Publications by authors named "Geert van Loo"

100 Publications

Central Role of Dendritic Cells in Pulmonary Arterial Hypertension in Human and Mice.

Int J Mol Sci 2021 Feb 10;22(4). Epub 2021 Feb 10.

Department of Pulmonary Medicine, Erasmus MC, University Medical Center Rotterdam, 3015 GD Rotterdam, The Netherlands.

The pathogenesis of idiopathic pulmonary arterial hypertension (IPAH) is not fully understood, but evidence is accumulating that immune dysfunction plays a significant role. We previously reported that 31-week-old mice develop pulmonary hypertension (PH) symptoms. These mice harbor a targeted deletion of the TNFα-induced protein-3 () gene, encoding the NF-κB regulatory protein A20, specifically in type I conventional dendritic cells (cDC1s). Here, we studied the involvement of dendritic cells (DCs) in PH in more detail. We found various immune cells, including DCs, in the hearts of mice, particularly in the right ventricle (RV). Secondly, in young mice, innate immune activation through airway exposure to toll-like receptor ligands essentially did not result in elevated RV pressures, although we did observe significant RV hypertrophy. Thirdly, PH symptoms in mice were not enhanced by concomitant mutation of bone morphogenetic protein receptor type 2 (), which is the most affected gene in PAH patients. Finally, in human IPAH lung tissue we found co-localization of DCs and CD8+ T cells, representing the main cell type activated by cDC1s. Taken together, these findings support a unique role of cDC1s in PAH pathogenesis, independent of general immune activation or a mutation in the gene.
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http://dx.doi.org/10.3390/ijms22041756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7916474PMC
February 2021

IL1β Promotes Immune Suppression in the Tumor Microenvironment Independent of the Inflammasome and Gasdermin D.

Cancer Immunol Res 2021 Mar 23;9(3):309-323. Epub 2020 Dec 23.

Myeloid Cell Immunology Lab, VIB Center for Inflammation Research, Brussels, Belgium.

IL1β is a central mediator of inflammation. Secretion of IL1β typically requires proteolytic maturation by the inflammasome and formation of membrane pores by gasdermin D (GSDMD). Emerging evidence suggests an important role for IL1β in promoting cancer progression in patients, but the underlying mechanisms are ill-defined. Here, we have shown a key role for IL1β in driving tumor progression in two distinct mouse tumor models. Notably, activation of the inflammasome, caspase-8, as well as the pore-forming proteins GSDMD and mixed lineage kinase domain-like protein in the host were dispensable for the release of intratumoral bioactive IL1β. Inflammasome-independent IL1β release promoted systemic neutrophil expansion and fostered accumulation of T-cell-suppressive neutrophils in the tumor. Moreover, IL1β was essential for neutrophil infiltration triggered by antiangiogenic therapy, thereby contributing to treatment-induced immunosuppression. Deletion of IL1β allowed intratumoral accumulation of CD8 effector T cells that subsequently activated tumor-associated macrophages. Depletion of either CD8 T cells or macrophages abolished tumor growth inhibition in IL1β-deficient mice, demonstrating a crucial role for CD8 T-cell-macrophage cross-talk in the antitumor immune response. Overall, these results support a tumor-promoting role for IL1β through establishing an immunosuppressive microenvironment and show that inflammasome activation is not essential for release of this cytokine in tumors.
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http://dx.doi.org/10.1158/2326-6066.CIR-20-0431DOI Listing
March 2021

Ileal immune tonus is a prognosis marker of proximal colon cancer in mice and patients.

Cell Death Differ 2020 Dec 1. Epub 2020 Dec 1.

Gustave Roussy Cancer Campus (GRCC), Villejuif, France.

Ileal epithelial cell apoptosis and the local microbiota modulate the effects of oxaliplatin against proximal colon cancer by modulating tumor immunosurveillance. Here, we identified an ileal immune profile associated with the prognosis of colon cancer and responses to chemotherapy. The whole immune ileal transcriptome was upregulated in poor-prognosis patients with proximal colon cancer, while the colonic immunity of healthy and neoplastic areas was downregulated (except for the Th17 fingerprint) in such patients. Similar observations were made across experimental models of implanted and spontaneous murine colon cancer, showing a relationship between carcinogenesis and ileal inflammation. Conversely, oxaliplatin-based chemotherapy could restore a favorable, attenuated ileal immune fingerprint in responders. These results suggest that chemotherapy inversely shapes the immune profile of the ileum-tumor axis, influencing clinical outcome.
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http://dx.doi.org/10.1038/s41418-020-00684-wDOI Listing
December 2020

DNGR1-Cre-mediated Deletion of /A20 in Conventional Dendritic Cells Induces Pulmonary Hypertension in Mice.

Am J Respir Cell Mol Biol 2020 11;63(5):665-680

Department of Pulmonary Medicine.

