Publications by authors named "Eicke Latz"

272 Publications

Deciphering How NLRP3 Incites the Stromal Response in Kawasaki Vasculitis.

Circ Res 2021 Oct 14;129(9):840-842. Epub 2021 Oct 14.

Institute of Innate Immunity, University Hospitals, University of Bonn, Bonn, Germany (A.C., E.L.).

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http://dx.doi.org/10.1161/CIRCRESAHA.121.320131DOI Listing
October 2021

The multifaceted therapeutic value of targeting ATP-citrate lyase in atherosclerosis.

Trends Mol Med 2021 Oct 8. Epub 2021 Oct 8.

Department of Molecular Cell Biology and Immunology, Amsterdam Cardiovascular Sciences, Amsterdam Gastroenterology Endocrinology Metabolism, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands. Electronic address:

ATP-citrate lyase (Acly) is the target of the new class low-density lipoprotein-cholesterol (LDL-C)-lowering drug bempedoic acid (BA). Acly is a key metabolic enzyme synthesizing acetyl-CoA as the building block of cholesterol and fatty acids. Treatment with BA lowers circulating lipid levels and reduces systemic inflammation, suggesting a dual benefit of this drug for atherosclerosis therapy. Recent studies have shown that targeting Acly in macrophages can attenuate inflammatory responses and decrease atherosclerotic plaque vulnerability. Therefore, it could be beneficial to extend the application of Acly inhibition from solely lipid-lowering by liver-specific inhibition to also targeting macrophages in atherosclerosis. Here, we outline the possibilities of targeting Acly and describe the future needs to translate these findings to the clinic.
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http://dx.doi.org/10.1016/j.molmed.2021.09.004DOI Listing
October 2021

Early IFN-α signatures and persistent dysfunction are distinguishing features of NK cells in severe COVID-19.

Immunity 2021 Sep 4. Epub 2021 Sep 4.

Institute of Innate Immunity, Medical Faculty, University of Bonn, Bonn, Germany.

Longitudinal analyses of the innate immune system, including the earliest time points, are essential to understand the immunopathogenesis and clinical course of coronavirus disease (COVID-19). Here, we performed a detailed characterization of natural killer (NK) cells in 205 patients (403 samples; days 2 to 41 after symptom onset) from four independent cohorts using single-cell transcriptomics and proteomics together with functional studies. We found elevated interferon (IFN)-α plasma levels in early severe COVD-19 alongside increased NK cell expression of IFN-stimulated genes (ISGs) and genes involved in IFN-α signaling, while upregulation of tumor necrosis factor (TNF)-induced genes was observed in moderate diseases. NK cells exert anti-SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) activity but are functionally impaired in severe COVID-19. Further, NK cell dysfunction may be relevant for the development of fibrotic lung disease in severe COVID-19, as NK cells exhibited impaired anti-fibrotic activity. Our study indicates preferential IFN-α and TNF responses in severe and moderate COVID-19, respectively, and associates a prolonged IFN-α-induced NK cell response with poorer disease outcome.
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http://dx.doi.org/10.1016/j.immuni.2021.09.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8416549PMC
September 2021

The inflammasome: from bench to bedside.

Authors:
Eicke Latz

Nat Rev Immunol 2021 Oct;21(10):622-623

Institute of Innate Immunity, University of Bonn, Bonn, Germany.

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http://dx.doi.org/10.1038/s41577-021-00600-4DOI Listing
October 2021

Microglia jointly degrade fibrillar alpha-synuclein cargo by distribution through tunneling nanotubes.

Cell 2021 Sep 22;184(20):5089-5106.e21. Epub 2021 Sep 22.

Department of Neurodegenerative Disease and Geriatric Psychiatry/Neurology, University of Bonn Medical Center, 53127 Bonn, Germany; German Center for Neurodegenerative Diseases (DZNE), 53127 Bonn, Germany; Divison of Infectious Diseases and Immunology, University of Massachusetts Medical School, 01605 Worcester, MA, USA. Electronic address:

Microglia are the CNS resident immune cells that react to misfolded proteins through pattern recognition receptor ligation and activation of inflammatory pathways. Here, we studied how microglia handle and cope with α-synuclein (α-syn) fibrils and their clearance. We found that microglia exposed to α-syn establish a cellular network through the formation of F-actin-dependent intercellular connections, which transfer α-syn from overloaded microglia to neighboring naive microglia where the α-syn cargo got rapidly and effectively degraded. Lowering the α-syn burden attenuated the inflammatory profile of microglia and improved their survival. This degradation strategy was compromised in cells carrying the LRRK2 G2019S mutation. We confirmed the intercellular transfer of α-syn assemblies in microglia using organotypic slice cultures, 2-photon microscopy, and neuropathology of patients. Together, these data identify a mechanism by which microglia create an "on-demand" functional network in order to improve pathogenic α-syn clearance.
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http://dx.doi.org/10.1016/j.cell.2021.09.007DOI Listing
September 2021

BTK operates a phospho-tyrosine switch to regulate NLRP3 inflammasome activity.

J Exp Med 2021 Nov 23;218(11). Epub 2021 Sep 23.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA.

