SwePub - sökning: WFRF:(Gekara Nelson O.)

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1.	Bavdek, Andrej, et al. (författare) Sterol and pH interdependence in the binding, oligomerization, and pore formation of Listeriolysin O. 2007 Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 46:14, s. 4425-4437 Tidskriftsartikel (refereegranskat)abstract Listeriolysin O (LLO) is the most important virulence factor of the intracellular pathogen Listeria monocytogenes. Its main task is to enable escape of bacteria from the phagosomal vacuole into the cytoplasm. LLO belongs to the cholesterol-dependent cytolysin (CDC) family but differs from other members, as it exhibits optimal activity at low pH. Its pore forming ability at higher pH values has been largely disregarded in Listeria pathogenesis. Here we show that high cholesterol concentrations in the membrane restore the low activity of LLO at high pH values. LLO binds to lipid membranes, at physiological or even slightly basic pH values, in a cholesterol-dependent fashion. Binding, insertion into lipid monolayers, and permeabilization of calcein-loaded liposomes are maximal above approximately 35 mol % cholesterol, a concentration range typically found in lipid rafts. The narrow transition region of cholesterol concentration separating low and high activity indicates that cholesterol not only allows the binding of LLO to membranes but also affects other steps in pore formation. We were able to detect some of these by surface plasmon resonance-based assays. In particular, we show that LLO recognition of cholesterol is determined by the most exposed 3beta-hydroxy group of cholesterol. In addition, LLO binds and permeabilizes J774 cells and human erythrocytes in a cholesterol-dependent fashion at physiological or slightly basic pH values. The results clearly show that LLO activity at physiological pH cannot be neglected and that its action at sites distal to cell entry may have important physiological consequences for Listeria pathogenesis.
2.	Dietrich, Nicole, et al. (författare) Mast cells elicit proinflammatory but not type I interferon responses upon activation of TLRs by bacteria. 2010 Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 107:19, s. 8748-8753 Tidskriftsartikel (refereegranskat)abstract Balanced induction of proinflammatory and type I IFN responses upon activation of Toll-like receptors (TLRs) determines the outcome of microbial infections and the pathogenesis of autoimmune and other inflammatory diseases. Mast cells, key components of the innate immune system, are known for their debilitating role in allergy and autoimmunity. However, their role in antimicrobial host defenses is being acknowledged increasingly. How mast cells interact with microbes and the nature of responses triggered thereby is not well characterized. Here we show that in response to TLR activation by Gram-positive and -negative bacteria or their components, mast cells elicit proinflammatory but not type I IFN responses. We demonstrate that in mast cells, bound bacteria and TLR ligands remain trapped at the cell surface and do not undergo internalization, a prerequisite for type I IFN induction. Such cells, however, can elicit type I IFNs in response to vesicular stomatitis virus which accesses the cytosolic retinoic acid-inducible gene I receptor. Although important for antiviral immunity, a strong I IFN response is known to contribute to pathogenesis of several bacterial pathogens such as Listeria monocytogenes. Interestingly, we observed that the mast cell-dependent neutrophil mobilization upon L. monocytogenes infection is highly impaired by IFN-beta. Thus, the fact that mast cells, although endowed with the capacity to elicit type I IFNs in response to viral infection, elicit only proinflammatory responses upon bacterial infection shows that mast cells, key effector cells of the innate immune system, are well adjusted for optimal antibacterial and antiviral responses.
3.	Dietrich, Nicole, et al. (författare) Murine toll-like receptor 2 activation induces type I interferon responses from endolysosomal compartments. 2010 Ingår i: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 5:4, s. e10250- Tidskriftsartikel (refereegranskat)abstract The results indicate that TLR2 activation induces pro-inflammatory and type I interferon responses from distinct subcellular sites: the plasma membrane and endolysosomal compartments respectively. Apart from identifying and characterizing a novel pathway for induction of type I interferons, the present study offers new insights into how TLR signaling discriminates and regulates the nature of responses to be elicited against extracellular and endocytosed microbes. These findings may also have clinical implication. Excessive production of pro-inflammatory cytokines and type I IFNs following activation of TLRs is a central pathologic event in several hyper-inflammatory conditions. The discovery that the induction of pro-inflammatory and type I IFN responses can be uncoupled through pharmacological manipulation of endolysosomal acidification suggests new avenues for potential therapeutic intervention against inflammations and sepsis.
4.	Erttmann, Saskia F., et al. (författare) Bacteria Induce Prolonged PMN Survival via a Phosphatidylcholine-Specific Phospholipase C- and Protein Kinase C-Dependent Mechanism 2014 Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:1, s. e87859- Tidskriftsartikel (refereegranskat)abstract Polymorphonuclear leukocytes (PMNs) are essential for the human innate immune defense, limiting expansion of invading microorganisms. PMN turnover is controlled by apoptosis, but the regulating signaling pathways remain elusive, largely due to inherent differences between mice and humans that undermine use of mouse models for understanding human PMN biology. Here, we aim to elucidate signal transduction mediating survival of human peripheral blood PMNs in response to bacteria, such as Yersinia pseudotuberculosis, an enteropathogen that causes the gastro-intestinal disease yersiniosis, as well as Escherichia coli and Staphylococcus aureus. Determinations of cell death reveal that uninfected control cells undergo apoptosis, while PMNs infected with either Gram-positive or -negative bacteria show profoundly increased survival. Infected cells exhibit decreased caspase 3 and 8 activities, increased mitochondrial integrity and are resistant to apoptosis induced by a death receptor ligand. This bacteria-induced response is accompanied by pro-inflammatory cytokine production including interleukin-8 and tumor necrosis factor-a competent to attract additional PMNs. Using agonists and pharmacological inhibitors, we show participation of Toll-like receptor 2 and 4, and interestingly, that protein kinase C (PKC) and phosphatidylcholine-specific phospholipase C (PC-PLC), but not tyrosine kinases or phosphatidylinositol-specific phospholipase C (PI-PLC) are key players in this dual PMN response. Our findings indicate the importance of prolonged PMN survival in response to bacteria, where general signaling pathways ensure complete exploitation of PMN anti-microbial capacity.