Chronic perivascular inflammation is a prominent feature in the lungs of idiopathic pulmonary arterial hypertension. Although the proportions of conventional dendritic cells (cDCs) and plasmacytoid DCs are increased in idiopathic pulmonary arterial hypertension lungs, it remains unknown whether activated cDCs play a pathogenic role. The gene encodes the ubiquitin-binding protein A20, which is a negative regulator of NF-κB, critically involved in DC activation. Targeting of Tnfaip3/A20 in cDCs was achieved by (DNGR1)-Cre-mediated excision of the gene in mice. Mice were evaluated for signs of pulmonary hypertension (PH) using right heart catheterization, echocardiography, and measurement of the Fulton index. Inflammation was assessed by immunohistochemistry and flow cytometry. Pulmonary cDCs and monocyte-derived DCs from 31-week-old mice showed modulated expression of cell surface activation markers compared with mice. mice developed elevated right ventricular systolic pressure and right ventricular hypertrophy. The lungs of these mice displayed increased vascular remodeling and perivascular and peribronchial immune cell infiltration resembling tertiary lymphoid organs. Proportions of activated T cells and expression of IL-1β, IL-6, and IL-10 were enhanced in the lungs of mice. Autoreactive IgA and IgG1 was detected in BAL and autoreactive IgA recognizing pulmonary endothelial antigens was present in the serum of mice. All signs of PH were ameliorated in mice by antiIL-6 antibody treatment. These results indicate that activation of the NF-κB pathway in DCs, through deletion of A20/, leads to experimental PH with accompanied pulmonary inflammation in an IL-6-dependent fashion.
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http://dx.doi.org/10.1165/rcmb.2019-0443OCDOI Listing
November 2020

Tnfaip3 expression in pulmonary conventional type 1 Langerin-expressing dendritic cells regulates T helper 2-mediated airway inflammation in mice.

Allergy 2020 10 14;75(10):2587-2598. Epub 2020 Jun 14.

Department of Pulmonary Medicine, Erasmus MC, Rotterdam, The Netherlands.

Background: Conventional type 1 dendritic cells (cDC1s) control anti-viral and anti-tumor immunity by inducing antigen-specific cytotoxic CD8 T-cell responses. Controversy exists whether cDC1s also control CD4 T helper 2 (Th2) cell responses, since suppressive and activating roles have been reported. DC activation status, controlled by the transcription factor NF-κB, might determine the precise outcome of Th-cell differentiation upon encounter with cDC1s. To investigate the role of activated cDC1s in Th2-driven immune responses, pulmonary cDC1s were activated by targeted deletion of A20/Tnfaip3, a negative regulator of NF-κB signaling.

Methods: To target pulmonary cDC1s, Cd207 (Langerin)-mediated excision of A20/Tnfaip3 was used, generating Tnfaip3 xCd207 (Tnfaip3 ) mice. Mice were exposed to house dust mite (HDM) to provoke Th2-mediated immune responses.

Results: Mice harboring Tnfaip3-deficient cDC1s did not develop Th2-driven eosinophilic airway inflammation upon HDM exposure, but rather showed elevated numbers of IFNγ-expressing CD8 T cells. In addition, Tnfaip3 mice harbored increased numbers of IL-12-expressing cDC1s and elevated PD-L1 expression in all pulmonary DC subsets. Blocking either IL-12 or IFNγ in Tnfaip3 mice restored Th2 responses, whereas administration of recombinant IFNγ during HDM sensitization in C57Bl/6 mice blocked Th2 development.

Conclusions: These findings indicate that the activation status of cDC1s, shown by their specific expression of co-inhibitory molecules and cytokines, critically contributes to the development of Th2 cell-mediated disorders, most likely by influencing IFNγ production in CD8 T cells.
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http://dx.doi.org/10.1111/all.14334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7687104PMC
October 2020

A20 and Cell Death-driven Inflammation.

Trends Immunol 2020 May 30;41(5):421-435. Epub 2020 Mar 30.

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

A20 is a potent anti-inflammatory molecule, and mutations in TNFAIP3, the gene encoding A20, are associated with a wide panel of inflammatory pathologies, both in human and mouse. The anti-inflammatory properties of A20 are commonly attributed to its ability to suppress inflammatory NF-κB signaling by functioning as a ubiquitin-editing enzyme. However, A20 also protects cells from death, independently of NF-κB regulation, and recent work has demonstrated that cell death may drive some of the inflammatory conditions caused by A20 deficiency. Adding to the fact that the protective role of A20 does not primarily rely on its catalytic activities, these findings shed new light on A20 biology.
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http://dx.doi.org/10.1016/j.it.2020.03.001DOI Listing
May 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

OTULIN Prevents Liver Inflammation and Hepatocellular Carcinoma by Inhibiting FADD- and RIPK1 Kinase-Mediated Hepatocyte Apoptosis.

Cell Rep 2020 02;30(7):2237-2247.e6

VIB Center for Inflammation Research, 9052 Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, 9052 Ghent, Belgium. Electronic address:

Inflammatory signaling pathways are tightly regulated to avoid chronic inflammation and the development of disease. OTULIN is a deubiquitinating enzyme that controls inflammation by cleaving linear ubiquitin chains generated by the linear ubiquitin chain assembly complex. Here, we show that ablation of OTULIN in liver parenchymal cells in mice causes severe liver disease which is characterized by liver inflammation, hepatocyte apoptosis, and compensatory hepatocyte proliferation, leading to steatohepatitis, fibrosis, and hepatocellular carcinoma (HCC). Genetic ablation of Fas-associated death domain (FADD) completely rescues and knockin expression of kinase inactive receptor-interacting protein kinase 1 (RIPK1) significantly protects mice from developing liver disease, demonstrating that apoptosis of OTULIN-deficient hepatocytes triggers disease pathogenesis in this model. Finally, we demonstrate that type I interferons contribute to disease in hepatocyte-specific OTULIN-deficient mice. Our study reveals the critical importance of OTULIN in protecting hepatocytes from death, thereby preventing the development of chronic liver inflammation and HCC.
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http://dx.doi.org/10.1016/j.celrep.2020.01.028DOI Listing
February 2020

Correction: A20 protects cells from TNF-induced apoptosis through linear ubiquitin-dependent and -independent mechanisms.