Activity of the NLRP3 inflammasome, a critical mediator of inflammation, is controlled by accessory proteins, posttranslational modifications, cellular localization, and oligomerization. How these factors relate is unclear. We show that a well-established drug target, Bruton's tyrosine kinase (BTK), affects several levels of NLRP3 regulation. BTK directly interacts with NLRP3 in immune cells and phosphorylates four conserved tyrosine residues upon inflammasome activation, in vitro and in vivo. Furthermore, BTK promotes NLRP3 relocalization, oligomerization, ASC polymerization, and full inflammasome assembly, probably by charge neutralization, upon modification of a polybasic linker known to direct NLRP3 Golgi association and inflammasome nucleation. As NLRP3 tyrosine modification by BTK also positively regulates IL-1β release, we propose BTK as a multifunctional positive regulator of NLRP3 regulation and BTK phosphorylation of NLRP3 as a novel and therapeutically tractable step in the control of inflammation.
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http://dx.doi.org/10.1084/jem.20201656DOI Listing
November 2021

Proteopathic tau primes and activates interleukin-1β via myeloid-cell-specific MyD88- and NLRP3-ASC-inflammasome pathway.

Cell Rep 2021 Sep;36(12):109720

Department of Molecular Genetics and Microbiology, University of New Mexico, Albuquerque, NM 87131, USA; Department of Neurology, University of New Mexico, Albuquerque, NM 87131, USA. Electronic address:

Pathological hyperphosphorylation and aggregation of tau (pTau) and neuroinflammation, driven by interleukin-1β (IL-1β), are the major hallmarks of tauopathies. Here, we show that pTau primes and activates IL-1β. First, RNA-sequence analysis suggests paired-helical filaments (PHFs) from human tauopathy brain primes nuclear factor κB (NF-κB), chemokine, and IL-1β signaling clusters in human primary microglia. Treating microglia with pTau-containing neuronal media, exosomes, or PHFs causes IL-1β activation, which is NLRP3, ASC, and caspase-1 dependent. Suppression of pTau or ASC reduces tau pathology and inflammasome activation in rTg4510 and hTau mice, respectively. Although the deletion of MyD88 prevents both IL-1β expression and activation in the hTau mouse model of tauopathy, ASC deficiency in myeloid cells reduces pTau-induced IL-1β activation and improves cognitive function in hTau mice. Finally, pTau burden co-exists with elevated IL-1β and ASC in autopsy brains of human tauopathies. Together, our results suggest pTau activates IL-1β via MyD88- and NLRP3-ASC-dependent pathways in myeloid cells, including microglia.
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http://dx.doi.org/10.1016/j.celrep.2021.109720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8491766PMC
September 2021

The Specific NLRP3 Antagonist IFM-514 Decreases Fibrosis and Inflammation in Experimental Murine Non-Alcoholic Steatohepatitis.

Front Mol Biosci 2021 13;8:715765. Epub 2021 Aug 13.

Translational Hepatology, Department of Internal Medicine I, Universitätsklinikum/ Goethe-Universität, Frankfurt, Germany.

Activation of the inflammasome NLRP3 (NOD-, LRR- and pyrin domain containing 3) contributes to the development of non-alcoholic fatty liver disease (NAFLD) and progression to non-alcoholic steatohepatitis (NASH). Therefore, this study explored the therapeutic effects of a novel and selective NLRP3 antagonist in a murine dietary model of NASH. Groups of 12-week-old mice were fed ad lib for 7 weeks with a methionine/choline deficient (MCD) and western diet (WD). After 3 weeks of diet-induced injury, mice were injected i. p. with the NLRP3 antagonist IFM-514 (100 mg/kg body weight) or vehicle (0.5% carmellose) every day, 5 days/week for a further 4 weeks. Several markers of inflammation, fibrosis and steatosis were evaluated. Whole transcriptome sequencing and panel RNA expression analysis (NanoString) were performed. IFM-514 inhibited IL-1 production in mice challenged with 20 mg/kg lipopolysaccharide, and in mouse and human inflammatory cells . IFM-514 inhibited hepatic inflammation in the non-alcoholic steatohepatitis model assessed by H&E staining and in the hepatic gene expression of inflammasome-related proinflammatory cytokines. This effect was associated with significant reduction in caspase-1 activation. Similarly, IFM-514 was efficacious in MDC-fed mice, markedly reducing portal pressure, Sirius red staining and 4-hydroxyproline content compared to vehicle-treated mice. Moreover, IFM-514 significantly reduced hepatic steatosis in MCD-fed mice, as evidenced by NAFLD scores, oil red O staining, hepatic triglycerides and gene expression. In WD treated animals, similar trends in inflammation and fibrosis were observed, although not sufficient IFM-514 levels were reached. Overall, IFM-514 reduced liver inflammation and fibrosis, with mild effects on liver steatosis in experimental murine NASH. Blocking of NLRP3 may be an attractive therapeutic approach for NASH patients.
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http://dx.doi.org/10.3389/fmolb.2021.715765DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8425476PMC
August 2021

Microglial NLRP3 Inflammasome Activation upon TLR2 and TLR5 Ligation by Distinct α-Synuclein Assemblies.