5.	Erttmann, Saskia F., et al. (författare) Hydrogen peroxide release by bacteria suppresses inflammasome-dependent innate immunity 2019 Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 10 Tidskriftsartikel (refereegranskat)abstract Hydrogen peroxide (H2O2) has a major function in host-microbial interactions. Although most studies have focused on the endogenous H2O2 produced by immune cells to kill microbes, bacteria can also produce H2O2. How microbial H2O2 influences the dynamics of host-microbial interactions is unclear. Here we show that H2O2 released by Streptococcus pneumoniae inhibits inflammasomes, key components of the innate immune system, contributing to the pathogen colonization of the host. We also show that the oral commensal H2O2-producing bacteria Streptococcus oralis can block inflammasome activation. This study uncovers an unexpected role of H2O2 in immune suppression and demonstrates how, through this mechanism, bacteria might restrain the immune system to co-exist with the host.
6.	Erttmann, Saskia F., et al. (författare) Loss of the DNA Damage Repair Kinase ATM Impairs Inflammasome-Dependent Anti-Bacterial Innate Immunity 2016 Ingår i: Immunity. - : Elsevier BV. - 1074-7613 .- 1097-4180. ; 45:1, s. 106-118 Tidskriftsartikel (refereegranskat)abstract The ATM kinase is a central component of the DNA damage repair machinery and redox balance. ATM dysfunction results in the multisystem disease ataxia-telangiectasia (AT). A major cause of mortality in AT is respiratory bacterial infections. Whether ATM deficiency causes innate immune defects that might contribute to bacterial infections is not known. Here we have shown that loss of ATM impairs inflammasome- dependent anti-bacterial innate immunity. Cells from AT patients or Atm(-/-) mice exhibited diminished interleukin-1 beta (IL-1 beta) production in response to bacteria. In vivo, Atm(-/-) mice were more susceptible to pulmonary S. pneumoniae infection in a manner consistent with inflammasome defects. Our data indicate that such defects were due to oxidative inhibition of inflammasome complex assembly. This study reveals an unanticipated function of reactive oxygen species (ROS) in negative regulation of inflammasomes and proposes a theory for the notable susceptibility of AT patients to pulmonary bacterial infection.
7.	Erttmann, Saskia F., et al. (författare) Protocol for isolation of microbiota-derived membrane vesicles from mouse blood and colon 2023 Ingår i: STAR Protocols. - : CellPress. - 2666-1667. ; 4:1 Tidskriftsartikel (refereegranskat)abstract Bacterial membrane vesicles have emerged as gadgets allowing remote communication between the microbiota and distal host organs. Here we describe a protocol for enriching vesicles from serum and colon that could widely be adapted for other tissues. We detail pre-clearing of serum or colon fluids using 0.2-μm syringe filters and their concentration by centrifugal filter devices. We also describe vesicle isolation with qEV size exclusion columns and finally the concentration of isolated vesicle fractions for downstream analyses. For complete details on the use and execution of this protocol, please refer to Erttmann et al. (2022).1
8.	Erttmann, Saskia F., et al. (författare) The gut microbiota prime systemic antiviral immunity via the cGAS-STING-IFN-I axis 2022 Ingår i: Immunity. - : Elsevier BV. - 1074-7613 .- 1097-4180. ; 55:5, s. 847-861 Tidskriftsartikel (refereegranskat)abstract The microbiota are vital for immune homeostasis and provide a competitive barrier to bacterial and fungal pathogens. Here, we investigated how gut commensals modulate systemic immunity and response to viral infection. Antibiotic suppression of the gut microbiota reduced systemic tonic type I interferon (IFN-I) and antiviral priming. The microbiota-driven tonic IFN-I-response was dependent on cGAS-STING but not on TLR signaling or direct host-bacteria interactions. Instead, membrane vesicles (MVs) from extracellular bacteria activated the cGAS-STING-IFN-I axis by delivering bacterial DNA into distal host cells. DNA-containing MVs from the gut microbiota were found in circulation and promoted the clearance of both DNA (herpes simplex virus type 1) and RNA (vesicular stomatitis virus) viruses in a cGAS-dependent manner. In summary, this study establishes an important role for the microbiota in peripheral cGAS-STING activation, which promotes host resistance to systemic viral infections. Moreover, it uncovers an underappreciated risk of antibiotic use during viral infections.
9.	Gekara, Nelson O. (författare) DNA damage-induced immune response : Micronuclei provide key platform 2017 Ingår i: Journal of Cell Biology. - : ROCKEFELLER UNIV PRESS. - 0021-9525 .- 1540-8140. ; 216:10, s. 2999-3001 Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract DNA damage-induced activation of the cytoplasmic DNA sensor cGAS influences the outcome of infections, autoinflammation, and cancer. Recent studies by Harding et al. (2017. Nature. http://dx.doi.org/10.1038/nature23470), Mackenzie et al. (2017. Nature. http://dx.doi.org/10.1038/nature23449), and Bartsch et al. (2017. Human Molecular Genetics. https://doi.org/10.1093/hmg/ddx283) demonstrate a role for micronuclei formation in DNA damage-induced immune activation.
10.	Gekara, Nelson O, et al. (författare) Listeria monocytogenes desensitizes immune cells to subsequent Ca2+ signaling via listeriolysin O-induced depletion of intracellular Ca2+ stores. 2008 Ingår i: Infection and Immunity. - 0019-9567 .- 1098-5522. ; 76:2, s. 857-862 Tidskriftsartikel (refereegranskat)abstract Listeriolysin O (LLO), the pore-forming toxin of Listeria monocytogenes, is a prototype of the cholesterol-dependent cytolysins (CDCs) secreted by several pathogenic and nonpathogenic gram-positive bacteria. In addition to mediating the escape of the bacterium into the cytosol, this toxin is generally believed to be a central player in host-pathogen interactions during L. monocytogenes infection. LLO triggers the influx of Ca(2+) into host cells as well as the release of Ca(2+) from intracellular stores. Thus, many of the cellular responses induced by LLO are related to calcium signaling. Interestingly, in this study, we report that prolonged exposure to LLO desensitizes cells to Ca(2+) mobilization upon subsequent stimulations with LLO. Cells preexposed to LLO-positive L. monocytogenes but not to the LLO-deficient Deltahly mutant were found to be highly refractory to Ca(2+) induction in response to receptor-mediated stimulation. Such cells also exhibited diminished Ca(2+) signals in response to stimulation with LLO and thapsigargin. The presented results suggest that this phenomenon is due to the depletion of intracellular Ca(2+) stores. The ability of LLO to desensitize immune cells provides a significant hint about the possible role played by CDCs in the evasion of the immune system by bacterial pathogens.
