Publications by authors named "Veit Hornung"

176 Publications

Phosphoproteome profiling uncovers a key role for CDKs in TNF signaling.

Nat Commun 2021 Oct 18;12(1):6053. Epub 2021 Oct 18.

Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, 82152, Germany.

Tumor necrosis factor (TNF) is one of the few cytokines successfully targeted by therapies against inflammatory diseases. However, blocking this well studied and pleiotropic ligand can cause dramatic side-effects. Here, we reason that a systems-level proteomic analysis of TNF signaling could dissect its diverse functions and offer a base for developing more targeted therapies. Therefore, we combine phosphoproteomics time course experiments with subcellular localization and kinase inhibitor analysis to identify functional modules of protein phosphorylation. The majority of regulated phosphorylation events can be assigned to an upstream kinase by inhibiting master kinases. Spatial proteomics reveals phosphorylation-dependent translocations of hundreds of proteins upon TNF stimulation. Phosphoproteome analysis of TNF-induced apoptosis and necroptosis uncovers a key role for transcriptional cyclin-dependent kinase activity to promote cytokine production and prevent excessive cell death downstream of the TNF signaling receptor. This resource of TNF-induced pathways and sites can be explored at http://tnfviewer.biochem.mpg.de/ .
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http://dx.doi.org/10.1038/s41467-021-26289-6DOI Listing
October 2021

Inflammasomes in T cells.

J Mol Biol 2021 Sep 29:167275. Epub 2021 Sep 29.

Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany. Electronic address:

The concept of non-self recognition through germ-line encoded pattern recognition receptors (PRRs) has been well-established for professional innate immune cells. However, there is growing evidence that also T cells employ PRRs and associated effector functions in response to certain non-self or damage signals. Inflammasomes constitute a special subgroup of PRRs that is hardwired to a signaling cascade that culminates in the activation of caspase-1. Active caspase-1 processes pro-inflammatory cytokines of the IL-1 family and also triggers a lytic programmed cell death pathway known as pyroptosis. An increasing body of literature suggests that inflammasomes are also functional in T cells. On the one hand, conventional inflammasome signaling cascades have been described that operate similarly to pathways characterized in innate immune cells. On the other hand, unconventional functions have been suggested, in which certain inflammasome components play a role in unrelated processes, such as cell fate decisions and functions of T helper cells. In this review, we discuss our current knowledge on inflammasome functions in T cells and the biological implications of these findings for health and disease.
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http://dx.doi.org/10.1016/j.jmb.2021.167275DOI Listing
September 2021

In-depth profiling of COVID-19 risk factors and preventive measures in healthcare workers.

Infection 2021 Aug 11. Epub 2021 Aug 11.

Faculty of Medicine, National Reference Center for Retroviruses, Max Von Pettenkofer Institute and Gene Center, Virology, LMU München, Munich, Germany.

Purpose: To determine risk factors for coronavirus disease 2019 (COVID-19) in healthcare workers (HCWs), characterize symptoms, and evaluate preventive measures against SARS-CoV-2 spread in hospitals.

Methods: In a cross-sectional study conducted between May 27 and August 12, 2020, after the first wave of the COVID-19 pandemic, we obtained serological, epidemiological, occupational as well as COVID-19-related data at a quaternary care, multicenter hospital in Munich, Germany.

Results: 7554 HCWs participated, 2.2% of whom tested positive for anti-SARS-CoV-2 antibodies. Multivariate analysis revealed increased COVID-19 risk for nurses (3.1% seropositivity, 95% CI 2.5-3.9%, p = 0.012), staff working on COVID-19 units (4.6% seropositivity, 95% CI 3.2-6.5%, p = 0.032), males (2.4% seropositivity, 95% CI 1.8-3.2%, p = 0.019), and HCWs reporting high-risk exposures to infected patients (5.5% seropositivity, 95% CI 4.0-7.5%, p = 0.0022) or outside of work (12.0% seropositivity, 95% CI 8.0-17.4%, p < 0.0001). Smoking was a protective factor (1.1% seropositivity, 95% CI 0.7-1.8% p = 0.00018) and the symptom taste disorder was strongly associated with COVID-19 (29.8% seropositivity, 95% CI 24.3-35.8%, p < 0.0001). An unbiased decision tree identified subgroups with different risk profiles. Working from home as a preventive measure did not protect against SARS-CoV-2 infection. A PCR-testing strategy focused on symptoms and high-risk exposures detected all larger COVID-19 outbreaks.

Conclusion: Awareness of the identified COVID-19 risk factors and successful surveillance strategies are key to protecting HCWs against SARS-CoV-2, especially in settings with limited vaccination capacities or reduced vaccine efficacy.
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http://dx.doi.org/10.1007/s15010-021-01672-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8354838PMC
August 2021

An autoimmune disease risk variant: A trans master regulatory effect mediated by IRF1 under immune stimulation?

PLoS Genet 2021 07 27;17(7):e1009684. Epub 2021 Jul 27.

New York Genome Center, New York, New York, United States of America.