J Immunol 2021 Oct 10;207(8):2143-2154. Epub 2021 Sep 10.

Department of Neurodegenerative Disease and Gerontopsychiatry/Neurology, University of Bonn Medical Center, Bonn, Germany;

Parkinson's disease (PD) is the second most common age-related neurodegenerative disorder and is characterized by the formation of cellular inclusions inside neurons that are rich in an abnormal form of the protein α-synuclein (α-syn). Microglia are the CNS resident immune cells that react to misfolded proteins through pattern recognition receptor ligation and activation of signaling transduction pathways. Here, we studied activation of primary microglia isolated from wild-type mouse by distinct α-syn forms and their clearance. Internalization of α-syn monomers and oligomers efficiently activated the NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome via TLR2 and TLR5 ligation, thereby acting on different signaling checkpoints. We found that primary microglia effectively engulf α-syn but hesitate in its degradation. NLRP3 inhibition by the selective inhibitor CRID3 sodium salt and NLRP3 deficiency improved the overall clearance of α-syn oligomers. Together, these data show that distinct α-syn forms exert different microglial NLRP3 inflammasome activation properties, thereby compromising its degradation, which can be prevented by NLRP3 inhibition.
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http://dx.doi.org/10.4049/jimmunol.2100035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8490941PMC
October 2021

IL-18 (Interleukin-18) Produced by Renal Tubular Epithelial Cells Promotes Renal Inflammation and Injury During Deoxycorticosterone/Salt-Induced Hypertension in Mice.

Hypertension 2021 Nov 7;78(5):1296-1309. Epub 2021 Sep 7.

Centre for Cardiovascular Biology and Disease Research and Department of Physiology, Anatomy and Microbiology, School of Life Sciences, La Trobe University, Bundoora, Australia (J.M.T., B.M.H., M.J., P.S., N.S., H.D., T.V.A., C.G.S., A.V., G.R.D.).

[Figure: see text].
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.120.16437DOI Listing
November 2021

CAD increases the long noncoding RNA in small extracellular vesicles and regulates endothelial cell function via vesicular shuttling.

Mol Ther Nucleic Acids 2021 Sep 4;25:388-405. Epub 2021 Jun 4.

Heart Center Bonn, Molecular Cardiology, Department of Internal Medicine II, University Hospital Bonn, Rheinische Friedrich-Wilhelms University of Bonn Venusberg-Campus 1, 53127 Bonn, Germany.

Long noncoding RNAs (lncRNAs) have emerged as biomarkers and regulators of cardiovascular disease. However, the expression pattern of circulating extracellular vesicle (EV)-incorporated lncRNAs in patients with coronary artery disease (CAD) is still poorly investigated. A human lncRNA array revealed that certain EV-lncRNAs are significantly dysregulated in CAD patients. Circulating small EVs (sEVs) from patients with (n = 30) or without (n = 30) CAD were used to quantify (also known as antisense RNA 1 []), , , and RNA levels. (p = 0.002) and (p = 0.02) were significantly increased in patients with CAD, compared to non-CAD patients. Fluorescent labeling and quantitative real-time PCR of sEVs demonstrated that functional was transported into the recipient cells. Mechanistically, the RNA-binding protein, heterogeneous nuclear ribonucleoprotein K (hnRNPK), interacts with , regulating its loading into sEVs. Knockdown of abrogated the EV-mediated effects on endothelial cell (EC) migration, proliferation, tube formation, and sprouting. Angiogenesis-related gene profiling showed that the expression of vascular endothelial growth factor A () RNA was significantly increased in EV recipient cells. Protein stability and RNA immunoprecipitation indicated that the -hnRNPK axis regulates the stability and binding of mRNA to hnRNPK. Loss of in EVs abolished the EV-mediated promotion of VEGFA gene and protein expression. Intercellular transfer of EV-incorporated promotes a pro-angiogenic phenotype via a VEGFA-dependent mechanism.
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http://dx.doi.org/10.1016/j.omtn.2021.05.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8403722PMC
September 2021

Inflammasome-induced extracellular vesicles harbour distinct RNA signatures and alter bystander macrophage responses.

J Extracell Vesicles 2021 Aug 2;10(10):e12127. Epub 2021 Aug 2.

Institute of Innate Immunity University Hospital University of Bonn Bonn Germany.

Infectious organisms and damage of cells can activate inflammasomes, which mediate tissue inflammation and adaptive immunity. These mechanisms evolved to curb the spread of microbes and to induce repair of the damaged tissue. Chronic activation of inflammasomes, however, contributes to non-resolving inflammatory responses that lead to immuno-pathologies. Inflammasome-activated cells undergo an inflammatory cell death associated with the release of potent pro-inflammatory cytokines and poorly characterized extracellular vesicles (EVs). Since inflammasome-induced EVs could signal inflammasome pathway activation in patients with chronic inflammation and modulate bystander cell activation, we performed a systems analysis of the ribonucleic acid (RNA) content and function of two EV classes. We show that EVs released from inflammasome-activated macrophages carry a specific RNA signature and contain interferon β (IFNβ). EV-associated IFNβ induces an interferon signature in bystander cells and results in dampening of NLRP3 inflammasome responses. EVs could, therefore, serve as biomarkers for inflammasome activation and act to prevent systemic hyper-inflammatory states by restricting NLRP3 activation in bystander cells.
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http://dx.doi.org/10.1002/jev2.12127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8329986PMC
August 2021

Topical inflammasome inhibition with disulfiram prevents irritant contact dermatitis.