Cell Death Dis 2020 Jan 23;11(1):60. Epub 2020 Jan 23.

Center for Inflammation Research, VIB, Ghent, Belgium.

The original version of this article contained an error in the name of one of the co-authors (Wim Declercq). This has been corrected in the PDF and HTML versions.
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http://dx.doi.org/10.1038/s41419-020-2260-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978509PMC
January 2020

Epithelial HMGB1 Delays Skin Wound Healing and Drives Tumor Initiation by Priming Neutrophils for NET Formation.

Cell Rep 2019 Nov;29(9):2689-2701.e4

VIB Center for Inflammation Research, 9052 Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, 9052 Ghent, Belgium. Electronic address:

Regenerative responses predispose tissues to tumor formation by largely unknown mechanisms. High-mobility group box 1 (HMGB1) is a danger-associated molecular pattern contributing to inflammatory pathologies. We show that HMGB1 derived from keratinocytes, but not myeloid cells, delays cutaneous wound healing and drives tumor formation. In wounds of mice lacking HMGB1 selectively in keratinocytes, a marked reduction in neutrophil extracellular trap (NET) formation is observed. Pharmacological targeting of HMGB1 or NETs prevents skin tumorigenesis and accelerates wound regeneration. HMGB1-dependent NET formation and skin tumorigenesis is orchestrated by tumor necrosis factor (TNF) and requires RIPK1 kinase activity. NETs are present in the microenvironment of keratinocyte-derived tumors in mice and lesional and tumor skin of patients suffering from recessive dystrophic epidermolysis bullosa, a disease in which skin blistering predisposes to tumorigenesis. We conclude that tumorigenicity of the wound microenvironment depends on epithelial-derived HMGB1 regulating NET formation, thereby establishing a mechanism linking reparative inflammation to tumor initiation.
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http://dx.doi.org/10.1016/j.celrep.2019.10.104DOI Listing
November 2019

El Tor Biotype Activates the Caspase-11-Independent Canonical Nlrp3 and Pyrin Inflammasomes.

Front Immunol 2019 29;10:2463. Epub 2019 Oct 29.

Department of Internal Medicine and Pediatrics, Ghent University, Ghent, Belgium.

is a Gram-negative enteropathogen causing potentially life-threatening cholera disease outbreaks, for which the World Health Organization currently registers 2-4 million cases and ~100.000 cholera-associated deaths annually worldwide. Genomic research revealed that the strains causing this ongoing cholera pandemic are members of the El Tor biotype, which fully replaced the Classical biotype that caused former cholera pandemics. While both of these biotypes express the characteristic Cholera Toxin (CT), the El Tor biotype additionally expresses the accessory toxins hemolysin (hlyA) and multifunctional auto-processing repeat-in-toxin (MARTX). Previous studies demonstrated that the Classical biotype of triggers caspase-11-dependent non-canonical inflammasome activation in macrophages following CT-mediated cytosolic delivery of LPS. In contrast to the Classical biotype, we here show that El Tor induces IL-1β maturation and secretion in a caspase-11- and CT-independent manner. Instead, we show that El Tor engages the canonical Nlrp3 inflammasome for IL-1β secretion through its accessory hlyA toxin. We further reveal the capacity of this enteropathogen to engage the canonical Pyrin inflammasome as an accessory mechanism for IL-1β secretion in conditions when the pro-inflammatory hlyA-Nlrp3 axis is blocked. Thus, we show that the El Tor biotype does not trigger caspase-11 activation, but instead triggers parallel Nlrp3- and Pyrin-dependent pathways toward canonical inflammasome activation to induce IL-1β-mediated inflammatory responses. These findings further unravel the complex inflammasome activating mechanisms that can be triggered when macrophages face the full arsenal of El Tor toxins, and as such increase our understanding of host-pathogen interactions in the context of the biotype associated with the ongoing cholera pandemic.
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http://dx.doi.org/10.3389/fimmu.2019.02463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6828848PMC
November 2020

A20 phosphorylation controls A20 function.

Nat Immunol 2019 10;20(10):1261-1262

VIB Center for Inflammation Research, Ghent, Belgium.

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http://dx.doi.org/10.1038/s41590-019-0481-3DOI Listing
October 2019

A20 protects cells from TNF-induced apoptosis through linear ubiquitin-dependent and -independent mechanisms.

Cell Death Dis 2019 09 18;10(10):692. Epub 2019 Sep 18.

Center for Inflammation Research, VIB, Ghent, Belgium.

The cytokine TNF promotes inflammation either directly by activating the MAPK and NF-κB signaling pathways, or indirectly by triggering cell death. A20 is a potent anti-inflammatory molecule, and mutations in the gene encoding A20 are associated with a wide panel of inflammatory pathologies, both in human and in the mouse. Binding of TNF to TNFR1 triggers the NF-κB-dependent expression of A20 as part of a negative feedback mechanism preventing sustained NF-κB activation. Apart from acting as an NF-κB inhibitor, A20 is also well-known for its ability to counteract the cytotoxic potential of TNF. However, the mechanism by which A20 mediates this function and the exact cell death modality that it represses have remained incompletely understood. In the present study, we provide in vitro and in vivo evidences that deletion of A20 induces RIPK1 kinase-dependent and -independent apoptosis upon single TNF stimulation. We show that constitutively expressed A20 is recruited to TNFR1 signaling complex (Complex I) via its seventh zinc finger (ZF7) domain, in a cIAP1/2-dependent manner, within minutes after TNF sensing. We demonstrate that Complex I-recruited A20 protects cells from apoptosis by stabilizing the linear (M1) ubiquitin network associated to Complex I, a process independent of its E3 ubiquitin ligase and deubiquitylase (DUB) activities and which is counteracted by the DUB CYLD, both in vitro and in vivo. In absence of linear ubiquitylation, A20 is still recruited to Complex I via its ZF4 and ZF7 domains, but this time protects the cells from death by deploying its DUB activity. Together, our results therefore demonstrate two distinct molecular mechanisms by which constitutively expressed A20 protect cells from TNF-induced apoptosis.
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http://dx.doi.org/10.1038/s41419-019-1937-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6751190PMC
September 2019

A20 at the Crossroads of Cell Death, Inflammation, and Autoimmunity.