11.	Gekara, Nelson O., et al. (författare) Listeria monocytogenes induces T cell receptor unresponsiveness through pore-forming toxin listeriolysin O 2010 Ingår i: Journal of Infectious Diseases. - : Oxford University Press. - 0022-1899 .- 1537-6613. ; 202:11, s. 1698-1707 Tidskriftsartikel (refereegranskat)abstract Background: The success of many pathogens relies on their ability to circumvent the innate and adaptive immune defenses. How bacterial pathogens subvert adaptive immune defenses is not clear. Cholesterol-dependent cytolysins (CDCs) represent an expansive family of homologous pore-forming toxins that are produced by more than 20 gram-positive bacterial species. Listeriolysin O (LLO), a prototype CDC, is the main virulence factor of Listeria monocytogenes.Methods: We employed flow cytometric and microarray techniques to analyze the effect of LLO on T cell activation in vitro and in vivo.Results: In vivo and in vitro proliferation of CD4+T cells upon T cell receptor (TCR) activation was highly diminished in the presence of LLO or wild-type L. monocytogenes but not in the presence of LLO-deficient L. monocytogenes. This block in T cell proliferation was specific to T cell activation via the TCR and not by phorbol 12-myristate 13-acetate-ionomycin, which bypasses the proximal TCR signaling event. The results of microarray analysis suggest that LLO-induced T cell unresponsiveness is due to the induction of a calcium-nuclear factor of activated T cells-dependent transcriptional program that drives the expression of negative regulators of TCR signaling.Conclusion: These findings provide important insights into how bacterial toxins silence adaptive immune responses and thus enable prolonged survival of the pathogen in the host.
12.	Gekara, Nelson O, et al. (författare) Mast cells initiate early anti-Listeria host defences. 2008 Ingår i: Cellular Microbiology. - : Hindawi Limited. - 1462-5814 .- 1462-5822. ; 10:1, s. 225-236 Tidskriftsartikel (refereegranskat)abstract The Gram-positive bacterium Listeria monocytogenes (L. m.) is the aetiological agent of listeriosis. The early phase listeriosis is characterized by strong innate host responses that play a major role in bacterial clearance. This is emphasized by the fact that mice deficient in T and B cells have a remarkable ability to control infection. Mast cells, among the principal effectors of innate immunity, have largely been studied in the context of hyper-reactive conditions such as allergy and autoimmune diseases. In the present study, we evaluated the significance of mast cells during the early phase of listeriosis. Compared with controls, mice depleted of mast cells showed hundred-fold higher bacterial burden in spleen and liver and were significantly impaired in neutrophil mobilization. Although L. m. interacts with and triggers mast cell degranulation, bacteria were hardly found within such cells. Mainly neutrophils and macrophages phagozytosed L. m. Thus, mast cells control infection not via direct bacterial uptake, but by initiating neutrophils influx to the site of infection. We show that this is initiated by pre-synthesized TNF-alpha, rapidly secreted by mast cell upon activation by L. m. We also show that upon recruitment, neutrophils also become activated and additionally secrete TNF-alpha thus amplifying the anti-L. m. inflammatory response.
13.	Gekara, Nelson O, et al. (författare) Signals triggered by a bacterial pore-forming toxin contribute to toll-like receptor redundancy in gram-positive bacterial recognition. 2009 Ingår i: Journal of Infectious Diseases. - : Oxford University Press (OUP). - 0022-1899 .- 1537-6613. ; 199:1, s. 124-33 Tidskriftsartikel (refereegranskat)abstract The results illustrate that signals triggered by LLO contribute to TLR2 redundancy in recognition of L. monocytogenes. Under normal conditions, multiple and, sometimes, redundant pathways cooperate to induce a rapid antimicrobial defense. When one signaling pathway-in this case, TLR2-is removed from the system, the other pathways are still capable of mounting a sufficient response to ensure survival of the host.
14.	Gekara, Nelson O, et al. (författare) The cholesterol-dependent cytolysin listeriolysin O aggregates rafts via oligomerization. 2005 Ingår i: Cellular Microbiology. - : Hindawi Limited. - 1462-5814 .- 1462-5822. ; 7:9, s. 1345-1356 Tidskriftsartikel (refereegranskat)abstract The pore-forming toxin listeriolysin O (LLO) is the main virulence factor of Listeria monocytogenes. LLO is known to act as a pseudo cytokine/chemokine, which induces a broad spectrum of host responses that ultimately influences the outcome of listeriosis. In the present study we demonstrate that LLO is a potent aggregator of lipid rafts. LLO was found to aggregate the raft associated molecules GM1, the GPI-anchored proteins CD14 and CD16 as well as the tyrosine kinase Lyn. Abrogation of the cytolytic activity of LLO by cholesterol pretreatment was found not to interfere with LLO's ability to aggregate rafts or trigger tyrosine phosphorylation in cells. However, a monoclonal antibody that blocks the oligomerization of LLO was found to inhibit rafts' aggregation as well as the induction of tyrosine phosphorylation. This implies that rafts aggregation by LLO which is independent of cytolytic activity, is due to the oligomerization of its membrane bound toxin monomers. Thus, LLO most likely induces signalling through the coaggregation of rafts' associated receptors, kinases and adaptors.