Clin Transl Allergy 2021 Jul 22;11(5):e12045. Epub 2021 Jul 22.

Dermatological Allergology, Allergie-Centrum-Charité, Department of Dermatology, Venereology and Allergology Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health Berlin Germany.

Background: The pathogenesis of contact dermatitis, a common inflammatory skin disease with limited treatment options, is held to be driven by inflammasome activation induced by allergens and irritants. We here aim to identify inflammasome-targeting treatment strategies for irritant contact dermatitis.

Methods: A high content screen with 41,184 small molecules was performed using fluorescent Apoptosis associated speck-like protein containing a CARD (ASC) speck formation as a readout for inflammasome activation. Hit compounds were validated for inhibition of interleukin (IL)-1β secretion. Of these, the approved thiuramdisulfide derivative disulfiram was selected and tested in a patch test model of irritant contact dermatitis in 25 healthy volunteers. Topical application of disulfiram, mometasone or vehicle was followed by application of sodiumdodecylsulfate (SDS) for 24 h each. Eczema induction was quantified by mexameter and laser speckle imaging. Corneocyte sampling of lesional skin was performed to assess inflammasome-mediated cytokines IL-1β and IL-18.

Results: Disulfiram induced a dose-dependent inhibition of ASC speck formation and IL-1β release in cellular assays in vitro. In vivo, treatment with disulfiram, but not with vehicle and less mometasone, inhibited SDS-induced eczema. This was demonstrated by significantly lower erythema and total perfusion values assessed by mexameter and laser speckle imaging for disulfiram compared to vehicle ( < 0.001) and/or mometasone ( < 0.001). Also, corneocyte IL-18 levels were significantly reduced after application of disulfiram compared to vehicle ( < 0.001).

Conclusion: We show that disulfiram is a dose-dependent inhibitor of inflammasome pathway activation in vitro and inhibitor of SDS-induced eczema in vivo. Topical application of disulfiram represents a potential treatment option for irritant contact dermatitis.
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http://dx.doi.org/10.1002/clt2.12045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8297992PMC
July 2021

Regulation of ubiquitin and ubiquitin-like modifiers by phosphorylation.

FEBS J 2021 Jul 2. Epub 2021 Jul 2.

Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN, USA.

The regulatory influence of ubiquitin is vast, encompassing all cellular processes, by virtue of its central roles in protein degradation, membrane trafficking, and cell signaling. But how does ubiquitin, a 76 amino acid peptide, carry out such diverse, complex functions in eukaryotic cells? Part of the answer is rooted in the high degree of complexity associated with ubiquitin polymers, which can be 'read' and processed differently depending on topology and cellular context. However, recent evidence indicates that post-translational modifications on ubiquitin itself enhance the complexity of the ubiquitin code. Here, we review recent discoveries related to the regulation of the ubiquitin code by phosphorylation. We summarize what is currently known about phosphorylation of ubiquitin at Ser65, Ser57, and Thr12, and we discuss the potential for phosphoregulation of ubiquitin at other sites. We also discuss accumulating evidence that ubiquitin-like modifiers, such as SUMO, are likewise regulated by phosphorylation. A complete understanding of these regulatory codes and their complex lexicon will require dissection of mechanisms that govern phosphorylation of ubiquitin and ubiquitin-like proteins, particularly in the context of cellular stress and disease.
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http://dx.doi.org/10.1111/febs.16101DOI Listing
July 2021

Contact-dependent inhibition of HIV-1 replication in human tonsil cultures by polymorphonuclear neutrophils.

Cell Rep Med 2021 Jun 15;2(6):100317. Epub 2021 Jun 15.

Department of Infectious Diseases, Integrative Virology, University Hospital Heidelberg, 69120 Heidelberg, Germany.

Polymorphonuclear neutrophils (PMNs), the most abundant white blood cells, are recruited rapidly to sites of infection to exert potent anti-microbial activity. Information regarding their role in infection with human immunodeficiency virus (HIV) is limited. Here we report that addition of PMNs to HIV-infected cultures of human tonsil tissue or peripheral blood mononuclear cells causes immediate and long-lasting suppression of HIV-1 spread and virus-induced depletion of CD4 T cells. This inhibition of HIV-1 spread strictly requires PMN contact with infected cells and is not mediated by soluble factors. 2-Photon (2PM) imaging visualized contacts of PMNs with HIV-1-infected CD4 T cells in tonsil tissue that do not result in lysis or uptake of infected cells. The anti-HIV activity of PMNs also does not involve degranulation, formation of neutrophil extracellular traps, or integrin-dependent cell communication. These results reveal that PMNs efficiently blunt HIV-1 replication in primary target cells and tissue by an unconventional mechanism.
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http://dx.doi.org/10.1016/j.xcrm.2021.100317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8233696PMC
June 2021

Skewed endosomal RNA responses from TLR7 to TLR3 in RNase T2-deficient macrophages.