Cold Spring Harb Perspect Biol 2020 01 2;12(1). Epub 2020 Jan 2.

VIB Center for Inflammation Research, 9052 Ghent, Belgium.

A20 is a potent anti-inflammatory protein, acting by inhibiting nuclear factor κB (NF-κB) signaling and inflammatory gene expression and/or by preventing cell death. Mutations in the gene have been associated with a plethora of inflammatory and autoimmune pathologies in humans and in mice. Although the anti-inflammatory role of A20 is well accepted, fundamental mechanistic questions regarding its mode of action remain unclear. Here, we review new findings that further clarify the molecular and cellular mechanisms by which A20 controls inflammatory signaling and cell death, and discuss new evidence for its involvement in inflammatory and autoimmune disease development.
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http://dx.doi.org/10.1101/cshperspect.a036418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6942121PMC
January 2020

The ubiquitin-editing enzyme A20 controls NK cell homeostasis through regulation of mTOR activity and TNF.

J Exp Med 2019 09 11;216(9):2010-2023. Epub 2019 Jul 11.

Laboratory for Endoplasmic Reticulum Stress and Inflammation, VIB Center for Inflammation Research, Ghent, Belgium

The ubiquitin-editing enzyme A20 is a well-known regulator of immune cell function and homeostasis. In addition, A20 protects cells from death in an ill-defined manner. While most studies focus on its role in the TNF-receptor complex, we here identify a novel component in the A20-mediated decision between life and death. Loss of A20 in NK cells led to spontaneous NK cell death and severe NK cell lymphopenia. The few remaining NK cells showed an immature, hyperactivated phenotype, hallmarked by the basal release of cytokines and cytotoxic molecules. NK-A20 cells were hypersensitive to TNF-induced cell death and could be rescued, at least partially, by a combined deficiency with TNF. Unexpectedly, rapamycin, a well-established inhibitor of mTOR, also strongly protected NK-A20 cells from death, and further studies revealed that A20 restricts mTOR activation in NK cells. This study therefore maps A20 as a crucial regulator of mTOR signaling and underscores the need for a tightly balanced mTOR pathway in NK cell homeostasis.
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http://dx.doi.org/10.1084/jem.20182164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719426PMC
September 2019

The anti-inflammatory protein TNFAIP3/A20 binds the WD40 domain of ATG16L1 to control the autophagic response, NFKB/NF-κB activation and intestinal homeostasis.

Autophagy 2019 09 16;15(9):1657-1659. Epub 2019 Jun 16.

a Instituto de Biología Molecular y Celular del Cáncer (CSIC-Universidad de Salamanca), Centro de Investigación del Cáncer, Campus Miguel de Unamuno, Universidad de Salamanca , Salamanca , Spain.

The C-terminal domain of ATG16L1 includes 7 WD40-type repeats (WD40 domain, WDD) and is not required for canonical macroautophagy/autophagy. Instead, the WDD allows ATG16L1 to induce LC3/Atg8 lipidation in single-membrane compartments, although a detailed functional characterization of this region is still missing. In a recent report we identify the anti-inflammatory molecule TNFAIP3/A20 as a binding partner of the WDD. Such physical interaction allows mutual downregulation of the expression levels of both proteins, so that the absence of one of them causes upregulation of the other. This cross-regulation provides a molecular basis for a striking genetic interaction in mice where elimination of both molecules in the intestinal epithelium generates an aggressive inflammatory phenotype. studies reveal unexpected features of the functional interplay between ATG16L1 and TNFAIP3. ATG16L1 requires TNFAIP3 to sustain the canonical autophagic flux measured by SQSTM1/p62 degradation. The WDD mediates lysosomal degradation of TNFAIP3 promoted by ATG16L1, and also regulates the NFKB/NF-κB response. Therefore, our data reveal new roles of the WDD and TNFAIP3 in the regulation of autophagy, protein stability and inflammatory signaling. More generally, we identify the interaction between ATG16L1 and TNFAIP3 as a signaling hub that integrates different pathways with important implications for intestinal homeostasis.
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http://dx.doi.org/10.1080/15548627.2019.1628549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6693451PMC
September 2019

DNGR1-mediated deletion of A20/Tnfaip3 in dendritic cells alters T and B-cell homeostasis and promotes autoimmune liver pathology.

J Autoimmun 2019 08 9;102:167-178. Epub 2019 Jul 9.