Functional mechanisms remain unknown for most genetic loci associated to complex human traits and diseases. In this study, we first mapped trans-eQTLs in a data set of primary monocytes stimulated with LPS, and discovered that a risk variant for autoimmune disease, rs17622517 in an intron of C5ORF56, affects the expression of the transcription factor IRF1 20 kb away. The cis-regulatory effect specific to IRF1 is active under early immune stimulus, with a large number of trans-eQTL effects across the genome under late LPS response. Using CRISPRi silencing, we showed that perturbation of the SNP locus downregulates IRF1 and causes widespread transcriptional effects. Genome editing by CRISPR had suggestive recapitulation of the LPS-specific trans-eQTL signal and lent support for the rs17622517 site being functional. Our results suggest that this common genetic variant affects inter-individual response to immune stimuli via regulation of IRF1. For this autoimmune GWAS locus, our work provides evidence of the functional variant, demonstrates a condition-specific enhancer effect, identifies IRF1 as the likely causal gene in cis, and indicates that overactivation of the downstream immune-related pathway may be the cellular mechanism increasing disease risk. This work not only provides rare experimental validation of a master-regulatory trans-eQTL, but also demonstrates the power of eQTL mapping to build mechanistic hypotheses amenable for experimental follow-up using the CRISPR toolkit.
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http://dx.doi.org/10.1371/journal.pgen.1009684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8345867PMC
July 2021

Molecular mechanisms of nonself nucleic acid recognition by the innate immune system.

Eur J Immunol 2021 08 5;51(8):1897-1910. Epub 2021 Jul 5.

Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany.

Nucleic acids (NAs) represent one of the most important classes of molecules recognized by the innate immune system. However, NAs are not limited to pathogens, but are also present within the host. As such, the immune system has evolved an elaborate set of pathogen recognition receptors (PRRs) that employ various strategies to recognize distinct types of NAs, while reliably distinguishing between self and nonself. The here-employed strategies encompass the positioning of NA-sensing PRRs in certain subcellular compartments that potentially come in contact with pathogens but not host NAs, the existence of counterregulatory measures that keep endogenous NAs below a certain threshold, and also the specific identification of certain nonself patterns. Here, we review recent advances in the molecular mechanisms of NA recognition by TLRs, RLRs, and the cGAS-STING axis. We highlight the differences in NA-PRR interfaces that confer specificity and selectivity toward an NA ligand, as well as the NA-dependent induced conformational changes required for signal transduction.
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http://dx.doi.org/10.1002/eji.202049116DOI Listing
August 2021

DPP9 restrains NLRP1 activation.

Nat Struct Mol Biol 2021 04;28(4):333-336

Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany.

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http://dx.doi.org/10.1038/s41594-021-00580-yDOI Listing
April 2021

Post-injury immunosuppression and secondary infections are caused by an AIM2 inflammasome-driven signaling cascade.

Immunity 2021 04 4;54(4):648-659.e8. Epub 2021 Mar 4.

Institute for Stroke and Dementia Research (ISD), University Hospital, LMU Munich, Munich, Germany; Munich Cluster for Systems Neurology (SyNergy), Munich, Germany. Electronic address:

Loss of lymphocytes, particularly T cell apoptosis, is a central pathological event after severe tissue injury that is associated with increased susceptibility for life-threatening infections. The precise immunological mechanisms leading to T cell death after acute injury are largely unknown. Here, we identified a monocyte-T cell interaction driving bystander cell death of T cells in ischemic stroke and burn injury. Specifically, we found that stroke induced a FasL-expressing monocyte population, which led to extrinsic T cell apoptosis. This phenomenon was driven by AIM2 inflammasome-dependent interleukin-1β (IL-1β) secretion after sensing cell-free DNA. Pharmacological inhibition of this pathway improved T cell survival and reduced post-stroke bacterial infections. As such, this study describes inflammasome-dependent monocyte activation as a previously unstudied cause of T cell death after injury and challenges the current paradigms of post-injury lymphopenia.
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http://dx.doi.org/10.1016/j.immuni.2021.02.004DOI Listing
April 2021

GGCX mutations show different responses to vitamin K thereby determining the severity of the hemorrhagic phenotype in VKCFD1 patients.

J Thromb Haemost 2021 06 4;19(6):1412-1424. Epub 2021 May 4.

Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Bonn, Germany.

Background: Vitamin K dependent coagulation factor deficiency type 1 (VKCFD1) is a rare hereditary bleeding disorder caused by mutations in γ-glutamyl carboxylase (GGCX). VKCFD1 patients are treated life-long with high doses of vitamin K in order to correct the bleeding phenotype. However, normalization of clotting factor activities cannot be achieved for all VKCFD1 patients.

Objective: The current study aims to investigate the responsiveness to vitamin K for all reported GGCX mutations with respect to clotting factors in order to optimize treatment.

Methods: This study developed an assay using genetically engineered GGCX cells, in which GGCX mutations were analyzed with respect to their ability to γ-carboxylate vitamin K dependent pro-coagulatory and anti-coagulatory clotting factors by ELISA. Additionally, factor VII activity was measured in order to proof protein functionality. For specific GGCX mutations immunofluorescent staining was performed to assess the intracellular localization of clotting factors with respect to GGCX wild-type and mutations.

Results: All GGCX mutations were categorized into responder and low responder mutations, thereby determining the efficiency of vitamin K supplementation. Most VKCFD1 patients have at least one vitamin K responsive GGCX allele that is able to γ-carboxylate clotting factors. In few patients, the hemorrhagic phenotype cannot be reversed by vitamin K administration because GGCX mutations on both alleles affect either structural or catalytically important sites thereby resulting in residual ability to γ-carboxylate clotting factors.