Int Immunol 2021 08;33(9):479-490

Division of Innate Immunity, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

RNase T2, a ubiquitously expressed RNase, degrades RNAs in the endosomal compartments. RNA sensors, double-stranded RNA (dsRNA)-sensing Toll-like receptor 3 (TLR3) and single-stranded RNA (ssRNA)-sensing TLR7, are localized in the endosomal compartment in mouse macrophages. We here studied the role of RNase T2 in TLR3 and TLR7 responses in macrophages. Macrophages expressed RNase T2 and a member of the RNase A family RNase 4. RNase T2 was also expressed in plasmacytoid and conventional dendritic cells. Treatment with dsRNAs or type I interferon (IFN) up-regulated expression of RNase T2 but not RNase 4. RNase T2-deficiency in macrophages up-regulated TLR3 responses but impaired TLR7 responses. Mechanistically, RNase T2 degraded both dsRNAs and ssRNAs in vitro, and its mutants showed a positive correlation between RNA degradation and the rescue of altered TLR3 and TLR7 responses. H122A and C188R RNase T2 mutations, not H69A and E118V mutations, impaired both RNA degradation and the rescue of altered TLR3 and TLR7 responses. RNase T2 in bone marrow-derived macrophages was broadly distributed from early endosomes to lysosomes, and colocalized with the internalized TLR3 ligand poly(I:C). These results suggest that RNase T2-dependent RNA degradation in endosomes/lysosomes negatively and positively regulates TLR3 and TLR7 responses, respectively, in macrophages.
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http://dx.doi.org/10.1093/intimm/dxab033DOI Listing
August 2021

Development of Fluorescent and Biotin Probes Targeting NLRP3.

Front Chem 2021 22;9:642273. Epub 2021 Apr 22.

Pharmaceutical Institute, University of Bonn, Bonn, Germany.

Extracellular signals drive the nucleation of the NLRP3 inflammasome which leads to the release of cytokines and causes inflammatory events. Hence, the inflammasome has gained enormous momentum in biomedical basic research. The detailed mechanisms of inflammasome generation and regulation remain to be elucidated. Our study was directed toward the design, convergent synthesis, and initial biochemical evaluation of activity-based probes addressing NLRP3. For this purpose, probes were assembled from a CRID3/MCC950-related NLRP3-binding unit, a linker portion and a coumarin 343 fluorophore or biotin. The affinity of our probes to NLRP3 was demonstrated through SPR measurements and their cellular activity was confirmed by reduction of the interleukin 1β release from stimulated bone marrow-derived macrophages. The initial characterizations of NLRP3-targeting probes highlighted the coumarin probe as a suitable tool compound for the cellular and biochemical analysis of the NLRP3 inflammasome.
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http://dx.doi.org/10.3389/fchem.2021.642273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8115122PMC
April 2021

Non-linear optical imaging of atherosclerotic plaques in the context of SIV and HIV infection prominently detects crystalline cholesterol esters.

PLoS One 2021 13;16(5):e0251599. Epub 2021 May 13.

Lipid Metabolism, Center for Computational and Integrative Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.

Chronic HIV infection may exacerbate atherosclerotic vascular disease, which at advanced stages presents as necrotic plaques rich in crystalline cholesterol. Such lesions can catastrophically rupture precipitating myocardial infarct and stroke, now important causes of mortality in those living with HIV. However, in this population little is known about plaque structure relative to crystalline content and its chemical composition. Here, we first interrogated plaque crystal structure and composition in atherosclerotic SIV-infected macaques using non-linear optical microscopy. By stimulated Raman scattering and second harmonic generation approaches both amorphous and crystalline plaque lipid was detected and the crystal spectral profile indicated a cholesterol ester (CE) dominated composition. Versus controls, SIV+ samples had a greater number of cholesterol crystals (CCs), with the difference, in part, accounted for by crystals of a smaller length. Given the ester finding, we profiled HIV+ plaques and also observed a CE crystalline spectral signature. We further profiled plaques from Ldlr-/- mice fed a high fat diet, and likewise, found CE-dominate crystals. Finally, macrophage exposure to CCs or AcLDL induced auto-fluorescent puncta that co-stained with the LC3B autophagy sensor. In aggregate, we show that atheromatous plaques from mice, macaques and humans, display necrotic cores dominated by esterified CCs, and that plaque macrophages may induce autophagic vesicle formation upon encountering CCs. These findings help inform our knowledge of plaque core lipid evolution and how the process may incite systemic inflammation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0251599PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8118308PMC
October 2021

Cytosolic d-type CpG-oligonucleotides induce a type I interferon response by activating the cGAS-STING signaling pathway.

Eur J Immunol 2021 07 12;51(7):1686-1697. Epub 2021 May 12.

Department of Anesthesiology and Intensive Care Medicine, University Hospital Bonn, Bonn, Germany.