15.	Gekara, Nelson O., et al. (författare) The innate immune DNA sensor cGAS : A membrane, cytosolic, or nuclear protein? 2019 Ingår i: Science Signaling. - Washington : American Association for the Advancement of Science. - 1945-0877 .- 1937-9145. ; 12:581 Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract Cyclic cGMP-AMP synthase (cGAS) alerts the innate immune system to the presence of foreign or damaged self-DNA inside the cell and is critical for the outcome of infections, inflammatory diseases, and cancer. Two studies now demonstrate that cGAS activation is regulated by differential subcellular localization through its non-enzymatic, N-terminal domain.
16.	Gekara, Nelson O, et al. (författare) The multiple mechanisms of Ca2+ signalling by listeriolysin O, the cholesterol-dependent cytolysin of Listeria monocytogenes. 2007 Ingår i: Cellular Microbiology. - : Hindawi Limited. - 1462-5814 .- 1462-5822. ; 9:8, s. 2008-2021 Tidskriftsartikel (refereegranskat)abstract Cholesterol-dependent cytolysins (CDCs) represent a large family of conserved pore-forming toxins produced by several Gram-positive bacteria such as Listeria monocytogenes, Streptococcus pyrogenes and Bacillus anthracis. These toxins trigger a broad range of cellular responses that greatly influence pathogenesis. Using mast cells, we demonstrate that listeriolysin O (LLO), a prototype of CDCs produced by L. monocytogenes, triggers cellular responses such as degranulation and cytokine synthesis in a Ca(2+)-dependent manner. Ca(2+) signalling by LLO is due to Ca(2+) influx from extracellular milieu and release of from intracellular stores. We show that LLO-induced release of Ca(2+) from intracellular stores occurs via at least two mechanisms: (i) activation of intracellular Ca(2+) channels and (ii) a Ca(2+) channels independent mechanism. The former involves PLC-IP(3)R operated Ca(2+) channels activated via G-proteins and protein tyrosine kinases. For the latter, we propose a novel mechanism of intracellular Ca(2+) release involving injury of intracellular Ca(2+) stores such as the endoplasmic reticulum. In addition to Ca(2+) signalling, the discovery that LLO causes damage to an intracellular organelle provides a new perspective in our understanding of how CDCs affect target cells during infection by the respective bacterial pathogens.
17.	Härtlova, Anetta, et al. (författare) DNA Damage Primes the Type I Interferon System via the Cytosolic DNA Sensor STING to Promote Anti-Microbial Innate Immunity. 2015 Ingår i: Immunity. - : Elsevier BV. - 1097-4180 .- 1074-7613. ; 42:2, s. 332-43 Tidskriftsartikel (refereegranskat)abstract Dysfunction in Ataxia-telangiectasia mutated (ATM), a central component of the DNA repair machinery, results in Ataxia Telangiectasia (AT), a cancer-prone disease with a variety of inflammatory manifestations. By analyzing AT patient samples and Atm(-/-) mice, we found that unrepaired DNA lesions induce type I interferons (IFNs), resulting in enhanced anti-viral and anti-bacterial responses in Atm(-/-) mice. Priming of the type I interferon system by DNA damage involved release of DNA into the cytoplasm where it activated the cytosolic DNA sensing STING-mediated pathway, which in turn enhanced responses to innate stimuli by activating the expression of Toll-like receptors, RIG-I-like receptors, cytoplasmic DNA sensors, and their downstream signaling partners. This study provides a potential explanation for the inflammatory phenotype of AT patients and establishes damaged DNA as a cell intrinsic danger signal that primes the innate immune system for a rapid and amplified response to microbial and environmental threats.
18.	Jiang, Hui, et al. (författare) Chromatin-bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death 2019 Ingår i: EMBO Journal. - : EMBO. - 0261-4189 .- 1460-2075. ; 38:21 Tidskriftsartikel (refereegranskat)abstract DNA repair via homologous recombination (HR) is indispensable for genome integrity and cell survival but if unrestrained can result in undesired chromosomal rearrangements. The regulatory mechanisms of HR are not fully understood. Cyclic GMP-AMP synthase (cGAS) is best known as a cytosolic innate immune sensor critical for the outcome of infections, inflammatory diseases, and cancer. Here, we report that cGAS is primarily a chromatin-bound protein that inhibits DNA repair by HR, thereby accelerating genome destabilization, micronucleus generation, and cell death under conditions of genomic stress. This function is independent of the canonical STING-dependent innate immune activation and is physiologically relevant for irradiation-induced depletion of bone marrow cells in mice. Mechanistically, we demonstrate that inhibition of HR repair by cGAS is linked to its ability to self-oligomerize, causing compaction of bound template dsDNA into a higher-ordered state less amenable to strand invasion by RAD51-coated ssDNA filaments. This previously unknown role of cGAS has implications for understanding its involvement in genome instability-associated disorders including cancer.
19.	Jiang, Hui, et al. (författare) Comet and micronucleus assays for analyzing DNA damage and genome integrity 2019 Ingår i: DNA Sensors and Inflammasomes. - : Elsevier. - 9780128183595 - 9780128183601 ; , s. 299-307 Bokkapitel (refereegranskat)abstract Detection of DNA damage in cells is fundamental for the study of DNA repair and genome-instability associated processes including carcinogenesis. Many studies often rely on cytotoxicity assays to estimate genotoxicity. However, measurements of cytotoxicity, a delayed outcome requiring high threshold genotoxicity to induce, does not provide information about the subtle, early genotoxic effects relevant for mechanistic understanding of DNA repair processes. Here describe how to combine two simple procedures for monitoring the presence of DNA damage in individual eukaryotic cells using: (1) the Comet assay for measuring initial DNA breaks and (2) the Micronucleus assay for detecting delayed outcome DNA breaks in dividing cells. We discuss the principles, experimental design considerations and troubleshooting tips for optimizing these methods. They require standard molecular biology instruments and a fluorescent microscope.