Conclusion: With these new functional data we can predict the hemorrhagic outcome of each VKCFD1 genotype, thus recommending treatments with either vitamin K or prothrombin complex concentrate.
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http://dx.doi.org/10.1111/jth.15238DOI Listing
June 2021

Deletion of Alzheimer's disease-associated CD33 results in an inflammatory human microglia phenotype.

Glia 2021 Jun 4;69(6):1393-1412. Epub 2021 Feb 4.

Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty and University Hospital Bonn, Bonn, Germany.

Genome-wide association studies demonstrated that polymorphisms in the CD33/sialic acid-binding immunoglobulin-like lectin 3 gene are associated with late-onset Alzheimer's disease (AD). CD33 is expressed on myeloid immune cells and mediates inhibitory signaling through protein tyrosine phosphatases, but the exact function of CD33 in microglia is still unknown. Here, we analyzed CD33 knockout human THP1 macrophages and human induced pluripotent stem cell-derived microglia for immunoreceptor tyrosine-based activation motif pathway activation, cytokine transcription, phagocytosis, and phagocytosis-associated oxidative burst. Transcriptome analysis of the macrophage lines showed that knockout of CD33 as well as knockdown of the CD33 signaling-associated protein tyrosine phosphatase, nonreceptor type 6 (PTPN6) led to constitutive activation of inflammation-related pathways. Moreover, deletion of CD33 or expression of Exon 2-deleted CD33 (CD33 /CD33m) led to increased phosphorylation of the kinases spleen tyrosine kinase (SYK) and extracellular signal-regulated kinase 1 and 2 (ERK1 and 2). Transcript analysis by quantitative real-time polymerase chain reaction confirmed increased levels of interleukin (IL) 1B, IL8, and IL10 after knockout of CD33 in macrophages and microglia. In addition, upregulation of the gene transcripts of the AD-associated phosphatase INPP5D was observed after knockout of CD33. Functional analysis of macrophages and microglia showed that phagocytosis of aggregated amyloid-β and bacterial particles were increased after knockout of CD33 or CD33 expression and knockdown of PTPN6. Furthermore, the phagocytic oxidative burst during uptake of amyloid-β or bacterial particles was increased after CD33 knockout but not in CD33 -expressing microglia. In summary, deletion of CD33 or expression of CD33 in human macrophages and microglia resulted in putative beneficial phagocytosis of amyloid β , but potentially detrimental oxidative burst and inflammation, which was absent in CD33 -expressing microglia.
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http://dx.doi.org/10.1002/glia.23968DOI Listing
June 2021

Human NLRP1 is a sensor for double-stranded RNA.

Science 2021 01 26;371(6528). Epub 2020 Nov 26.

Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany.

Inflammasomes function as intracellular sensors of pathogen infection or cellular perturbation and thereby play a central role in numerous diseases. Given the high abundance of NLRP1 in epithelial barrier tissues, we screened a diverse panel of viruses for inflammasome activation in keratinocytes. We identified Semliki Forest virus (SFV), a positive-strand RNA virus, as a potent activator of human but not murine NLRP1B. SFV replication and the associated formation of double-stranded (ds) RNA was required to engage the NLRP1 inflammasome. Moreover, delivery of long dsRNA was sufficient to trigger activation. Biochemical studies revealed that NLRP1 binds dsRNA through its leucine-rich repeat domain, resulting in its NACHT domain gaining adenosine triphosphatase activity. Altogether, these results establish human NLRP1 as a direct sensor for dsRNA and thus RNA virus infection.
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http://dx.doi.org/10.1126/science.abd0811DOI Listing
January 2021

Irgm2 and Gate-16 put a break on caspase-11 activation.

EMBO Rep 2020 11 1;21(11):e51787. Epub 2020 Nov 1.

Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany.

In infections caused by gram-negative bacteria, the bacterial cell wall component lipopolysaccharide (LPS) acts as a potent pathogen-associated molecular pattern (PAMP) that triggers the innate immune system. This is accomplished by two pattern recognition receptor systems. Toll-like receptor 4 (TLR4) senses extracellular LPS and induces a broad pro-inflammatory transcriptional program and also antiviral interferons. A complementary system detects intracellular LPS. As such, upon its release into the cytoplasm, LPS can directly engage the protease caspase-4 (caspase-11 in the murine system) and thereby trigger a pro-inflammatory cell death program known as pyroptosis (Rathinam et al, 2019). This is mediated by active caspase-4 cleaving its substrate gasdermin D (GSDMD). The thereby released N-terminal fragment of GSDMD inserts into the cell membrane and forms a cytotoxic pore. As a consequence, the cell ruptures and releases its pro-inflammatory content. In addition, the GSDMD pore results in potassium efflux that can activate the NLRP3 inflammasome. NLRP3 in turn activates caspase-1, which matures pro-IL-1β and pro-IL-18, further perpetuating the inflammatory nature of this cell death. Given its unconventional mode of NLRP3 activation, this pathway has been coined the non-canonical inflammasome.
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http://dx.doi.org/10.15252/embr.202051787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7645257PMC
November 2020

C-tag TNF: a reporter system to study TNF shedding.

J Biol Chem 2020 12 20;295(52):18065-18075. Epub 2020 Oct 20.

Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany. Electronic address:

TNF is a highly pro-inflammatory cytokine that contributes not only to the regulation of immune responses but also to the development of severe inflammatory diseases. TNF is synthesized as a transmembrane protein, which is further matured via proteolytic cleavage by metalloproteases such as ADAM17, a process known as shedding. At present, TNF is mainly detected by measuring the precursor or the mature cytokine of bulk cell populations by techniques such as ELISA or immunoblotting. However, these methods do not provide information on the exact timing and extent of TNF cleavage at single-cell resolution and they do not allow the live visualization of shedding events. Here, we generated C-tag TNF as a genetically encoded reporter to study TNF shedding at the single-cell level. The functionality of the C-tag TNF reporter is based on the exposure of a cryptic epitope on the C terminus of the transmembrane portion of pro-TNF on cleavage. In both denatured and nondenatured samples, this epitope can be detected by a nanobody in a highly sensitive and specific manner only upon TNF shedding. As such, C-tag TNF can successfully be used for the detection of TNF cleavage in flow cytometry and live-cell imaging applications. We furthermore demonstrate its applicability in a forward genetic screen geared toward the identification of genetic regulators of TNF maturation. In summary, the C-tag TNF reporter can be employed to gain novel insights into the complex regulation of ADAM-dependent TNF shedding.
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http://dx.doi.org/10.1074/jbc.RA120.015248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7939438PMC
December 2020

Structural basis for sequestration and autoinhibition of cGAS by chromatin.

Nature 2020 11 10;587(7835):678-682. Epub 2020 Sep 10.

Gene Center, Ludwig-Maximilians-Universität, Munich, Germany.

Cyclic GMP-AMP synthase (cGAS) is an innate immune sensor for cytosolic microbial DNA. After binding DNA, cGAS synthesizes the messenger 2'3'-cyclic GMP-AMP (cGAMP), which triggers cell-autonomous defence and the production of type I interferons and pro-inflammatory cytokines via the activation of STING. In addition to responding to cytosolic microbial DNA, cGAS also recognizes mislocalized cytosolic self-DNA and has been implicated in autoimmunity and sterile inflammation. Specificity towards pathogen- or damage-associated DNA was thought to be caused by cytosolic confinement. However, recent findings place cGAS robustly in the nucleus, where tight tethering of chromatin is important to prevent autoreactivity to self-DNA. Here we show how cGAS is sequestered and inhibited by chromatin. We provide a cryo-electron microscopy structure of the cGAS catalytic domain bound to a nucleosome, which shows that cGAS does not interact with the nucleosomal DNA, but instead interacts with histone 2A-histone 2B, and is tightly anchored to the 'acidic patch'. The interaction buries the cGAS DNA-binding site B, and blocks the formation of active cGAS dimers. The acidic patch robustly outcompetes agonistic DNA for binding to cGAS, which suggests that nucleosome sequestration can efficiently inhibit cGAS, even when accessible DNA is nearby, such as in actively transcribed genomic regions. Our results show how nuclear cGAS is sequestered by chromatin and provides a mechanism for preventing autoreactivity to nuclear self-DNA.
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http://dx.doi.org/10.1038/s41586-020-2748-0DOI Listing
November 2020

CARD8 inflammasome activation triggers pyroptosis in human T cells.

EMBO J 2020 10 25;39(19):e105071. Epub 2020 Aug 25.

Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany.

Inflammasomes execute a unique type of cell death known as pyroptosis. Mostly characterized in myeloid cells, caspase-1 activation downstream of an inflammasome sensor results in the cleavage and activation of gasdermin D (GSDMD), which then forms a lytic pore in the plasma membrane. Recently, CARD8 was identified as a novel inflammasome sensor that triggers pyroptosis in myeloid leukemia cells upon inhibition of dipeptidyl-peptidases (DPP). Here, we show that blocking DPPs using Val-boroPro triggers a lytic form of cell death in primary human CD4 and CD8 T cells, while other prototypical inflammasome stimuli were not active. This cell death displays morphological and biochemical hallmarks of pyroptosis. By genetically dissecting candidate components in primary T cells, we identify this response to be dependent on the CARD8-caspase-1-GSDMD axis. Moreover, DPP9 constitutes the relevant DPP restraining CARD8 activation. Interestingly, this CARD8-induced pyroptosis pathway can only be engaged in resting, but not in activated T cells. Altogether, these results broaden the relevance of inflammasome signaling and associated pyroptotic cell death to T cells, central players of the adaptive immune system.
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http://dx.doi.org/10.15252/embj.2020105071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7527815PMC
October 2020

Reduced mitochondrial resilience enables non-canonical induction of apoptosis after TNF receptor signaling in virus-infected hepatocytes.

J Hepatol 2020 12 26;73(6):1347-1359. Epub 2020 Jun 26.

Institute of Pathology, Technical University of Munich, Munich, Germany.

Background & Aims: Selective elimination of virus-infected hepatocytes occurs through virus-specific CD8 T cells recognizing peptide-loaded MHC molecules. Herein, we report that virus-infected hepatocytes are also selectively eliminated through a cell-autonomous mechanism.

Methods: We generated recombinant adenoviruses and genetically modified mouse models to identify the molecular mechanisms determining TNF-induced hepatocyte apoptosis in vivo and used in vivo bioluminescence imaging, immunohistochemistry, immunoblot analysis, RNAseq/proteome/phosphoproteome analyses, bioinformatic analyses, mitochondrial function tests.