Department of Pulmonary Medicine, Erasmus MC, PO Box 2040, 3000 CA, Rotterdam, the Netherlands. Electronic address:

Dendritic cells (DCs) are central regulators of tolerance versus immunity. The outcome depends amongst others on DC subset and activation status. Whereas CD11b type 2 conventional DCs (cDC2s) initiate proinflammatory helper T (Th)-cell responses, CD103 cDC1s are crucial for regulatory T-cell (Treg) induction and CD8 T-cell activation. DC activation is controlled by the transcription factor NF-κB. Ablation of A20/Tnfaip3, a critical regulator of NF-κB activation, in DCs leads to constitutive DC activation and development of systemic autoimmunity. We hypothesized that the activation status of cDCs controls the development of autoimmunity. To target cDCs, DNGR1(Clec9a)-cre-mediated excision of A20/Tnfaip3 was used through generation of Tnfaip3xClec9a (Tnfaip3) mice. Immune cell activation was evaluated at 31-weeks of age. We found that DNGR1-cre-mediated deletion of A20/Tnfaip3 resulted in liver pathology characterized by inflammatory infiltrates adjacent to the portal triads. Both cDC subsets as well as monocyte-derived DCs (moDCs) in Tnfaip3 livers harbored an activated phenotype. Specifically, the costimulatory molecule CD40 in liver cDCs and moDCs was regulated by A20/Tnfaip3 expression. Livers from Tnfaip3 mice had augmented proportions of Th1, Th17, Treg, and follicular Th (Tfh)-cells compared to control mice, accompanied by an increase in IgA-producing plasma cells. Serum IgA from Tnfaip3 mice recognized self-proteins, specifically cytoplasmic proteins in liver periportal regions. These data show that enhanced activation of cDCs and moDCs, due to A20/Tnfaip3 ablation, promotes the development of organ-specific autoimmunity but not systemic autoimmunity. This model could be useful to examine the pathobiological processes contributing to autoimmune liver diseases.
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http://dx.doi.org/10.1016/j.jaut.2019.05.007DOI Listing
August 2019

A20 prevents inflammasome-dependent arthritis by inhibiting macrophage necroptosis through its ZnF7 ubiquitin-binding domain.

Nat Cell Biol 2019 06 13;21(6):731-742. Epub 2019 May 13.

Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD) and Center for Molecular Medicine, University of Cologne, Cologne, Germany.

Deficiency in the deubiquitinating enzyme A20 causes severe inflammation in mice, and impaired A20 function is associated with human inflammatory diseases. A20 has been implicated in negatively regulating NF-κB signalling, cell death and inflammasome activation; however, the mechanisms by which A20 inhibits inflammation in vivo remain poorly understood. Genetic studies in mice revealed that its deubiquitinase activity is not essential for A20 anti-inflammatory function. Here we show that A20 prevents inflammasome-dependent arthritis by inhibiting macrophage necroptosis and that this function depends on its zinc finger 7 (ZnF7). We provide genetic evidence that RIPK1 kinase-dependent, RIPK3-MLKL-mediated necroptosis drives inflammasome activation in A20-deficient macrophages and causes inflammatory arthritis in mice. Single-cell imaging revealed that RIPK3-dependent death caused inflammasome-dependent IL-1β release from lipopolysaccharide-stimulated A20-deficient macrophages. Importantly, mutation of the A20 ZnF7 ubiquitin binding domain caused arthritis in mice, arguing that ZnF7-dependent inhibition of necroptosis is critical for A20 anti-inflammatory function in vivo.
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http://dx.doi.org/10.1038/s41556-019-0324-3DOI Listing
June 2019

Nlrp3 inflammasome activation and Gasdermin D-driven pyroptosis are immunopathogenic upon gastrointestinal norovirus infection.

PLoS Pathog 2019 04 24;15(4):e1007709. Epub 2019 Apr 24.

Department of Internal Medicine and Pediatrics, Ghent University, Ghent, Belgium.

Norovirus infection is the leading cause of food-borne gastroenteritis worldwide, being responsible for over 200,000 deaths annually. Studies with murine norovirus (MNV) showed that protective STAT1 signaling controls viral replication and pathogenesis, but the immune mechanisms that noroviruses exploit to induce pathology are elusive. Here, we show that gastrointestinal MNV infection leads to widespread IL-1β maturation in MNV-susceptible STAT1-deficient mice. MNV activates the canonical Nlrp3 inflammasome in macrophages, leading to maturation of IL-1β and to Gasdermin D (GSDMD)-dependent pyroptosis. STAT1-deficient macrophages displayed increased MAVS-mediated expression of pro-IL-1β, facilitating elevated Nlrp3-dependent release of mature IL-1β upon MNV infection. Accordingly, MNV-infected Stat1-/- mice showed Nlrp3-dependent maturation of IL-1β as well as Nlrp3-dependent pyroptosis as assessed by in vivo cleavage of GSDMD to its active N-terminal fragment. While MNV-induced diarrheic responses were not affected, Stat1-/- mice additionally lacking either Nlrp3 or GSDMD displayed lower levels of the fecal inflammatory marker Lipocalin-2 as well as delayed lethality after gastrointestinal MNV infection. Together, these results uncover new insights into the mechanisms of norovirus-induced inflammation and cell death, thereby revealing Nlrp3 inflammasome activation and ensuing GSDMD-driven pyroptosis as contributors to MNV-induced immunopathology in susceptible STAT1-deficient mice.
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http://dx.doi.org/10.1371/journal.ppat.1007709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6502405PMC
April 2019

Physical and functional interaction between A20 and ATG16L1-WD40 domain in the control of intestinal homeostasis.

Nat Commun 2019 04 23;10(1):1834. Epub 2019 Apr 23.

VIB Center for Inflammation Research, 9052, Ghent, Belgium.