20.	Jiang, Hui, et al. (författare) Nuclear AIM2-Like Receptors Drive Genotoxic Tissue Injury by Inhibiting DNA Repair 2021 Ingår i: Advanced Science. - : Wiley. - 2198-3844. ; 8:22 Tidskriftsartikel (refereegranskat)abstract Radiation is an essential preparative procedure for bone marrow (BM) transplantation and cancer treatment. The therapeutic efficacy of radiation and associated toxicity varies from patient to patient, making it difficult to prescribe an optimal patient-specific irradiation dose. The molecular determinants of radiation response remain unclear. AIM2-like receptors (ALRs) are key players in innate immunity and determine the course of infections, inflammatory diseases, senescence, and cancer. Here it is reported that mice lacking ALRs are resistant to irradiation-induced BM injury. It is shown that nuclear ALRs are inhibitors of DNA repair, thereby accelerate genome destabilization, micronuclei generation, and cell death, and that this novel function is uncoupled from their role in innate immunity. Mechanistically, ALRs bind to and interfere with chromatin decompaction vital for DNA repair. The finding uncovers ALRs as targets for new interventions against genotoxic tissue injury and as possible biomarkers for predicting the outcome of radio/chemotherapy.
21.	Kurhade, Chaitanya, et al. (författare) Type I Interferon response in olfactory bulb, the site of tick-borne flavivirus accumulation, is primarily regulated by IPS-1 2016 Ingår i: Journal of Neuroinflammation. - : Springer Science and Business Media LLC. - 1742-2094. ; 13 Tidskriftsartikel (refereegranskat)abstract Background: Although type I interferons (IFNs)—key effectors of antiviral innate immunity are known to be induced via different pattern recognition receptors (PRRs), the cellular source and the relative contribution of different PRRs in host protection against viral infection is often unclear. IPS-1 is a downstream adaptor for retinoid-inducible gene I (RIG-I)-like receptor signaling. In this study, we investigate the relative contribution of IPS-1 in the innate immune response in the different brain regions during infection with tick-borne encephalitis virus (TBEV), a flavivirus that causes a variety of severe symptoms like hemorrhagic fevers, encephalitis, and meningitis in the human host.Methods: IPS-1 knockout mice were infected with TBEV/Langat virus (LGTV), and viral burden in the peripheral and the central nervous systems, type I IFN induction, brain infiltrating cells, and inflammatory response was analyzed.Results: We show that IPS-1 is indispensable for controlling TBEV and LGTV infections in the peripheral and central nervous system. Our data indicate that IPS-1 regulates neuropathogenicity in mice. IFN response is differentially regulated in distinct regions of the central nervous system (CNS) influencing viral tropism, as LGTV replication was mainly restricted to olfactory bulb in wild-type (WT) mice. In contrast to the other brain regions, IFN upregulation in the olfactory bulb was dependent on IPS-1 signaling. IPS-1 regulates basal levels of antiviral interferon-stimulated genes (ISGs) like viperin and IRF-1 which contributes to the establishment of early viral replication which inhibits STAT1 activation. This diminishes the antiviral response even in the presence of high IFN-β levels. Consequently, the absence of IPS-1 causes uncontrolled virus replication, in turn resulting in apoptosis, activation of microglia and astrocytes, elevated proinflammatory response, and recruitment of inflammatory cells into the CNS.Conclusions: We show that LGTV replication is restricted to the olfactory bulb and that IPS-1 is a very important player in the olfactory bulb in shaping the innate immune response by inhibiting early viral replication and viral spread throughout the central nervous system. In the absence of IPS-1, higher viral replication leads to the evasion of antiviral response by inhibiting interferon signaling. Our data suggest that the local microenvironment of distinct brain regions is critical to determine virus permissiveness.
22.	Lindqvist, Richard, et al. (författare) Fast type I interferon response protects astrocytes from flavivirus infection and virus-induced cytopathic effects 2016 Ingår i: Journal of Neuroinflammation. - : Springer Science and Business Media LLC. - 1742-2094. ; 13 Tidskriftsartikel (refereegranskat)abstract Background: Neurotropic flaviviruses such as tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and Zika virus (ZIKV) are causative agents of severe brain-related diseases including meningitis, encephalitis, and microcephaly. We have previously shown that local type I interferon response within the central nervous system (CNS) is involved in the protection of mice against tick-borne flavivirus infection. However, the cells responsible for mounting this protective response are not defined. Methods: Primary astrocytes were isolated from wild-type (WT) and interferon alpha receptor knock out (IFNAR(-/-)) mice and infected with neurotropic flaviviruses. Viral replication and spread, IFN induction and response, and cellular viability were analyzed. Transcriptional levels in primary astrocytes treated with interferon or supernatant from virus-infected cells were analyzed by RNA sequencing and evaluated by different bioinformatics tools. Results: Here, we show that astrocytes control viral replication of different TBEV strains, JEV, WNV, and ZIKV. In contrast to fibroblast, astrocytes mount a rapid interferon response and restrict viral spread. Furthermore, basal expression levels of key interferon-stimulated genes are high in astrocytes compared to mouse embryonic fibroblasts. Bioinformatic analysis of RNA-sequencing data reveals that astrocytes have established a basal antiviral state which contributes to the rapid viral recognition and upregulation of interferons. The most highly upregulated pathways in neighboring cells were linked to type I interferon response and innate immunity. The restriction in viral growth was dependent on interferon signaling, since loss of the interferon receptor, or its blockade in wild-type cells, resulted in high viral replication and virus-induced cytopathic effects. Astrocyte supernatant from TBEV-infected cells can restrict TBEV growth in astrocytes already 6 h post infection, the effect on neurons is highly reinforced, and astrocyte supernatant from 3 h post infection is already protective. Conclusions: These findings suggest that the combination of an intrinsic constitutive antiviral response and the fast induction of type I IFN production by astrocytes play an important role in self-protection of astrocytes and suppression of flavivirus replication in the CNS.