Results: We found that TNF precisely eliminated only virus-infected hepatocytes independently of local inflammation and activation of immune sensory receptors. TNF receptor I was equally relevant for NF-kB activation in healthy and infected hepatocytes, but selectively mediated apoptosis in infected hepatocytes. Caspase 8 activation downstream of TNF receptor signaling was dispensable for apoptosis in virus-infected hepatocytes, indicating an unknown non-canonical cell-intrinsic pathway promoting apoptosis in hepatocytes. We identified a unique state of mitochondrial vulnerability in virus-infected hepatocytes as the cause for this non-canonical induction of apoptosis through TNF. Mitochondria from virus-infected hepatocytes showed normal biophysical and bioenergetic functions but were characterized by reduced resilience to calcium challenge. In the presence of unchanged TNF-induced signaling, reactive oxygen species-mediated calcium release from the endoplasmic reticulum caused mitochondrial permeability transition and apoptosis, which identified a link between extrinsic death receptor signaling and cell-intrinsic mitochondrial-mediated caspase activation.

Conclusion: Our findings reveal a novel concept in immune surveillance by identifying a cell-autonomous defense mechanism that selectively eliminates virus-infected hepatocytes through mitochondrial permeability transition.

Lay Summary: The liver is known for its unique immune functions. Herein, we identify a novel mechanism by which virus-infected hepatocytes can selectively eliminate themselves through reduced mitochondrial resilience to calcium challenge.
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http://dx.doi.org/10.1016/j.jhep.2020.06.026DOI Listing
December 2020

Hepatitis B Virus DNA is a Substrate for the cGAS/STING Pathway but is not Sensed in Infected Hepatocytes.

Viruses 2020 05 29;12(6). Epub 2020 May 29.

Host-Pathogen Interactions, Paul-Ehrlich-Institut, 63225 Langen, Germany.

Hepatitis B virus HBV) chronic infection is a critical risk factor for hepatocellular carcinoma. The innate immune response to HBV infection is a matter of debate. In particular, viral escape mechanisms are poorly understood. Our study reveals that HBV RNAs are not immunostimulatory in immunocompetent myeloid cells. In contrast, HBV DNA from viral particles and DNA replication intermediates are immunostimulatory and sensed by cyclic GMP-AMP Synthase (cGAS) and Stimulator of Interferon Genes (STING). We show that primary human hepatocytes express DNA sensors to reduced levels compared to myeloid cells. Nevertheless, hepatocytes can respond to HBV relaxed-circular DNA (rcDNA), when transfected in sufficient amounts, but not to HBV infection. Finally, our data suggest that HBV infection does not actively inhibit the DNA-sensing pathway. In conclusion, in infected hepatocytes, HBV passively evades recognition by cellular sensors of nucleic acids by (i) producing non-immunostimulatory RNAs, (ii) avoiding sensing of its DNAs by cGAS/STING without active inhibition of the pathway.
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http://dx.doi.org/10.3390/v12060592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7354540PMC
May 2020

Molecular mechanisms and cellular functions of cGAS-STING signalling.

Nat Rev Mol Cell Biol 2020 09 18;21(9):501-521. Epub 2020 May 18.

Department of Biochemistry, Ludwig-Maximilians-Universität, Munich, Germany.

The cGAS-STING signalling axis, comprising the synthase for the second messenger cyclic GMP-AMP (cGAS) and the cyclic GMP-AMP receptor stimulator of interferon genes (STING), detects pathogenic DNA to trigger an innate immune reaction involving a strong type I interferon response against microbial infections. Notably however, besides sensing microbial DNA, the DNA sensor cGAS can also be activated by endogenous DNA, including extranuclear chromatin resulting from genotoxic stress and DNA released from mitochondria, placing cGAS-STING as an important axis in autoimmunity, sterile inflammatory responses and cellular senescence. Initial models assumed that co-localization of cGAS and DNA in the cytosol defines the specificity of the pathway for non-self, but recent work revealed that cGAS is also present in the nucleus and at the plasma membrane, and such subcellular compartmentalization was linked to signalling specificity of cGAS. Further confounding the simple view of cGAS-STING signalling as a response mechanism to infectious agents, both cGAS and STING were shown to have additional functions, independent of interferon response. These involve non-catalytic roles of cGAS in regulating DNA repair and signalling via STING to NF-κB and MAPK as well as STING-mediated induction of autophagy and lysosome-dependent cell death. We have also learnt that cGAS dimers can multimerize and undergo liquid-liquid phase separation to form biomolecular condensates that could importantly regulate cGAS activation. Here, we review the molecular mechanisms and cellular functions underlying cGAS-STING activation and signalling, particularly highlighting the newly emerging diversity of this signalling pathway and discussing how the specificity towards normal, damage-induced and infection-associated DNA could be achieved.
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http://dx.doi.org/10.1038/s41580-020-0244-xDOI Listing
September 2020

New Approaches for Absolute Quantification of Stable-Isotope-Labeled Peptide Standards for Targeted Proteomics Based on a UV Active Tag.

Proteomics 2020 05 20;20(10):e2000007. Epub 2020 May 20.

JPT Peptide Technologies GmbH, Volmerstrasse 5, Berlin, 12489, Germany.