Cytosolic DNA receptor cyclic GMP-AMP (cGAMP) synthase (cGAS) has been shown to be critically involved in the detection of cytosolic, self- and non-self-DNA, initiating a type I IFN response through the adaptor protein Stimulator of Interferon Genes (STING) and interferon regulatory factor 3 (IRF3). Current studies propose that canonical binding of dsDNA by cGAS depends on DNA length, but not on base sequence. In contrast, activation of TLR9 is sequence dependent. It requires unmethylated CpG dinucleotides in microbial DNA, which is mimicked by synthetic oligodeoxynucleotides (ODN). Here, we provide evidence that d-type ODN (D-ODN), but not K-type ODN (K-ODN), bind to human cGAS and activate downstream signaling. Transfection of D-ODN into a TLR9-deficient, human monocytic cell line (THP-1) induced phosphorylation of IRF3 and secretion of IFN. This response was absent in cells with CRISPR/Cas9-mediated cGAS- or STING-deficiency. Utilizing a protein pulldown approach, we further demonstrate direct binding of D-ODN to cGAS. Induction of a type I IFN response by D-ODN was confirmed in human primary monocytes and monocyte-derived macrophages. These results are relevant to our understanding of self-nonself-discrimination by cGAS and to the pharmacologic effects of ODN, which currently are investigated in clinical studies.
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http://dx.doi.org/10.1002/eji.202048810DOI Listing
July 2021

Necroptosis, pyroptosis and apoptosis: an intricate game of cell death.

Cell Mol Immunol 2021 05 30;18(5):1106-1121. Epub 2021 Mar 30.

Institute of Innate Immunity, University Hospitals Bonn, University of Bonn, Bonn, NRW, Germany.

Cell death is a fundamental physiological process in all living organisms. Its roles extend from embryonic development, organ maintenance, and aging to the coordination of immune responses and autoimmunity. In recent years, our understanding of the mechanisms orchestrating cellular death and its consequences on immunity and homeostasis has increased substantially. Different modalities of what has become known as 'programmed cell death' have been described, and some key players in these processes have been identified. We have learned more about the intricacies that fine tune the activity of common players and ultimately shape the different types of cell death. These studies have highlighted the complex mechanisms tipping the balance between different cell fates. Here, we summarize the latest discoveries in the three most well understood modalities of cell death, namely, apoptosis, necroptosis, and pyroptosis, highlighting common and unique pathways and their effect on the surrounding cells and the organism as a whole.
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http://dx.doi.org/10.1038/s41423-020-00630-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8008022PMC
May 2021

N-protein presents early in blood, dried blood and saliva during asymptomatic and symptomatic SARS-CoV-2 infection.

Nat Commun 2021 03 26;12(1):1931. Epub 2021 Mar 26.

Quanterix Corporation, Billerica, MA, USA.

The COVID-19 pandemic continues to have an unprecedented impact on societies and economies worldwide. There remains an ongoing need for high-performance SARS-CoV-2 tests which may be broadly deployed for infection monitoring. Here we report a highly sensitive single molecule array (Simoa) immunoassay in development for detection of SARS-CoV-2 nucleocapsid protein (N-protein) in venous and capillary blood and saliva. In all matrices in the studies conducted to date we observe >98% negative percent agreement and >90% positive percent agreement with molecular testing for days 1-7 in symptomatic, asymptomatic, and pre-symptomatic PCR+ individuals. N-protein load decreases as anti-SARS-CoV-2 spike-IgG increases, and N-protein levels correlate with RT-PCR Ct-values in saliva, and between matched saliva and capillary blood samples. This Simoa SARS-CoV-2 N-protein assay effectively detects SARS-CoV-2 infection via measurement of antigen levels in blood or saliva, using non-invasive, swab-independent collection methods, offering potential for at home and point of care sample collection.
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http://dx.doi.org/10.1038/s41467-021-22072-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7997897PMC
March 2021

Inhibition of Caspase-1 with Tetracycline Ameliorates Acute Lung Injury.

Am J Respir Crit Care Med 2021 07;204(1):53-63

Department of Anesthesiology and Intensive Care Medicine.

Acute respiratory distress syndrome (ARDS) is a heterogeneous syndrome with a mortality of up to 40%. Precision medicine approaches targeting patients on the basis of their molecular phenotypes of ARDS might help to identify effective pharmacotherapies. The inflammasome-caspase-1 pathway contributes to the development of ARDS via IL-1β and IL-18 production. Recent studies indicate that tetracycline can be used to treat inflammatory diseases mediated by IL-1β and IL-18, although the molecular mechanism by which tetracycline inhibits inflammasome-caspase-1 signaling remains unknown. To identify patients with ARDS characterized by IL-1β and IL-18 expression and investigate the ability of tetracycline to inhibit inflammasome-caspase-1 signaling in ARDS. IL-1β and IL-18 concentrations were quantified in BAL fluid from patients with ARDS. Tetracycline's effects on lung injury and inflammation were assessed in two mouse models of direct (pulmonary) acute lung injury, and its effects on IL-1β and IL-18 production were assessed by alveolar leukocytes from patients with direct ARDS . Murine macrophages were used to further characterize the effect of tetracycline on the inflammasome-caspase-1 pathway. BAL fluid concentrations of IL-1β and IL-18 are significantly higher in patients with direct ARDS than those with indirect (nonpulmonary) ARDS. In experimental acute lung injury, tetracycline significantly diminished lung injury and pulmonary inflammation by selectively inhibiting caspase-1-dependent IL-1β and IL-18 production, leading to improved survival. Tetracycline also reduced the production of IL-1β and IL-18 by alveolar leukocytes from patients with direct ARDS. Tetracycline may be effective in the treatment of direct ARDS in patients with elevated caspase-1 activity. Clinical Trial registered with www.clinicaltrials.gov (NCT04079426).
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http://dx.doi.org/10.1164/rccm.202005-1916OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8437127PMC
July 2021