Prevention of inflammatory bowel disease (IBD) relies on tight control of inflammatory, cell death and autophagic mechanisms, but how these pathways are integrated at the molecular level is still unclear. Here we show that the anti-inflammatory protein A20 and the critical autophagic mediator Atg16l1 physically interact and synergize to regulate the stability of the intestinal epithelial barrier. A proteomic screen using the WD40 domain of ATG16L1 (WDD) identified A20 as a WDD-interacting protein. Loss of A20 and Atg16l1 in mouse intestinal epithelium induces spontaneous IBD-like pathology, as characterized by severe inflammation and increased intestinal epithelial cell death in both small and large intestine. Mechanistically, absence of A20 promotes Atg16l1 accumulation, while elimination of Atg16l1 or expression of WDD-deficient Atg16l1 stabilizes A20. Collectively our data show that A20 and Atg16l1 cooperatively control intestinal homeostasis by acting at the intersection of inflammatory, autophagy and cell death pathways.
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http://dx.doi.org/10.1038/s41467-019-09667-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6478926PMC
April 2019

Inflammasomes in neuroinflammatory and neurodegenerative diseases.

EMBO Mol Med 2019 06;11(6)

VIB Center for Inflammation Research, Ghent, Belgium

Neuroinflammation and neurodegeneration often result from the aberrant deposition of aggregated host proteins, including amyloid-β, α-synuclein, and prions, that can activate inflammasomes. Inflammasomes function as intracellular sensors of both microbial pathogens and foreign as well as host-derived danger signals. Upon activation, they induce an innate immune response by secreting the inflammatory cytokines interleukin (IL)-1β and IL-18, and additionally by inducing pyroptosis, a lytic cell death mode that releases additional inflammatory mediators. Microglia are the prominent innate immune cells in the brain for inflammasome activation. However, additional CNS-resident cell types including astrocytes and neurons, as well as infiltrating myeloid cells from the periphery, express and activate inflammasomes. In this review, we will discuss current understanding of the role of inflammasomes in common degenerative diseases of the brain and highlight inflammasome-targeted strategies that may potentially treat these diseases.
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http://dx.doi.org/10.15252/emmm.201810248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6554670PMC
June 2019

Author Correction: Spatial and temporal heterogeneity of mouse and human microglia at single-cell resolution.

Nature 2019 Apr;568(7751):E4

Institute of Neuropathology, Medical Faculty, University of Freiburg, Freiburg, Germany.

In this Letter, Dominic Grün and Sagar have been added to the author list (affiliated with Max-Planck-Institute of Immunology and Epigenetics (MPI-IE), Freiburg, Germany). The author list, 'Author contribution' and 'Acknowledgements' sections have been corrected online. See accompanying Amendment.
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http://dx.doi.org/10.1038/s41586-019-1045-2DOI Listing
April 2019

Nucleic Acid Induced Interferon and Inflammasome Responses in Regulating Host Defense to Gastrointestinal Viruses.

Int Rev Cell Mol Biol 2019 3;345:137-171. Epub 2018 Oct 3.

Department of Internal Medicine, Ghent University, Ghent, Belgium; VIB-UGent Center for Inflammation Research, VIB, Ghent, Belgium; Ghent Gut Inflammation Group (GGIG), Ghent University, Ghent, Belgium. Electronic address:

The gut bacterial and fungal communities residing in the gastrointestinal tract have undisputed far-reaching effects in regulating host health. In the meantime, however, metagenomic sequencing efforts are revealing enteric viruses as the most abundant dimension of the intestinal gut ecosystem, and the first gut virome-wide association studies showed that inflammatory bowel disease as well as type 1 diabetes could be linked to the presence or absence of particular viral inhabitants in the intestine. In line with the genetic component of these human diseases, mouse model studies demonstrated how beneficial functions of a resident virus can switch to detrimental inflammatory effects in a genetically predisposed host. Such viral-induced intestinal immune disturbances are also recapitulated by several gastrointestinal infectious viruses such as rotavirus and human norovirus. This wide range of viral effects on intestinal immunity emphasizes the need for understanding the innate immune responses to gastrointestinal viruses. Numerous nucleic acid sensors such as DexD/H helicases and AIM2 serve as cytosolic viral guardians to induce antiviral interferon and/or pro-inflammatory inflammasome responses. In both cases, pioneering examples are emerging in which RNA helicases cooperate with particular Nod-like receptors to trigger these cellular responses to enteric viruses. Here we summarize the reported beneficial versus detrimental effects of enteric viruses in the intestinal immune system, and we zoom in on the mechanisms through which sensing of nucleic acids from these enteric viruses trigger interferon and inflammasome responses.
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http://dx.doi.org/10.1016/bs.ircmb.2018.08.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7104954PMC
January 2020

Spatial and temporal heterogeneity of mouse and human microglia at single-cell resolution.

Nature 2019 02 13;566(7744):388-392. Epub 2019 Feb 13.

Institute of Neuropathology, Medical Faculty, University of Freiburg, Freiburg, Germany.