23.	Lopez Chiloeches, Maria, et al. (författare) Genotoxin-producing Salmonella enterica induces tissue-specific types of DNA damage and DNA damage response outcomes 2023 Ingår i: Frontiers in Immunology. - : Frontiers Media S.A.. - 1664-3224. ; 14 Tidskriftsartikel (refereegranskat)abstract Introduction: Typhoid toxin-expressing Salmonella enterica causes DNA damage in the intestinal mucosa in vivo, activating the DNA damage response (DDR) in the absence of inflammation. To understand whether the tissue microenvironment constrains the infection outcome, we compared the immune response and DDR patterns in the colon and liver of mice infected with a genotoxigenic strain or its isogenic control strain.Methods: In situ spatial transcriptomic and immunofluorescence have been used to assess DNA damage makers, activation of the DDR, innate immunity markers in a multiparametric analysis.Result: The presence of the typhoid toxin protected from colonic bacteria-induced inflammation, despite nuclear localization of p53, enhanced co-expression of type-I interferons (IfnbI) and the inflammasome sensor Aim2, both classic features of DNA-break-induced DDR activation. These effects were not observed in the livers of either infected group. Instead, in this tissue, the inflammatory response and DDR were associated with high oxidative stress-induced DNA damage.Conclusions: Our work highlights the relevance of the tissue microenvironment in enabling the typhoid toxin to suppress the host inflammatory response in vivo.
24.	Łyszkiewicz, Marcin, et al. (författare) SIGN-R1+MHC II+ cells of the splenic marginal zone : a novel type of resident dendritic cells 2011 Ingår i: Journal of Leukocyte Biology. - : Oxford University Press (OUP). - 0741-5400 .- 1938-3673. ; 89:4, s. 607-615 Tidskriftsartikel (refereegranskat)abstract In the spleen, the MZ forms an interface between red and white pulp. Its major function is to trap blood-borne antigens and to reorient them to APCs and lymphocytes. SIGN-R1(+) cells are of the MZ inherent cell population, which for a long time, have been considered as macrophages. We now show that one subpopulation of SIGN-R1(+) cells that express MHC II molecules should be considered as a resident DC. Histological analysis indicated that SIGN-R1(+) cells have dendritic-like protrusions extending into T and B cell areas. Flow cytometry analysis revealed an expression profile of adhesion, costimulatory, and MHC molecules similar to cDCs but distinct from macrophages. Most importantly, SIGN-R1(+)MHC(+) cells were able to present antigen to naïve CD4 T cells, as well as to cross-present soluble, particulate antigens secreted by Listeria monocytogenes to CD8 T cells in vitro and in vivo. Our experiments identified SIGN-R1(+)MHC II(+) cells as professional APCs and indicate their nature as splenic resident DCs.
25.	Panda, Swarupa, 1985-, et al. (författare) Deubiquitinase MYSM1 Regulates Innate Immunity through Inactivation of TRAF3 and TRAF6 Complexes. 2015 Ingår i: Immunity. - : Elsevier BV. - 1097-4180 .- 1074-7613. ; 43:4, s. 647-59 Tidskriftsartikel (refereegranskat)abstract Pattern-recognition receptors (PRRs) including Toll-like receptors, RIG-I-like receptors, and cytoplasmic DNA receptors are essential for protection against pathogens but require tight control to avert inflammatory diseases. The mechanisms underlying this strict regulation are unclear. MYSM1 was previously described as a key component of epigenetic signaling machinery. We found that in response to microbial stimuli, MYSM1 accumulated in the cytoplasm where it interacted with and inactivated TRAF3 and TRAF6 complexes to terminate PRR pathways for pro-inflammatory and type I interferon responses. Consequently, Mysm1 deficiency in mice resulted in hyper-inflammation and enhanced viral clearance but also susceptibility to septic shock. We identified two motifs in MYSM1 that were essential for innate immune suppression: the SWIRM domain that interacted with TRAF3 and TRAF6 and the metalloproteinase domain that removed K63 polyubiquitins. This study identifies MYSM1 as a key negative regulator of the innate immune system that guards against an overzealous self-destructive immune response.
26.	Panda, Swarupa, 1985-, et al. (författare) The deubiquitinase MYSM1 dampens NOD2-mediated inflammation and tissue damage by inactivating the RIP2 complex 2018 Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 9 Tidskriftsartikel (refereegranskat)abstract NOD2 is essential for antimicrobial innate immunity and tissue homeostasis, but require tight regulation to avert pathology. A focal point of NOD2 signaling is RIP2, which upon polyubiquitination nucleates the NOD2:RIP2 complex, enabling signaling events leading to inflammation, yet the precise nature and the regulation of the polyubiquitins coordinating this process remain unclear. Here we show that NOD2 signaling involves conjugation of RIP2 with lysine 63 (K63), K48 and M1 polyubiquitin chains, as well as with non-canonical K27 chains. In addition, we identify MYSM1 as a proximal deubiquitinase that attenuates NOD2:RIP2 complex assembly by selectively removing the K63, K27 and M1 chains, but sparing the K48 chains. Consequently, MYSM1 deficient mice have unrestrained NOD2-mediated peritonitis, systemic inflammation and liver injury. This study provides a complete overview of the polyubiquitins in NOD2:RIP2 signaling and reveal MYSM1 as a central negative regulator restricting these polyubiquitins to prevent excessive inflammation.
