3. CXCL3 was correlated with poor survival in human patients with PDAC. Mechanistically, activation of the IL-33-ST2-MYC pathway attributed to high CXCL3 production. The highest level of CXCL3 was found in PDAC relative to other cancer types and its receptor CXCR2 was almost exclusively expressed in CAFs. Activation of CXCR2 by CXCL3 induced a CAF-to-myoCAF transition and α-smooth muscle actin (α-SMA) was uniquely upregulated by the CXCL3-CXCR2 signalling. Type III collagen was identified as the CXCL3-CXCR2-targeted adhesive molecule responsible for myoCAF-driven PDAC metastasis.

Conclusions: Our work provides novel mechanistic insights into understanding PDAC metastasis by the TAM-CAF interaction and targeting each of these signalling components would provide an attractive and new paradigm for treating pancreatic cancer.
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http://dx.doi.org/10.1136/gutjnl-2020-322744DOI Listing
February 2021

Immune responses and therapeutic options in psoriasis.

Cell Mol Life Sci 2021 Mar 2;78(6):2709-2727. Epub 2021 Jan 2.

Unit of Molecular Signal Transduction in Inflammation, Center for Inflammation Research, Ghent University - VIB, Technologiepark 71, B-9052, Ghent, Belgium.

Psoriasis is a chronic inflammatory disease of the skin that affects about 2-3% of the population and greatly impairs the quality of life of affected individuals. Psoriatic skin is characterized by excessive proliferation and aberrant differentiation of keratinocytes, as well as redness caused by increased dilation of the dermal blood vessels and infiltration of immune cells. Although the pathogenesis of psoriasis has not yet been completely elucidated, it is generally believed to arise from a complex interplay between hyperproliferating keratinocytes and infiltrating, activated immune cells. So far, the exact triggers that elicit this disease are still enigmatic, yet, it is clear that genetic predisposition significantly contributes to the development of psoriasis. In this review, we summarize current knowledge of important cellular and molecular mechanisms driving the initiation and amplification stages of psoriasis development, with a particular focus on cytokines and emerging evidence illustrating keratinocyte-intrinsic defects as key drivers of inflammation. We also discuss mouse models that have contributed to a better understanding of psoriasis pathogenesis and the preclinical development of novel therapeutics, including monoclonal antibodies against specific cytokines or cytokine receptors that have revolutionized the treatment of psoriasis. Future perspectives that may have the potential to push basic research and open up new avenues for therapeutic intervention are provided.
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http://dx.doi.org/10.1007/s00018-020-03726-1DOI Listing
March 2021

Long-Term MALT1 Inhibition in Adult Mice Without Severe Systemic Autoimmunity.

iScience 2020 Oct 12;23(10):101557. Epub 2020 Sep 12.

Center for Inflammation Research, VIB, Technologiepark-Zwijnaarde 71, 9052 Ghent, Belgium.

The protease MALT1 is a key regulator of NF-κB signaling and a novel therapeutic target in autoimmunity and cancer. Initial enthusiasm supported by preclinical results with MALT1 inhibitors was tempered by studies showing that germline MALT1 protease inactivation in mice results in reduced regulatory T cells and lethal multi-organ inflammation due to expansion of IFN-γ-producing T cells. However, we show that long-term MALT1 inactivation, starting in adulthood, is not associated with severe systemic inflammation, despite reduced regulatory T cells. In contrast, IL-2-, TNF-, and IFN-γ-producing CD4 T cells were strongly reduced. Limited formation of tertiary lymphoid structures was detectable in lungs and stomach, which did not affect overall health. Our data illustrate that MALT1 inhibition in prenatal or adult life has a different outcome and that long-term MALT1 inhibition in adulthood is not associated with severe side effects.
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http://dx.doi.org/10.1016/j.isci.2020.101557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7522757PMC
October 2020

Recognition by Dendritic Cells Negatively Regulates Allergic Lung Inflammation through a TLR2/MyD88 Pathway.