Targeted proteomics depends on the availability of stable isotope labeled (SIL) peptide standards, which for absolute protein quantification need to be absolutely quantified. In the present study, three new approaches for absolute quantification of SIL peptides are developed. All approaches rely on a quantification tag (Qtag) with a specific UV absorption. The Qtag is attached to the peptide during synthesis and is removed by tryptic digestion under standard proteomics workflow conditions. While one quantification method (method A) is designed to allow the fast and economic production of absolutely quantified SIL peptides, two other methods (methods B and C) are developed to enable the straightforward re-quantification of SIL peptides after reconstitution to control and monitor known problems related to peptide solubility, precipitation, and adhesion to vials. All methods yield consistent results when compared to each other and when compared to quantification by amino acid analysis. The precise quantitation methods are used to characterize the in vivo specificity of the H3 specific histone methyltransferase EZH2.
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http://dx.doi.org/10.1002/pmic.202000007DOI Listing
May 2020

Author Correction: AIM2 inflammasome-derived IL-1β induces postoperative ileus in mice.

Sci Rep 2020 Feb 21;10(1):3457. Epub 2020 Feb 21.

Department of Surgery, University of Bonn, Bonn, Germany.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-60391-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7035264PMC
February 2020

The NLRP3 inflammasome pathway is activated in sarcoidosis and involved in granuloma formation.

Eur Respir J 2020 03 26;55(3). Epub 2020 Mar 26.

Fraunhofer ITEM, Hannover, Germany

Sarcoidosis is a disease characterised by granuloma formation. There is an unmet need for new treatment strategies beyond corticosteroids. The NLRP3 inflammasome pathway is expressed in innate immune cells and senses danger signals to elicit inflammatory interleukin (IL)-1β; it has recently become a druggable target. This prompted us to test the role of the NLRP3 inflammasome and IL-1β pathway in granuloma formation and sarcoidosis.19 sarcoid patients and 19 healthy volunteers were recruited into this pilot study. NLRP3 inflammasome activity was measured in bronchoalveolar lavage (BAL) cells and lung and skin biopsies using immunohistochemistry, Western blot, reverse-transcriptase PCR and ELISA. For experiments we used the trehalose 6,6'-dimycolate-granuloma mouse model and evaluated lung granuloma burden in miR-223 knockout and NLRP3 knockout mice, as well as the treatment effects of MCC950 and anti-IL-1β antibody therapy.We found strong upregulation of the NLRP3 inflammasome pathway, evidenced by expression of activated NLRP3 inflammasome components, including cleaved caspase-1 and IL-1β in lung granuloma, and increased IL-1β release of BAL cells from sarcoid patients compared to healthy volunteers (p=0.006). mRNA levels of miR-223, a micro-RNA downregulating NLRP3, were decreased and NLRP3 mRNA correspondingly increased in alveolar macrophages from sarcoid patients (p<0.005). NLRP3 knockout mice showed decreased and miR-223 knockout mice increased granuloma formation compared to wild-type mice. Pharmacological interference using NLRP3 pathway inhibitor MCC950 or an anti-IL-1β antibody resulted in reduced granuloma formation (p<0.02).In conclusion, our data provide evidence of upregulated inflammasome and IL-1β pathway activation in sarcoidosis and suggest both as valid therapeutic targets.
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http://dx.doi.org/10.1183/13993003.00119-2019DOI Listing
March 2020

Cytosolic Gram-negative bacteria prevent apoptosis by inhibition of effector caspases through lipopolysaccharide.

Nat Microbiol 2020 02 23;5(2):354-367. Epub 2019 Dec 23.

Institute for Medical Microbiology, Immunology and Hygiene, Centre for Molecular Medicine Cologne and Cologne Excellence Cluster on Cellular Stress Responses in Ageing-Associated Diseases, University of Cologne, Cologne, Germany.

The cytosolic appearance and propagation of bacteria cause overwhelming cellular stress responses that induce apoptosis under normal conditions. Therefore, successful bacterial colonization depends on the ability of intracellular pathogens to block apoptosis and to safeguard bacterial replicative niches. Here, we show that the cytosolic Gram-negative bacterium Shigella flexneri stalls apoptosis by inhibiting effector caspase activity. Our data identified lipopolysaccharide (LPS) as a bona fide effector caspase inhibitor that directly binds caspases by involving its O-antigen (O Ag) moiety. Bacterial strains that lacked the O Ag or failed to replicate within the cytosol were incapable of blocking apoptosis and exhibited reduced virulence in a murine model of bacterial infection. Our findings demonstrate how Shigella inhibits pro-apoptotic caspase activity, effectively delays coordinated host-cell demise and supports its intracellular propagation. Next to the recently discovered pro-inflammatory role of cytosolic LPS, our data reveal a distinct mode of LPS action that, through the disruption of the early coordinated non-lytic cell death response, ultimately supports the inflammatory breakdown of infected cells at later time points.
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http://dx.doi.org/10.1038/s41564-019-0620-5DOI Listing
February 2020

Cytoplasmic RNA Sensor Pathways and Nitazoxanide Broadly Inhibit Intracellular Mycobacterium tuberculosis Growth.

iScience 2019 Dec 6;22:299-313. Epub 2019 Nov 6.