27.	Panda, Swarupa, 1985- (författare) The role and mechanism of ubiquitin system in innate immune regulation 2018 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract Pattern-recognition receptors (PRRs) include the cell surface or endosomal membrane localized Toll-like receptors (TLRs) and the cytoplasmic PRRs such as RIG-I-like receptors (RLRs), NOD-like receptors (NLRs) and cytoplasmic DNA receptors (CDRs). Triggering of PRRs culminates in the transcriptional induction of pro-inflammatory cytokines and type I interferons (IFNs) that coordinate protection against pathogens but require tight control to avert inflammatory diseases. The mechanisms underlying this strict regulation are unclear.Ubiquitiation is a reversible post-translational modification that controls nearly all cellular processes including the immune system. We identified, H2A deubiquitinase myb-like SWIM and MPN domains 1 (MYSM1) a previously described as a key component of epigenetic signaling machinery as a key negative regulator of the innate immune system that guards against an overzealous self-destructive immune response. In response to microbial stimuli, MYSM1 accumulated in the cytoplasm where it interacted with and inactivated TRAF3 and TRAF6 complexes to terminate TLR, RLR and CDR pathways for pro-inflammatory and type I interferone responses. Consequently, MYSM1 deficiency in mice resulted in hyper-inflammation and enhanced viral clearance but also susceptibility to septic shock.NOD2, belonging to the intracellular NLR family. A focal point of NOD2 signalling is RIP2, which upon polyubiquitination nucleates the NOD2:RIP2 complex, enabling signaling events leading to inflammation, yet the precise nature and the regulation of the polyubiquitins coordinating this process remains unclear. We show that NOD2 signaling involves conjugation of RIP2 with K63, K48 and M1 polyubiquitin chains as well as with non-canonical K27 chains. Furthermore, we identify MYSM1 as the proximal deubiquitinase that attenuates NOD2- RIP2 complex assembly by selectively removing the K63, M1 and K27 chains. Consequently, MYSM1 deficient mice have unrestrained NOD2-mediated peritonitis and liver injury. Henceforth, this study provide a complete description of the polyubiquitin modifications in the NOD2:RIP2 signalling and reveal MYSM1 as a central negative regulator that prevents excessive inflammation.In order to overcome the host barrier to infection, some pathogens elude the immune defense by hijacking the ubiquitin system. Francisella tularensis is one of the most infectious bacteria. It employs several mechanisms to evade detection by the innate immune system, but how remains obscure. Here, we showed that Francisella triggers but concomitantly inhibits the TLRs, RLRs and CDRs pathway by inhibiting K63-linked polyubiquitination and assembly of TRAF6 and TRAF3 complexes that control the transcriptional responses of PRRs.In summary, my work identify MYSM1 as a key negative regulator of the innate immune system. Although, mainly located in the nucleus MYSM1 rapidly amass in the cytoplasm, where it interacts with and inactivates the key PRR signalling complexes. Afterward, MYSM1 undergoes proteaosomal degradation to avert sustained immune suppression. Thus, MYSM1 is part of a highly versatile negative feedback regulatory mechanism, which in response to biological danger is swiftly activated in “on-and-off” manner to restore immune homeostasis. Furthermore, Francisella targets the ubiquitin system to inhibt multiple PRRs hence allowing this bacterium to invade and proliferate in the host without evoking a self-limiting innate immune response.
28.	Panda, Swarupa, 1985-, et al. (författare) TUBE and UbiCRest assays for elucidating polyubiquitin modifications in protein complexes 2019 Ingår i: DNA Sensors and Inflammasomes. - : Elsevier. - 9780128183595 - 9780128183601 ; , s. 339-350 Bokkapitel (refereegranskat)abstract Ubiquitination is a reversible posttranslational modification that regulates nearly all cellular processes. The ubiquitin polypeptide is conjugated via its C-terminus to amine groups of lysine residues on target protein. Additionally, ubiquitins moieties can be conjugated in tandem to the initial ubiquitin via any of its internal lysine residues or N terminal methionine residue, resulting in the formation of polyubiquitin chains with distinct biophysical properties and biological functions. Elucidating the types of polyubiquitin chains present in proteins is essential for understanding their function and mechanism of regulation. Traditionally, ubiqutin modifications have been elucidated by exogenously co-expressing proteins of interest with epitope-tagged ubiquitins mutated in specific lysine residues. However, this strategy is prone experimental artifacts. In this protocol, we describe how to elucidate endogenous ubiquitin modifications. This procedure combines TUBE (Tandem Ubiquitin Binding Entity)-based isolation of ubiquitin conjugates, digestion with linkage specific deubiquitinases and immunoblotting. This procedure is very robust can be applied to profile types and architectural organization polyubiquitin chains present on the any proteins of interest and has been instrumental in elucidating ubiquitin modifications in NOD2 signaling in our recent study (Panda & Gekara, 2018).
29.	Putzova, Daniela, et al. (författare) Subversion of innate immune responses by Francisella involves the disruption of TRAF3 and TRAF6 signalling complexes 2017 Ingår i: Cellular Microbiology. - Hoboken : Wiley-Blackwell. - 1462-5814 .- 1462-5822. ; 19:11 Tidskriftsartikel (refereegranskat)abstract The success of pathogens depends on their ability to circumvent immune defences. Francisella tularensis is one of the most infectious bacteria known. The remarkable virulence of Francisella is believed to be due to its capacity to evade or subvert the immune system, but how remains obscure. Here, we show that Francisella triggers but concomitantly inhibits the Toll-like receptor, RIG-I-like receptor, and cytoplasmic DNA pathways. Francisella subverts these pathways at least in part by inhibiting K63-linked polyubiquitination and assembly of TRAF6 and TRAF3 complexes that control the transcriptional responses of pattern recognition receptors. We show that this mode of inhibition requires a functional type VI secretion system and/or the presence of live bacteria in the cytoplasm. The ability of Francisella to enter the cytosol while simultaneously inhibiting multiple pattern recognition receptor pathways may account for the notable capacity of this bacterium to invade and proliferate in the host without evoking a self-limiting innate immune response.
30.	Samperio Ventayol, Pilar, et al. (författare) Bacterial detection by NAIP/NLRC4 elicits prompt contractions of intestinal epithelial cell layers 2021 Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 118:16 Tidskriftsartikel (refereegranskat)abstract The gut epithelium serves to maximize the surface for nutrient and fluid uptake, but at the same time must provide a tight barrier to pathogens and remove damaged intestinal epithelial cells (IECs) without jeopardizing barrier integrity. How the epithelium coordinates these tasks remains a question of significant interest. We used imaging and an optical flow analysis pipeline to study the dynamicity of untransformed murine and human intestinal epithelia, cultured atop flexible hydrogel supports. Infection with the pathogen Salmonella Typhimurium (S.Tm) within minutes elicited focal contractions with inward movements of up to similar to 1,000 IECs. Genetics approaches and chimeric epithelial monolayers revealed contractions to be triggered by the NAIP/NLRC4 inflammasome, which sensed type-III secretion system and flagellar ligands upon bacterial invasion, converting the local tissue into a contraction epicenter. Execution of the response required swift sublytic Gasdermin D pore formation, ion fluxes, and the propagation of a myosin contraction pulse across the tissue. Importantly, focal contractions preceded, and could be uncoupled from, the death and expulsion of infected IECs. In both two-dimensional monolayers and three-dimensional enteroids, multiple infection-elicited contractions coalesced to produce shrinkage of the epithelium as a whole. Monolayers deficient for Caspase-1(-11) or Gasdermin D failed to elicit focal contractions but were still capable of infected IEC death and expulsion. Strikingly, these monolayers lost their integrity to a markedly higher extent than wild-type counterparts. We propose that prompt NAIP/NLRC4/Caspase-1/Gasdermin D/myosin-dependent contractions allow the epithelium to densify its cell packing in infected regions, thereby preventing tissue disintegration due to the subsequent IEC death and expulsion process.