Am J Respir Cell Mol Biol 2021 01;64(1):39-49

Service Immune Response and.

is an opportunistic fungal pathogen responsible for a spectrum of clinical manifestations. Dendritic cells recognize pathogen-associated molecular patterns of via two main receptor families, Toll-like receptors (TLRs) and C-type lectin receptors (CLR). Here, the importance of TLR and CLR signaling in the regulation of T-helper cell type 2 (Th2) responses was analyzed using a mouse model based on the transfer of bone marrow-derived dendritic cells (BMDCs) pulsed with conidia. BMDCs were generated from mice deficient in either MyD88 or MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1). Both the MyD88 and MALT1 signaling pathway in BMDCs contributed to the production of inflammatory cytokines induced by conidia. Mice sensitized with MyD88 BMDCs pulsed with conidia showed an exacerbated allergic inflammation, with stronger eosinophil recruitment in the BAL and higher Th2 cytokine production compared with mice sensitized with wild-type or MALT1 BMDCs. This exacerbation was not observed when MyD88 BMDCs were pulsed with , a nonpathogenic mold. A lack of TLR2 signaling recapitulated the exacerbation of the Th2 response observed in the absence of MyD88 signaling, whereas TLR2 agonist dampened the response induced with and conidia. IL-10 production by BMDCs in response to was dependent on the expression of TLR2 and MyD88. IL-10 BMDCs exacerbated, whereas MyD88 BMDCs supplemented with exogenous IL-10 decreased the allergic pulmonary inflammation. These results indicate that TLR2/MyD88-specific recognition of PAMPs from conidia can upregulate IL-10 production and downregulate lung eosinophilia and the development of a Th2 response.
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http://dx.doi.org/10.1165/rcmb.2020-0083OCDOI Listing
January 2021

Zinc inhibits lethal inflammatory shock by preventing microbe-induced interferon signature in intestinal epithelium.

EMBO Mol Med 2020 10 11;12(10):e11917. Epub 2020 Sep 11.

Center for Inflammation Research, VIB, Ghent, Belgium.

The cytokine TNF drives inflammatory diseases, e.g., Crohn's disease. In a mouse model of TNF-induced systemic inflammatory response syndrome (SIRS), severe impact on intestinal epithelial cells (IECs) is observed. Zinc confers complete protection in this model. We found that zinc no longer protects in animals which lack glucocorticoids (GCs), or express mutant versions of their receptor GR in IECs, nor in mice which lack gut microbiota. RNA-seq studies in IECs showed that zinc caused reduction in expression of constitutive (STAT1-induced) interferon-stimulated response (ISRE) genes and interferon regulatory factor (IRF) genes. Since some of these genes are involved in TNF-induced cell death in intestinal crypt Paneth cells, and since zinc has direct effects on the composition of the gut microbiota (such as several Staphylococcus species) and on TNF-induced Paneth cell death, we postulate a new zinc-related anti-inflammatory mechanism. Zinc modulates the gut microbiota, causing less induction of ISRE/IRF genes in crypt cells, less TNF-induced necroptosis in Paneth cells, and less fatal evasion of gut bacteria into the system.
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http://dx.doi.org/10.15252/emmm.201911917DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7539219PMC
October 2020

Defining the combinatorial space of PKC::CARD-CC signal transduction nodes.

FEBS J 2021 03 1;288(5):1630-1647. Epub 2020 Sep 1.

Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.

Signal transduction typically displays a so-called bow-tie topology: Multiple receptors lead to multiple cellular responses but the signals all pass through a narrow waist of central signaling nodes. One such signaling node for several inflammatory and oncogenic signaling pathways is the CARD-CC/BCL10/MALT1 (CBM) complexes, which get activated by protein kinase C (PKC)-mediated phosphorylation of the caspase activation and recruitment domain (CARD)-coiled-coil domain (CC) component. In humans, there are four CARD-CC family proteins (CARD9, CARD10, CARD11, and CARD14) and 9 true PKC isozymes (α to ι). At this moment, less than a handful of PKC::CARD-CC relationships are known. In order to explore the biologically relevant combinatorial space out of all 36 potential permutations in this two-component signaling event, we made use of CARD10-deficient human embryonic kidney 293T cells for subsequent pairwise cotransfections of all CARD-CC family members and all activated PKCs. Upon analysis of NF-κB-dependent reporter gene expression, we could define specific PKC::CARD-CC relationships. Surprisingly, as many as 21 PKC::CARD-CC functional combinations were identified. CARD10 was responsive to most PKCs, while CARD14 was mainly activated by PKCδ. The CARD11 activation profile was most similar to that of CARD9. We also discovered the existence of mixed protein complexes between different CARD-CC proteins, which was shown to influence their PKC response profile. Finally, multiple PKCs were found to use a common phosphorylation site to activate CARD9, while additional phosphorylation sites contribute to CARD14 activation. Together, these data reveal the combinatorial space of PKC::CARD-CC signal transduction nodes, which will be valuable for future studies on the regulation of CBM signaling.
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http://dx.doi.org/10.1111/febs.15522DOI Listing
March 2021

Single-Chain Soluble Receptor Fusion Proteins as Versatile Cytokine Inhibitors.