Program in Cellular and Molecular Medicine, Children's Hospital Boston, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

To establish stable infection, Mycobacterium tuberculosis (MTb) must overcome host innate immune mechanisms, including those that sense pathogen-derived nucleic acids. Here, we show that the host cytosolic RNA sensing molecules RIG-I-like receptor (RLR) signaling proteins RIG-I and MDA5, their common adaptor protein MAVS, and the RNA-dependent kinase PKR each independently inhibit MTb growth in human cells. Furthermore, we show that MTb broadly stimulates RIG-I, MDA5, MAVS, and PKR gene expression and their biological activities. We also show that the oral FDA-approved drug nitazoxanide (NTZ) significantly inhibits intracellular MTb growth and amplifies MTb-stimulated RNA sensor gene expression and activity. This study establishes prototypic cytoplasmic RNA sensors as innate restriction factors for MTb growth in human cells and it shows that targeting this pathway is a potential host-directed approach to treat tuberculosis disease.
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http://dx.doi.org/10.1016/j.isci.2019.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6909047PMC
December 2019

Insights into Innate Sensing of Prototype Foamy Viruses in Myeloid Cells.

Viruses 2019 11 26;11(12). Epub 2019 Nov 26.

Institute of Virology, Medical Faculty "Carl Gustav Carus", Technische Universität Dresden, 01307 Dresden, Germany.

Foamy viruses (FVs) belong to the subfamily of retroviruses and are characterized by unique features in their replication strategy. This includes a reverse transcription (RTr) step of the packaged RNA genome late in replication, resulting in the release of particles with a fraction of them already containing an infectious viral DNA (vDNA) genome. Little is known about the immune responses against FVs in their hosts, which control infection and may be responsible for their apparent apathogenic nature. We studied the interaction of FVs with the innate immune system in myeloid cells, and characterized the viral pathogen-associated molecular patterns (PAMPs) and the cellular pattern recognition receptors and sensing pathways involved. Upon cytoplasmic access, full-length but not minimal vector genome containing FVs with active reverse transcriptase, induced an efficient innate immune response in various myeloid cells. It was dependent on cellular cGAS and STING and largely unaffected by RTr inhibition during viral entry. This suggests that RTr products, which are generated during FV morphogenesis in infected cells, and are therefore already present in FV particles taken up by immune cells, are the main PAMPs of FVs with full-length genomes sensed in a cGAS and STING-dependent manner by the innate immune system in host cells of the myeloid lineage.
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http://dx.doi.org/10.3390/v11121095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6950106PMC
November 2019

TLR8 Is a Sensor of RNase T2 Degradation Products.

Cell 2019 11;179(6):1264-1275.e13

Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany; Center for Integrated Protein Science (CiPSM), Ludwig-Maximilians-Universität München, Munich, Germany; Max Planck Institute of Biochemistry, Martinsried, Germany. Electronic address:

TLR8 is among the highest-expressed pattern-recognition receptors in the human myeloid compartment, yet its mode of action is poorly understood. TLR8 engages two distinct ligand binding sites to sense RNA degradation products, although it remains unclear how these ligands are formed in cellulo in the context of complex RNA molecule sensing. Here, we identified the lysosomal endoribonuclease RNase T2 as a non-redundant upstream component of TLR8-dependent RNA recognition. RNase T2 activity is required for rendering complex single-stranded, exogenous RNA molecules detectable for TLR8. This is due to RNase T2's preferential cleavage of single-stranded RNA molecules between purine and uridine residues, which critically contributes to the supply of catabolic uridine and the generation of purine-2',3'-cyclophosphate-terminated oligoribonucleotides. Thus-generated molecules constitute agonistic ligands for the first and second binding pocket of TLR8. Together, these results establish the identity and origin of the RNA-derived molecular pattern sensed by TLR8.
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http://dx.doi.org/10.1016/j.cell.2019.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116005PMC
November 2019

AIM2 inflammasome-derived IL-1β induces postoperative ileus in mice.

Sci Rep 2019 07 22;9(1):10602. Epub 2019 Jul 22.

Department of Surgery, University of Bonn, Bonn, Germany.

Postoperative ileus (POI) is an intestinal dysmotility frequently occurring after abdominal surgery. An orchestrated neuroimmune response within the muscularis externa (ME) involves activation of resident macrophages, enteric glia and infiltration of blood-derived leukocytes. Interleukin-1 receptor type-I (IL1R1) signalling on enteric glia has been shown to be involved in POI development. Herein we investigated the distinct role of the IL1R1 ligands interleukin (IL) -1α and IL-1β and focused on the mechanism of IL-1β production. IL-1α and IL-1β deficient mice were protected from POI. Bone-marrow transplantation studies indicated that IL-1α originated from radio-resistant cells while IL-1β was released from the radio-sensitive infiltrating leukocytes. Mouse strains deficient in inflammasome formation identified the absent in melanoma 2 (AIM2) inflammasome to be crucial for IL-1β production in POI. Mechanistically, antibiotic-treated mice revealed a prominent role of the microbiome in IL-1β production. Our study provides new insights into distinct roles of IL-1α and IL-1β signalling during POI. While IL-1α release is most likely an immediate passive response to the surgical trauma, IL-1β production depends on AIM2 inflammasome formation and the microbiome. Selective interaction in this pathway might be a promising target to prevent POI in surgical patients.
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http://dx.doi.org/10.1038/s41598-019-46968-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6646358PMC
July 2019

Human GBP1 is a microbe-specific gatekeeper of macrophage apoptosis and pyroptosis.

EMBO J 2019 07 3;38(13):e100926. Epub 2019 Jun 3.

Host-Toxoplasma Interaction Laboratory, The Francis Crick Institute, London, UK.