31.	Swacha, Patrycja, et al. (författare) Biochemical and microscopic analysis of inflammasome complex formation 2019 Ingår i: DNA Sensors and Inflammasomes. - : Elsevier. - 9780128183595 - 9780128183601 ; , s. 287-298 Bokkapitel (refereegranskat)abstract Inflammasomes are multiprotein signaling platforms responsible for the maturation of pro-IL-1β and pro-IL-18 as well as the induction of an inflammatory cell death termed pyroptosis. Most inflammasomes consist of an upstream sensor, in most cases an adaptor protein (ASC) and inflammatory caspases such as caspase-1. Upon activation, sensor proteins oligomerize with adaptor proteins, forming large complexes called specks. These complexes can be stabilized and detected by Western blotting or fluorescence microscopy providing a direct evidence of inflammasome activation. Here we describe protocols for two complementary methods for detecting inflammasome complexes: (1) biochemical isolation and detection of ASC oligomers by Western blot analysis and (2) microscopic visualization of active caspase-1—ASC complexes. These protocols have successfully been applied in our recent study to unveil new regulatory mechanisms for different inflammasomes including the DNA sensor AIM2 (Erttmann et al., 2016).
32.	Weber, Elvira, et al. (författare) Type I interferon protects mice from fatal neurotropic infection with Langat virus by systemic and local antiviral responses 2014 Ingår i: Journal of Virology. - : American Society for Microbiology. - 0022-538X .- 1098-5514. ; 89:21, s. 12202-12212 Tidskriftsartikel (refereegranskat)abstract Vector-borne flaviviruses, such as tick-borne encephalitis virus (TBEV), West Nile virus, and dengue virus, cause millions of infections in humans. TBEV causes a broad range of pathological symptoms, ranging from meningitis to severe encephalitis or even hemorrhagic fever, with high mortality. Despite the availability of an effective vaccine, the incidence of TBEV infections is increasing. Not much is known about the role of the innate immune system in the control of TBEV infections. Here, we show that the type I interferon (IFN) system is essential for protection against TBEV and Langat virus (LGTV) in mice. In the absence of a functional IFN system, mice rapidly develop neurological symptoms and succumb to LGTV and TBEV infections. Type I IFN system deficiency results in severe neuroinflammation in LGTV-infected mice, characterized by breakdown of the blood-brain barrier and infiltration of macrophages into the central nervous system (CNS). Using mice with tissue-specific IFN receptor deletions, we show that coordinated activation of the type I IFN system in peripheral tissues as well as in the CNS is indispensable for viral control and protection against virus induced inflammation and fatal encephalitis. IMPORTANCE: The type I interferon (IFN) system is important to control viral infections; however, the interactions between tick-borne encephalitis virus (TBEV) and the type I IFN system are poorly characterized. TBEV causes severe infections in humans that are characterized by fever and debilitating encephalitis, which can progress to chronic illness or death. No treatment options are available. An improved understanding of antiviral innate immune responses is pivotal for the development of effective therapeutics. We show that type I IFN, an effector molecule of the innate immune system, is responsible for the extended survival of TBEV and Langat virus (LGTV), an attenuated member of the TBE serogroup. IFN production and signaling appeared to be essential in two different phases during infection. The first phase is in the periphery, by reducing systemic LGTV replication and spreading into the central nervous system (CNS). In the second phase, the local IFN response in the CNS prevents virus-induced inflammation and the development of encephalitis.
33.	Webster, Steve J., et al. (författare) Detection of a microbial metabolite by STING regulates inflammasome activation in response to Chlamydia trachomatis infection 2017 Ingår i: PLoS Pathogens. - : Public Library Science. - 1553-7366 .- 1553-7374. ; 13:6 Tidskriftsartikel (refereegranskat)abstract The innate immune system is a critical component of host defence against microbial pathogens, but effective responses require an ability to distinguish between infectious and noninfectious insult to prevent inappropriate inflammation. Using the important obligate intracellular human pathogen Chlamydia trachomatis; an organism that causes significant immunopathology, we sought to determine critical host and pathogen factors that contribute to the induction of inflammasome activation. We assayed inflammasome activation by immunoblotting and ELISA to detect IL-1 beta processing and LDH release to determine pyroptosis. Using primary murine bone marrow derived macrophages or human monocyte derived dendritic cells, infected with live or attenuated Chlamydia trachomatis we report that the live organism activates both canonical and non-canonical inflammasomes, but only canonical inflammasomes controlled IL-1 beta processing which preceded pyroptosis. NADPH oxidase deficient macrophages were permissive to Chlamydia trachomatis replication and displayed elevated type-1 interferon and inflammasome activation. Conversely, attenuated, non-replicating Chlamydia trachomatis, primed but did not activate inflammasomes and stimulated reduced type-1 interferon responses. This suggested bacterial replication or metabolism as important factors that determine interferon responses and inflammasome activation. We identified STING but not cGAS as a central mediator of interferon regulated inflammasome activation. Interestingly, exogenous delivery of a Chlamydia trachomatis metabolite and STING ligand D cyclic di-AMP, recovered inflammasome activation to attenuated bacteria in a STING dependent manner thus indicating that a bacterial metabolite is a key factor initiating inflammasome activation through STING, independent of cGAS. These data suggest a potential mechanism of how the innate immune system can distinguish between infectious and non-infectious insult and instigate appropriate immune responses that could be therapeutically targeted.

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