Front Immunol 2020 13;11:1422. Epub 2020 Jul 13.

Center for Inflammation Research, Unit of Molecular Signal Transduction in Inflammation, VIB, Ghent, Belgium.

Cytokines are small secreted proteins that among many functions also play key roles in the orchestration of inflammation in host defense and disease. Over the past years, a large number of biologics have been developed to target cytokines in disease, amongst which soluble receptor fusion proteins have shown some promise in pre-clinical studies. We have previously shown proof-of-concept for the therapeutic targeting of interleukin (IL)-33 in airway inflammation using a newly developed biologic, termed IL-33trap, comprising the ectodomains of the cognate receptor ST2 and the co-receptor IL-1RAcP fused into a single-chain recombinant fusion protein. Here we extend the biophysical and biological characterization of IL-33trap variants, and show that IL-33trap is a stable protein with a monomeric profile both at physiological temperatures and during liquid storage at 4°C. Reducing the N-glycan heterogeneity and complexity of IL-33trap via GlycoDelete engineering neither affects its stability nor its inhibitory activity against IL-33. We also report that IL-33trap specifically targets biologically active IL-33 splice variants. Finally, we document the generation and antagonistic activity of a single-chain IL-4/13trap, which inhibits both IL-4 and IL-13 signaling. Collectively, these results illustrate that single-chain soluble receptor fusion proteins against IL-4, IL-13, and IL-33 are novel biologics that might not only be of interest for research purposes and further interrogation of the role of their target cytokines in physiology and disease, but may also complement monoclonal antibodies for the treatment of allergic and other inflammatory diseases.
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http://dx.doi.org/10.3389/fimmu.2020.01422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7370943PMC
April 2021

ST2 as checkpoint target for colorectal cancer immunotherapy.

JCI Insight 2020 05 7;5(9). Epub 2020 May 7.

Department of Medical and Molecular Genetics.

Immune checkpoint blockade immunotherapy delivers promising clinical results in colorectal cancer (CRC). However, only a fraction of cancer patients develop durable responses. The tumor microenvironment (TME) negatively impacts tumor immunity and subsequently clinical outcomes. Therefore, there is a need to identify other checkpoint targets associated with the TME. Early-onset factors secreted by stromal cells as well as tumor cells often help recruit immune cells to the TME, among which are alarmins such as IL-33. The only known receptor for IL-33 is stimulation 2 (ST2). Here we demonstrated that high ST2 expression is associated with poor survival and is correlated with low CD8+ T cell cytotoxicity in CRC patients. ST2 is particularly expressed in tumor-associated macrophages (TAMs). In preclinical models of CRC, we demonstrated that ST2-expressing TAMs (ST2+ TAMs) were recruited into the tumor via CXCR3 expression and exacerbated the immunosuppressive TME; and that combination of ST2 depletion using ST2-KO mice with anti-programmed death 1 treatment resulted in profound growth inhibition of CRC. Finally, using the IL-33trap fusion protein, we suppressed CRC tumor growth and decreased tumor-infiltrating ST2+ TAMs. Together, our findings suggest that ST2 could serve as a potential checkpoint target for CRC immunotherapy.
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http://dx.doi.org/10.1172/jci.insight.136073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7253019PMC
May 2020

MALT1 targeting suppresses CARD14-induced psoriatic dermatitis in mice.

EMBO Rep 2020 07 28;21(7):e49237. Epub 2020 Apr 28.

VIB Center for Inflammation Research, Ghent, Belgium.