Microglia have critical roles not only in neural development and homeostasis, but also in neurodegenerative and neuroinflammatory diseases of the central nervous system. These highly diverse and specialized functions may be executed by subsets of microglia that already exist in situ, or by specific subsets of microglia that develop from a homogeneous pool of cells on demand. However, little is known about the presence of spatially and temporally restricted subclasses of microglia in the central nervous system during development or disease. Here we combine massively parallel single-cell analysis, single-molecule fluorescence in situ hybridization, advanced immunohistochemistry and computational modelling to comprehensively characterize subclasses of microglia in multiple regions of the central nervous system during development and disease. Single-cell analysis of tissues of the central nervous system during homeostasis in mice revealed specific time- and region-dependent subtypes of microglia. Demyelinating and neurodegenerative diseases evoked context-dependent subtypes of microglia with distinct molecular hallmarks and diverse cellular kinetics. Corresponding clusters of microglia were also identified in healthy human brains, and the brains of patients with multiple sclerosis. Our data provide insights into the endogenous immune system of the central nervous system during development, homeostasis and disease, and may also provide new targets for the treatment of neurodegenerative and neuroinflammatory pathologies.
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http://dx.doi.org/10.1038/s41586-019-0924-xDOI Listing
February 2019

Candidalysin Crucially Contributes to Nlrp3 Inflammasome Activation by Candida albicans Hyphae.

mBio 2019 01 8;10(1). Epub 2019 Jan 8.

Department of Internal Medicine and Pediatrics, Ghent University, Ghent, Belgium

is an opportunistic fungal pathogen that can cause life-threatening infections, particularly in immunocompromised patients. induced activation of the Nlrp3 inflammasome, leading to secretion of bioactive interleukin 1β (IL-1β) is a crucial myeloid cell immune response needed for antifungal host defense. Being a pleiomorphic fungus, can provoke Nlrp3 inflammasome responses only upon morphological transformation to its hyphal appearance. However, the specific hyphal factors that enable to activate the Nlrp3 inflammasome in primary macrophages remain to be revealed. Here, we identify candidalysin, a peptide derived from the hypha-specific gene, as a fungal trigger for Nlrp3 inflammasome-mediated maturation and secretion of IL-1β from primary macrophages. Direct peptide administration experiments showed that candidalysin was sufficient for inducing secretion of mature IL-1β from macrophages in an Nlrp3 inflammasome-dependent manner. Conversely, infection experiments using candidalysin-deficient showed that candidalysin crucially contributed to the capacity of this fungus to induce maturation and secretion of IL-1β from primary macrophages. These complementary observations identify the expression of candidalysin as one of the molecular mechanisms by which hyphal transformation equips with its proinflammatory capacity to elicit the release of bioactive IL-1β from macrophages. Candidiasis is a potentially lethal condition that is caused by systemic dissemination of , a common fungal commensal residing mostly on mucosal surfaces. The transition of from an innocuous commensal to an opportunistic pathogen goes hand in hand with its morphological transformation from a fungus to a hyphal appearance. On the one hand, the latter manifestation enables to penetrate tissues, while on the other hand, the expression of many hypha-specific genes also endows it with the capacity to trigger particular cytokine responses. The Nlrp3 inflammasome is a crucial component of the innate immune system that provokes release of the IL-1β cytokine from myeloid cells upon encountering hyphae. Our study reveals the peptide candidalysin as one of the hypha-derived drivers of Nlrp3 inflammasome responses in primary macrophages and, thus, contributes to better understanding the fungal mechanisms that determine the pathogenicity of .
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http://dx.doi.org/10.1128/mBio.02221-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6325245PMC
January 2019

Microglia in Central Nervous System Inflammation and Multiple Sclerosis Pathology.

Trends Mol Med 2019 02 18;25(2):112-123. Epub 2018 Dec 18.

Vlaams Instituut voor Biotechnologie (VIB) Center for Inflammation Research, Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium. Electronic address:

Microglia are the resident macrophages of the central nervous system (CNS). They have important physiological functions in maintaining tissue homeostasis but also contribute to CNS pathology. Microglia respond to changes in the microenvironment, and the resulting reactive phenotype can be very diverse, with both neuroinflammatory and neuroprotective properties, illustrating the plasticity of these cells. Recent progress in understanding the autoimmune neuroinflammatory disease multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis suggests major roles for microglia in the disease, which have drastically changed our view on the function of microglia in MS.
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http://dx.doi.org/10.1016/j.molmed.2018.11.005DOI Listing
February 2019

A protein-protein interaction map of the TNF-induced NF-κB signal transduction pathway.

Sci Data 2018 12 18;5:180289. Epub 2018 Dec 18.

VIB Center for Medical Biotechnology, B-9000 Ghent, Belgium.

Tumor Necrosis Factor (TNF) has a crucial role in inflammation, cell proliferation and cell death. Dysregulation of TNF receptor 1 (TNFR1)-induced Nuclear Factor-kappa B (NF-κB) signaling leads to chronic inflammation and is associated with several human inflammatory pathologies. Hence, TNF neutralization suppresses inflammation and attenuates inflammatory pathology. However, despite its beneficial effects, anti-TNF therapy suffers from efficacy issues and severe immune side effects. There is thus an urging need to identify novel targets for pharmaceutical intervention in the NF-κB signaling pathway. Here, we present a protein-protein interaction dataset of the TNFR1-induced signaling pathway. For this, we used Virotrap, a novel method for studying protein complexes without disrupting the cellular integrity, on 12 central proteins controlling NF-κB and cell death signaling, both under resting conditions as well as upon TNF stimulation. Our dataset reveals dynamic interactions in TNFR1-induced NF-κB signaling and identifies both known as well as novel interactors that may help to further unravel the molecular mechanisms steering TNF-induced inflammatory signaling and pathology.
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http://dx.doi.org/10.1038/sdata.2018.289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298254PMC
December 2018

BACE2 distribution in major brain cell types and identification of novel substrates.

Life Sci Alliance 2018 Jan 15;1(1):e201800026. Epub 2018 Feb 15.

Department of Neurosciences, Katholieke Universiteit Leuven, Leuven, Belgium.