Interaction of TLR4 and TLR8 in the Innate Immune Response against Mycobacterium Tuberculosis.

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

Department of Internal Medicine III, Division of Infectious Diseases, University Hospital of Ulm, 89081 Ulm, Germany.

The interaction and crosstalk of Toll-like receptors (TLRs) is an established pathway in which the innate immune system recognises and fights pathogens. In a single nucleotide polymorphisms (SNP) analysis of an Indian cohort, we found evidence for both TLR4-399T and TRL8-1A conveying increased susceptibility towards tuberculosis (TB) in an interdependent manner, even though there is no established TLR4 ligand present in (Mtb), which is the causative pathogen of TB. Docking studies revealed that TLR4 and TLR8 can build a heterodimer, allowing interaction with TLR8 ligands. The conformational change of TLR4-399T might impair this interaction. With immunoprecipitation and mass spectrometry, we precipitated TLR4 with TLR8-targeted antibodies, indicating heterodimerisation. Confocal microscopy confirmed a high co-localisation frequency of TLR4 and TLR8 that further increased upon TLR8 stimulation. The heterodimerisation of TLR4 and TLR8 led to an induction of IL12p40, NF-κB, and IRF3. TLR4-399T in interaction with TLR8 induced an increased NF-κB response as compared to TLR4-399C, which was potentially caused by an alteration of subsequent immunological pathways involving type I IFNs. In summary, we present evidence that the heterodimerisation of TLR4 and TLR8 at the endosome is involved in Mtb recognition via TLR8 ligands, such as microbial RNA, which induces a Th1 response. These findings may lead to novel targets for therapeutic interventions and vaccine development regarding TB.
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http://dx.doi.org/10.3390/ijms22041560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7913854PMC
February 2021

Sensing soluble uric acid by Naip1-Nlrp3 platform.

Cell Death Dis 2021 02 5;12(2):158. Epub 2021 Feb 5.

Department of Immunology, Institute of Biomedical Sciences IV, University of São Paulo, São Paulo, SP, Brazil.

Uric acid (UA), a product of purine nucleotide degradation able to initiate an immune response, represents a breakpoint in the evolutionary history of humans, when uricase, the enzyme required for UA cleavage, was lost. Despite being inert in human cells, UA in its soluble form (sUA) can increase the level of interleukin-1β (IL-1β) in murine macrophages. We, therefore, hypothesized that the recognition of sUA is achieved by the Naip1-Nlrp3 inflammasome platform. Through structural modelling predictions and transcriptome and functional analyses, we found that murine Naip1 expression in human macrophages induces IL-1β expression, fatty acid production and an inflammation-related response upon sUA stimulation, a process reversed by the pharmacological and genetic inhibition of Nlrp3. Moreover, molecular interaction experiments showed that Naip1 directly recognizes sUA. Accordingly, Naip may be the sUA receptor lost through the human evolutionary process, and a better understanding of its recognition may lead to novel anti-hyperuricaemia therapies.
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http://dx.doi.org/10.1038/s41419-021-03445-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864962PMC
February 2021

Macrophage inflammatory state in Type 1 diabetes: triggered by NLRP3/iNOS pathway and attenuated by docosahexaenoic acid.

Clin Sci (Lond) 2021 01;135(1):19-34

Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Sao Paulo, Brazil.

Type 1 diabetes mellitus (T1D) is a chronic autoimmune disease characterized by insulin-producing pancreatic β-cell destruction and hyperglycemia. While monocytes and NOD-like receptor family-pyrin domain containing 3 (NLRP3) are associated with T1D onset and development, the specific receptors and factors involved in NLRP3 inflammasome activation remain unknown. Herein, we evaluated the inflammatory state of resident peritoneal macrophages (PMs) from genetically modified non-obese diabetic (NOD), NLRP3-KO, wild-type (WT) mice and in peripheral blood mononuclear cells (PBMCs) from human T1D patients. We also assessed the effect of docosahexaenoic acid (DHA) on the inflammatory status. Macrophages from STZ-induced T1D mice exhibited increased inflammatory cytokine/chemokine levels, nitric oxide (NO) secretion, NLRP3 and iNOS protein levels, and augmented glycolytic activity compared to control animals. In PMs from NOD and STZ-induced T1D mice, DHA reduced NO production and attenuated the inflammatory state. Furthermore, iNOS and IL-1β protein expression levels and NO production were lower in the PMs from diabetic NLRP3-KO mice than from WT mice. We also observed increased IL-1β secretion in PBMCs from T1D patients and immortalized murine macrophages treated with advanced glycation end products and palmitic acid. The present study demonstrated that the resident PMs are in a proinflammatory state characterized by increased NLRP3/iNOS pathway-mediated NO production, up-regulated proinflammatory cytokine/chemokine receptor expression and altered glycolytic activity. Notably, ex vivo treatment with DHA reverted the diabetes-induced changes and attenuated the macrophage inflammatory state. It is plausible that DHA supplementation could be employed as adjuvant therapy for treating individuals with T1D.
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http://dx.doi.org/10.1042/CS20201348DOI Listing
January 2021

Intestinal Dysbiosis Amplifies Acetaminophen-Induced Acute Liver Injury.