CARD14 gain-of-function mutations cause psoriasis in humans and mice. Together with BCL10 and the protease MALT1, mutant CARD14 forms a signaling node that mediates increased NF-κB signaling and proinflammatory gene expression in keratinocytes. However, it remains unclear whether psoriasis in response to CARD14 hyperactivation is keratinocyte-intrinsic or requires CARD14 signaling in other cells. Moreover, the in vivo effect of MALT1 targeting on mutant CARD14-induced psoriasis has not yet been documented. Here, we show that inducible keratinocyte-specific expression of CARD14 in mice rapidly induces epidermal thickening and inflammation as well as increased expression of several genes associated with psoriasis in humans. Keratinocyte-specific MALT1 deletion as well as oral treatment of mice with a specific MALT1 protease inhibitor strongly reduces psoriatic skin disease in CARD14 mice. Together, these data illustrate a keratinocyte-intrinsic causal role of enhanced CARD14/MALT1 signaling in the pathogenesis of psoriasis and show the potential of MALT1 inhibition for the treatment of psoriasis.
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http://dx.doi.org/10.15252/embr.201949237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7332803PMC
July 2020

Taking the STING Out of Sepsis?

Cell Host Microbe 2020 04;27(4):491-493

Center for Inflammation Research, VIB, Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium. Electronic address:

In this issue of Cell Host & Microbe, Zhang et al. use a sepsis mouse model to show that macrophage-specific release of coagulation factor F3 depends on pathogen detection and responses mediated by TMEM173/STING. The therapeutic power of targeting TMEM173/STING-F3 is evident in mice, but will it penetrate the sepsis bedside?
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http://dx.doi.org/10.1016/j.chom.2020.03.016DOI Listing
April 2020

Dominant-negative mutations in human IL6ST underlie hyper-IgE syndrome.

J Exp Med 2020 06;217(6)

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX.

Autosomal dominant hyper-IgE syndrome (AD-HIES) is typically caused by dominant-negative (DN) STAT3 mutations. Patients suffer from cold staphylococcal lesions and mucocutaneous candidiasis, severe allergy, and skeletal abnormalities. We report 12 patients from 8 unrelated kindreds with AD-HIES due to DN IL6ST mutations. We identified seven different truncating mutations, one of which was recurrent. The mutant alleles encode GP130 receptors bearing the transmembrane domain but lacking both the recycling motif and all four STAT3-recruiting tyrosine residues. Upon overexpression, the mutant proteins accumulate at the cell surface and are loss of function and DN for cellular responses to IL-6, IL-11, LIF, and OSM. Moreover, the patients' heterozygous leukocytes and fibroblasts respond poorly to IL-6 and IL-11. Consistently, patients with STAT3 and IL6ST mutations display infectious and allergic manifestations of IL-6R deficiency, and some of the skeletal abnormalities of IL-11R deficiency. DN STAT3 and IL6ST mutations thus appear to underlie clinical phenocopies through impairment of the IL-6 and IL-11 response pathways.
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http://dx.doi.org/10.1084/jem.20191804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7971136PMC
June 2020

Two distinct ubiquitin-binding motifs in A20 mediate its anti-inflammatory and cell-protective activities.

Nat Immunol 2020 04 16;21(4):381-387. Epub 2020 Mar 16.

VIB Center for Inflammation Research, Ghent, Belgium.

Protein ubiquitination regulates protein stability and modulates the composition of signaling complexes. A20 is a negative regulator of inflammatory signaling, but the molecular mechanisms involved are ill understood. Here, we generated Tnfaip3 gene-targeted A20 mutant mice bearing inactivating mutations in the zinc finger 7 (ZnF7) and ZnF4 ubiquitin-binding domains, revealing that binding to polyubiquitin is essential for A20 to suppress inflammatory disease. We demonstrate that a functional ZnF7 domain was required for recruiting A20 to the tumor necrosis factor receptor 1 (TNFR1) signaling complex and to suppress inflammatory signaling and cell death. The combined inactivation of ZnF4 and ZnF7 phenocopied the postnatal lethality and severe multiorgan inflammation of A20-deficient mice. Conditional tissue-specific expression of mutant A20 further revealed the key role of ubiquitin-binding in myeloid and intestinal epithelial cells. Collectively, these results demonstrate that the anti-inflammatory and cytoprotective functions of A20 are largely dependent on its ubiquitin-binding properties.
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http://dx.doi.org/10.1038/s41590-020-0621-9DOI Listing
April 2020

Phytohormones: Multifunctional nutraceuticals against metabolic syndrome and comorbid diseases.

Biochem Pharmacol 2020 05 20;175:113866. Epub 2020 Feb 20.