The guanylate binding protein (GBP) family of interferon-inducible GTPases promotes antimicrobial immunity and cell death. During bacterial infection, multiple mouse Gbps, human GBP2, and GBP5 support the activation of caspase-1-containing inflammasome complexes or caspase-4 which trigger pyroptosis. Whether GBPs regulate other forms of cell death is not known. The apicomplexan parasite Toxoplasma gondii causes macrophage death through unidentified mechanisms. Here we report that Toxoplasma-induced death of human macrophages requires GBP1 and its ability to target Toxoplasma parasitophorous vacuoles through its GTPase activity and prenylation. Mechanistically, GBP1 promoted Toxoplasma detection by AIM2, which induced GSDMD-independent, ASC-, and caspase-8-dependent apoptosis. Identical molecular determinants targeted GBP1 to Salmonella-containing vacuoles. GBP1 facilitated caspase-4 recruitment to Salmonella leading to its enhanced activation and pyroptosis. Notably, GBP1 could be bypassed by the delivery of Toxoplasma DNA or bacterial LPS into the cytosol, pointing to its role in liberating microbial molecules. GBP1 thus acts as a gatekeeper of cell death pathways, which respond specifically to infecting microbes. Our findings expand the immune roles of human GBPs in regulating not only pyroptosis, but also apoptosis.
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http://dx.doi.org/10.15252/embj.2018100926DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6600649PMC
July 2019

IRF1 Inhibits Antitumor Immunity through the Upregulation of PD-L1 in the Tumor Cell.

Cancer Immunol Res 2019 08 25;7(8):1258-1266. Epub 2019 Jun 25.

Cancer Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania.

Multiple studies have associated the transcription factor IRF1 with tumor-suppressive activities. Here, we report an opposite tumor cell-intrinsic function of IRF1 in promoting tumor growth. IRF1-deficient tumor cells showed reduced tumor growth in MC38 and CT26 colon carcinoma and B16 melanoma mouse models. This reduction in tumor growth was dependent on host CD8 T cells. Detailed profiling of tumor-infiltrating leukocytes did not show changes in the various T-cell and myeloid cell populations. However, CD8 T cells that had infiltrated IRF1-deficieint tumors exhibited enhanced cytotoxicity. IRF1-deficient tumor cells lost the ability to upregulate PD-L1 expression and and were more susceptible to T-cell-mediated killing. Induced expression of PD-L1 in IRF1-deficient tumor cells restored tumor growth. These results indicate differential activity of IRF1 in tumor escape.
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http://dx.doi.org/10.1158/2326-6066.CIR-18-0711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6677597PMC
August 2019

Immune homeostasis and regulation of the interferon pathway require myeloid-derived Regnase-3.

J Exp Med 2019 07 24;216(7):1700-1723. Epub 2019 May 24.

Institute for Diabetes and Obesity, Helmholtz Diabetes Center at Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany

The RNase Regnase-1 is a master RNA regulator in macrophages and T cells that degrades cellular and viral RNA upon NF-κB signaling. The roles of its family members, however, remain largely unknown. Here, we analyzed -deficient mice, which develop hypertrophic lymph nodes. We used various mice with immune cell-specific deletions of to demonstrate that Regnase-3 acts specifically within myeloid cells. deficiency systemically increased IFN signaling, which increased the proportion of immature B and innate immune cells, and suppressed follicle and germinal center formation. Expression analysis revealed that Regnase-3 and Regnase-1 share protein degradation pathways. Unlike Regnase-1, Regnase-3 expression is high specifically in macrophages and is transcriptionally controlled by IFN signaling. Although direct targets in macrophages remain unknown, Regnase-3 can bind, degrade, and regulate mRNAs, such as (), in vitro. These data indicate that Regnase-3, like Regnase-1, is an RNase essential for immune homeostasis but has diverged as key regulator in the IFN pathway in macrophages.
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http://dx.doi.org/10.1084/jem.20181762DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6605757PMC
July 2019

KMT9 monomethylates histone H4 lysine 12 and controls proliferation of prostate cancer cells.

Nat Struct Mol Biol 2019 05 6;26(5):361-371. Epub 2019 May 6.

Klinik für Urologie und Zentrale Klinische Forschung, Klinikum der Albert-Ludwigs-Universität Freiburg, Freiburg, Germany.

Histone lysine methylation is generally performed by SET domain methyltransferases and regulates chromatin structure and gene expression. Here, we identify human C21orf127 (HEMK2, N6AMT1, PrmC), a member of the seven-β-strand family of putative methyltransferases, as a novel histone lysine methyltransferase. C21orf127 functions as an obligate heterodimer with TRMT112, writing the methylation mark on lysine 12 of histone H4 (H4K12) in vitro and in vivo. We characterized H4K12 recognition by solving the crystal structure of human C21orf127-TRMT112, hereafter termed 'lysine methyltransferase 9' (KMT9), in complex with S-adenosyl-homocysteine and H4K12me1 peptide. Additional analyses revealed enrichment for KMT9 and H4K12me1 at the promoters of numerous genes encoding cell cycle regulators and control of cell cycle progression by KMT9. Importantly, KMT9 depletion severely affects the proliferation of androgen receptor-dependent, as well as that of castration- and enzalutamide-resistant prostate cancer cells and xenograft tumors. Our data link H4K12 methylation with KMT9-dependent regulation of androgen-independent prostate tumor cell proliferation, thereby providing a promising paradigm for the treatment of castration-resistant prostate cancer.
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http://dx.doi.org/10.1038/s41594-019-0219-9DOI Listing
May 2019
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