β-Site APP-cleaving enzyme 1 (BACE1) inhibition is considered one of the most promising therapeutic strategies for Alzheimer's disease, but current BACE1 inhibitors also block BACE2. As the localization and function of BACE2 in the brain remain unknown, it is difficult to predict whether relevant side effects can be caused by off-target inhibition of BACE2 and whether it is important to generate BACE1-specific inhibitors. Here, we show that BACE2 is expressed in discrete subsets of neurons and glia throughout the adult mouse brain. We uncover four new substrates processed by BACE2 in cultured glia: vascular cell adhesion molecule 1, delta and notch-like epidermal growth factor-related receptor, fibroblast growth factor receptor 1, and plexin domain containing 2. Although these substrates were not prominently cleaved by BACE2 in healthy adult mice, proinflammatory TNF induced a drastic increase in BACE2-mediated shedding of vascular cell adhesion molecule 1 in CSF. Thus, although under steady-state conditions the effect of BACE2 cross-inhibition by BACE1-directed inhibitors is rather subtle, it is important to consider that side effects might become apparent under physiopathological conditions that induce TNF expression.
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http://dx.doi.org/10.26508/lsa.201800026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6238391PMC
January 2018

House dust mite-driven neutrophilic airway inflammation in mice with TNFAIP3-deficient myeloid cells is IL-17-independent.

Clin Exp Allergy 2018 12 27;48(12):1705-1714. Epub 2018 Sep 27.

Department of Pulmonary Medicine, Erasmus MC, Rotterdam, The Netherlands.

Background: Asthma is a heterogeneous disease of the airways that involves several types of granulocytic inflammation. Recently, we have shown that the activation status of myeloid cells regulated by TNFAIP3/A20 is a crucial determinant of eosinophilic or neutrophilic airway inflammation. However, whether neutrophilic inflammation observed in this model is dependent on IL-17 remains unknown.

Objective: In this study, we investigated whether IL-17RA-signalling is essential for eosinophilic or neutrophilic inflammation in house dust mite (HDM)-driven airway inflammation.

Methods: Tnfaip3 xLyz2 (Tnfaip3 ) mice were crossed to Il17ra mice, generating Tnfaip3 Il17ra mice and subjected to an HDM-driven airway inflammation model.

Results: Both eosinophilic and neutrophilic inflammation observed in HDM-exposed WT and Tnfaip3 mice respectively were unaltered in the absence of IL-17RA. Production of IL-5, IL-13 and IFN-γ by CD4 T cells was similar between WT, Tnfaip3 and Il17ra mice, whereas mucus-producing cells in Tnfaip3 Il17ra mice were reduced compared to controls. Strikingly, spontaneous accumulation of pulmonary Th1, Th17 and γδ-17 T cells was observed in Tnfaip3 Il17ra mice, but not in the other genotypes. Th17 cell-associated cytokines such as GM-CSF and IL-22 were increased in the lungs of HDM-exposed Tnfaip3 Il17ra mice, compared to IL-17RA-sufficient controls. Moreover, neutrophilic chemo-attractants CXCL1, CXCL2, CXCL12 and Th17-promoting cytokines IL-1β and IL-6 were unaltered between Tnfaip3 and Tnfaip3 Il17ra mice.

Conclusion And Clinical Relevance: These findings show that neutrophilic airway inflammation induced by activated TNFAIP3/A20-deficient myeloid cells can develop in the absence of IL-17RA-signalling. Neutrophilic inflammation is likely maintained by similar quantities of pro-inflammatory cytokines IL-1β and IL-6 that can, independently of IL-17-signalling, induce the expression of neutrophil chemo-attractants.
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http://dx.doi.org/10.1111/cea.13262DOI Listing
December 2018

Keratinocyte Expression of A20/TNFAIP3 Controls Skin Inflammation Associated with Atopic Dermatitis and Psoriasis.

J Invest Dermatol 2019 01 14;139(1):135-145. Epub 2018 Aug 14.

Université de Lille, INSERM, Centre Hospitalier Universitaire de Lille, Institut Pasteur de Lille, U1011-EGID, Lille, France. Electronic address:

Keratinocytes are key players in chronic inflammatory skin diseases. A20 regulates NF-κB-dependent expression of proinflammatory genes and cell death, but the impact of its expression in keratinocytes on systemic inflammation and skin disorders has not been determined. Comparative transcriptomic analysis of microdissected epidermis showed that A20 is down-regulated in involved epidermis, but not in dermis, of psoriasis and atopic dermatitis patients, suggesting that loss of A20 expression in keratinocytes increases the vulnerability for psoriasis/atopic dermatitis induction. We have previously shown that epidermis-specific A20 knockout mice (A20) develop mild epidermal hyperplasia but no macroscopic skin inflammation. We now show that various cytokines and chemokines are up-regulated in A20 mouse skin. A20 mice also display systemic proinflammatory changes, even in the absence of skin immune cell infiltration, and an exacerbated disease severity upon induction of experimental psoriasis, atopic dermatitis, or skin barrier disruption. Keratinocytes showed increased proinflammatory gene expression in the absence of A20 in unstimulated and IL-17A-stimulated conditions, in part resulting from uncontrolled MyD88-dependent signaling. Our findings indicate that absence of A20 in keratinocytes leads to systemic inflammation at homeostatic conditions and is sufficient to exacerbate inflammatory skin disorders associated with different immune profiles by increasing cytokine and chemokine expression.
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http://dx.doi.org/10.1016/j.jid.2018.06.191DOI Listing
January 2019