VIB-UGent Center for Inflammation Research, VIB, Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium. Electronic address:

Metabolic syndrome is characterized by the co-occurrence of diverse symptoms initiating the development of type 2 diabetes, cardiovascular diseases, and a variety of comorbid diseases. The complex constellation of numerous comorbidities makes it difficult to develop common therapeutic approaches that ameliorate these pathological features simultaneously. The plant hormones abscisic acid, salicylic acid, auxin, and cytokinins, have shown promising anti-inflammatory and pro-metabolic effects that could mitigate several disorders relevant to metabolic syndrome. Intriguingly, besides plants, human cells and gut microbes also endogenously produce these molecules, indicating a role in the complex interplay between inflammatory responses associated with metabolic syndrome, the gut microbiome, and nutrition. Here, we introduce how bioactive phytohormones can be generated endogenously and through the gut microbiome. These molecules subsequently influence immune responses and metabolism. We also elaborate on how phytohormones can beneficially modulate metabolic syndrome comorbidities, and propose them as nutraceuticals.
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http://dx.doi.org/10.1016/j.bcp.2020.113866DOI Listing
May 2020

Fragility can be a good thing in cancer.

Nat Immunol 2020 01;21(1):11-13

Center for Inflammation Research, VIB, Ghent, Belgium.

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http://dx.doi.org/10.1038/s41590-019-0564-1DOI Listing
January 2020

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

MALT1-Deficient Mice Develop Atopic-Like Dermatitis Upon Aging.

Front Immunol 2019 1;10:2330. Epub 2019 Oct 1.

VIB Center for Inflammation Research, Ghent, Belgium.

MALT1 plays an important role in innate and adaptive immune signaling by acting as a scaffold protein that mediates NF-κB signaling. In addition, MALT1 is a cysteine protease that further fine tunes proinflammatory signaling by cleaving specific substrates. Deregulated MALT1 activity has been associated with immunodeficiency, autoimmunity, and cancer in mice and humans. Genetically engineered mice expressing catalytically inactive MALT1, still exerting its scaffold function, were previously shown to spontaneously develop autoimmunity due to a decrease in Tregs associated with increased effector T cell activation. In contrast, complete absence of MALT1 does not lead to autoimmunity, which has been explained by the impaired effector T cell activation due to the absence of MALT1-mediated signaling. However, here we report that MALT1-deficient mice develop atopic-like dermatitis upon aging, which is preceded by Th2 skewing, an increase in serum IgE, and a decrease in Treg frequency and surface expression of the Treg functionality marker CTLA-4.
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http://dx.doi.org/10.3389/fimmu.2019.02330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779721PMC
October 2020

MALT1 Proteolytic Activity Suppresses Autoimmunity in a T Cell Intrinsic Manner.

Front Immunol 2019 14;10:1898. Epub 2019 Aug 14.

VIB Center for Inflammation Research, Ghent, Belgium.

MALT1 is a central signaling component in innate and adaptive immunity by regulating NF-κB and other key signaling pathways in different cell types. Activities of MALT1 are mediated by its scaffold and protease functions. Because of its role in lymphocyte activation and proliferation, inhibition of MALT1 proteolytic activity is of high interest for therapeutic targeting in autoimmunity and certain lymphomas. However, recent studies showing that protease-dead knock-in (-PD) mice suffer from autoimmune disease have somewhat tempered the initial enthusiasm. Although it has been proposed that an imbalance between immune suppressive regulatory T cells (Tregs) and activated effector CD4 T cells plays a key role in the autoimmune phenotype of -PD mice, the specific contribution of MALT1 proteolytic activity in T cells remains unclear. Using T cell-conditional protease-dead knock-in (-PDT) mice, we here demonstrate that MALT1 has a T cell-intrinsic role in regulating the homeostasis and function of thymic and peripheral T cells. T cell-specific ablation of MALT1 proteolytic activity phenocopies mice in which MALT1 proteolytic activity has been genetically inactivated in all cell types. The -PDT mice have a reduced number of Tregs in the thymus and periphery, although the effect in the periphery is less pronounced compared to full-body -PD mice, indicating that also other cell types may promote Treg induction in a MALT1 protease-dependent manner. Despite the difference in peripheral Treg number, both T cell-specific and full-body -PD mice develop ataxia and multi-organ inflammation to a similar extent. Furthermore, reconstitution of the full-body -PD mice with T cell-specific expression of wild-type human eliminated all signs of autoimmunity. Together, these findings establish an important T cell-intrinsic role of MALT1 proteolytic activity in the suppression of autoimmune responses.
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http://dx.doi.org/10.3389/fimmu.2019.01898DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6702287PMC
October 2020

Deletion of Mucosa-Associated Lymphoid Tissue Lymphoma Translocation Protein 1 in Mouse T Cells Protects Against Development of Autoimmune Arthritis but Leads to Spontaneous Osteoporosis.