Cell Mol Gastroenterol Hepatol 2021 12;11(4):909-933. Epub 2020 Nov 12.

Department of Medicine III, University Hospital RWTH Aachen, Aachen, Germany. Electronic address:

Background & Aims: Acute liver failure (ALF) represents an unmet medical need in Western countries. Although the link between intestinal dysbiosis and chronic liver disease is well-established, there is little evidence for a functional role of gut-liver interaction during ALF. Here we hypothesized that intestinal dysbiosis may affect ALF.

Methods: To test this hypothesis, we assessed the association of proton pump inhibitor (PPI) or long-term antibiotics (ABx) intake, which have both been linked to intestinal dysbiosis, and occurrence of ALF in the 500,000 participants of the UK BioBank population-based cohort. For functional studies, male Nlrp6 mice were used as a dysbiotic mouse model and injected with a sublethal dose of acetaminophen (APAP) or lipopolysaccharide (LPS) to induce ALF.

Results: Multivariate Cox regression analyses revealed a significantly increased risk (odds ratio, 2.3-3) for developing ALF in UK BioBank participants with PPI or ABx. Similarly, dysbiotic Nlrp6 mice displayed exacerbated APAP- and LPS-induced liver injury, which was linked to significantly reduced gut and liver tissue microbiota diversity and correlated with increased intestinal permeability at baseline. Fecal microbiota transfer (FMT) from Nlrp6 mice into wild-type (WT) mice augmented liver injury on APAP treatment in recipient WT mice, resembling the inflammatory phenotype of Nlrp6 mice. Specifically, FMT skewed monocyte polarization in WT mice toward a Ly6C inflammatory phenotype, suggesting a critical function of these cells as sensors of gut-derived signals orchestrating the inflammatory response.

Conclusions: Our data show an important yet unknown function of intestinal microbiota during ALF. Intestinal dysbiosis was transferrable to healthy WT mice via FMT and aggravated liver injury. Our study highlights intestinal microbiota as a targetable risk factor for ALF.
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http://dx.doi.org/10.1016/j.jcmgh.2020.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900526PMC
November 2020

The RNA-binding protein hnRNPU regulates the sorting of microRNA-30c-5p into large extracellular vesicles.

J Extracell Vesicles 2020 Jul 2;9(1):1786967. Epub 2020 Jul 2.

Heart Center Bonn, Medical Department II, University Hospital Bonn, Venusberg-Campus 1, Bonn, Germany.

The transfer of microRNAs (miRs) via extracellular vesicles (EVs) is a functionally relevant mechanism of intercellular communication that regulates both organ homoeostasis and disease development. Little is known about the packaging of miRs into EVs. Previous studies have shown that certain miRs are exported by RNA-binding proteins into small EVs, while for other miRs and for large EVs, in general, the export mechanisms remain unclear. Therefore, a proteomic analysis of endothelial cell-derived large EVs was performed, which revealed that heterogeneous nuclear ribonucleoprotein U (hnRNPU) is abundantly present in EVs. EVs were characterized by electron microscopy, immunoblotting and nanoparticle tracking analysis. Taqman microRNA array and single qPCR experiments identified specific miR patterns to be exported into EVs in an hnRNPU-dependent way. The specific role of hnRNPU for vesicular miR-sorting was confirmed independently by gain- and loss-of-function experiments. In our study, miR-30c-5p was the miR whose export was most significantly regulated by hnRNPU. Mechanistically, binding analysis showed that the export of miRs into EVs depends on the binding efficiency of the respective miRs to hnRNPU. Among the exported miRs, a significant enrichment of the sequence motif AAMRUGCU was detected as a potential sorting signal. Experimentally, binding of miR-30c-5p to hnRNPU was confirmed independently by RNA-immunoprecipitation, electrophoretic mobility shift assay and reciprocally by miR-pulldown. Nuclear binding of miR-30c-5p to hnRNPU and subsequent stabilization was associated with a lower cytoplasmatic abundance and consequently reduced availability for vesicular export. hnRNPU-dependent miR-30c-5p export reduced cellular migration as well as pro-angiogenic gene expression in EV-recipient cells. In summary, hnRNPU retains miR-30c-5p and other miRs and thereby prevents their export into large EVs. The data presented provide a novel and functionally relevant mechanism of vesicular miR export.
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http://dx.doi.org/10.1080/20013078.2020.1786967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7480565PMC
July 2020
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