Arthritis Rheumatol 2019 12 9;71(12):2005-2015. Epub 2019 Nov 9.

VIB-UGent Center for Inflammation Research and Ghent University, Ghent, Belgium.

Objective: Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT-1) plays a crucial role in innate and adaptive immune signaling by modulating the threshold for activation of immune cells, including Treg cells. Therefore, MALT-1 is regarded to be an interesting therapeutic target in several immune-mediated diseases. The goal of this study was to examine the role of MALT-1 in experimental animal models of rheumatoid arthritis (RA).

Methods: MALT-1 activation was assessed by measuring cleavage of the deubiquitinase CYLD in lymphocytes from mice with collagen-induced arthritis (CIA). Furthermore, the impact of MALT-1 deficiency on arthritis was evaluated in Malt1 mice with CIA or with collagen antibody-induced arthritis (CAIA). T cell-specific MALT-1 deficiency was measured in mice with deletion of T cell-specific MALT-1 (Malt1 ), and the time-dependent effects of MALT-1 deficiency were assessed in mice with deletion of tamoxifen-inducible T cell-specific MALT-1 (Malt1 ). Bone density was determined in MALT-1-deficient mice using micro-computed tomography and femur-bending tests. Reconstitution of Treg cells was performed using adoptive transfer experiments.

Results: MALT-1 activation was observed in the lymphocytes of mice with CIA. T cell-specific MALT-1 deletion in the induction phase of arthritis (incidence of arthritis, 25% in control mice versus 0% in Malt1 mice; P < 0.05), but not in the effector phase of arthritis, completely protected mice against the development of CIA. Consistent with this finding, MALT-1 deficiency had no impact on CAIA, an effector phase model of RA. Finally, mice with MALT-1 deficiency showed a spontaneous decrease in bone density (mean ± SEM trabecular thickness, 46.3 ± 0.7 μm in control mice versus 40 ± 1.1 μm in Malt1 mice; P < 0.001), which was linked to the loss of Treg cells in these mice.

Conclusion: Overall, these data in murine models of RA highlight MALT-1 as a master regulator of T cell activation, which is relevant to the pathogenesis of autoimmune arthritis. Furthermore, these findings show that MALT-1 deficiency can lead to spontaneous osteoporosis, which is associated with impaired Treg cell numbers.
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http://dx.doi.org/10.1002/art.41029DOI Listing
December 2019

TNF-α inhibits glucocorticoid receptor-induced gene expression by reshaping the GR nuclear cofactor profile.

Proc Natl Acad Sci U S A 2019 06 10;116(26):12942-12951. Epub 2019 Jun 10.

Mouse Genetics in Inflammation, VIB Center for Inflammation Research, 9052 Ghent, Belgium;

Glucocorticoid resistance (GCR) is defined as an unresponsiveness to the therapeutic effects, including the antiinflammatory ones of glucocorticoids (GCs) and their receptor, the glucocorticoid receptor (GR). It is a problem in the management of inflammatory diseases and can be congenital as well as acquired. The strong proinflammatory cytokine TNF-alpha (TNF) induces an acute form of GCR, not only in mice, but also in several cell lines: e.g., in the hepatoma cell line BWTG3, as evidenced by impaired Dexamethasone (Dex)-stimulated direct GR-dependent gene up- and down-regulation. We report that TNF has a significant and broad impact on this transcriptional performance of GR, but no impact on nuclear translocation, dimerization, or DNA binding capacity of GR. Proteome-wide proximity-mapping (BioID), however, revealed that the GR interactome was strongly modulated by TNF. One GR cofactor that interacted significantly less with the receptor under GCR conditions is p300. NFκB activation and p300 knockdown both reduced direct transcriptional output of GR whereas p300 overexpression and NFκB inhibition reverted TNF-induced GCR, which is in support of a cofactor reshuffle model. This hypothesis was supported by FRET studies. This mechanism of GCR opens avenues for therapeutic interventions in GCR diseases.
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http://dx.doi.org/10.1073/pnas.1821565116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6600915PMC
June 2019
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