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Swacha, P., Gekara, N. O. & Erttmann, S. F. (2019). Biochemical and microscopic analysis of inflammasome complex formation. In: Jungsan Sohn (Ed.), DNA Sensors and Inflammasomes: (pp. 287-298). Elsevier, 625, Article ID S0076-6879(19)30173-9.
Open this publication in new window or tab >>Biochemical and microscopic analysis of inflammasome complex formation
2019 (English)In: DNA Sensors and Inflammasomes / [ed] Jungsan Sohn, Elsevier, 2019, Vol. 625, p. 287-298, article id S0076-6879(19)30173-9Chapter in book (Refereed)
Abstract [en]

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).

Place, publisher, year, edition, pages
Elsevier, 2019
Series
Methods in Enzymology, ISSN 0076-6879 ; 625
Keywords
Inflammasome complex, ASC specks, Caspase 1, AIM2, IL-1β, Innate immunity
National Category
Cell and Molecular Biology
Research subject
Immunology; cell research; Infectious Diseases
Identifiers
urn:nbn:se:umu:diva-162825 (URN)10.1016/bs.mie.2019.05.014 (DOI)000488782900018 ()31455532 (PubMedID)2-s2.0-85067031234 (Scopus ID)978-0-12-818359-5 (ISBN)
Available from: 2019-08-30 Created: 2019-08-30 Last updated: 2019-10-25Bibliographically approved
Jiang, H., Xue, X., Panda, S., Kawale, A., Hooy, R. M., Liang, F., . . . Gekara, N. O. (2019). Chromatin-bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death. EMBO Journal, Article ID e102718.
Open this publication in new window or tab >>Chromatin-bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death
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2019 (English)In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, article id e102718Article in journal (Refereed) Epub ahead of print
Abstract [en]

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.

Place, publisher, year, edition, pages
EMBOpress, 2019
Keywords
cancer, cell death, cGAS, chromatin compaction, DNA repair
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-164894 (URN)10.15252/embj.2019102718 (DOI)000487392000001 ()31544964 (PubMedID)
Funder
Swedish Research Council, 2015-02857Swedish Research Council, 2016-00890Swedish Cancer Society, CAN 2017/421NIH (National Institute of Health), R01 CA220123NIH (National Institute of Health), P30 CA054174NIH (National Institute of Health), R01 GM GM 129342-01-A1
Available from: 2019-11-06 Created: 2019-11-06 Last updated: 2019-11-07
Jiang, H., Panda, S. & Gekara, N. O. (2019). Comet and micronucleus assays for analyzing DNA damage and genome integrity. In: Sohn, J (Ed.), DNA SENSORS AND INFLAMMASOMES: (pp. 299-307). ELSEVIER ACADEMIC PRESS INC
Open this publication in new window or tab >>Comet and micronucleus assays for analyzing DNA damage and genome integrity
2019 (English)In: DNA SENSORS AND INFLAMMASOMES / [ed] Sohn, J, ELSEVIER ACADEMIC PRESS INC , 2019, p. 299-307Chapter in book (Refereed)
Abstract [en]

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.

Place, publisher, year, edition, pages
ELSEVIER ACADEMIC PRESS INC, 2019
Series
Methods in Enzymology, ISSN 0076-6879 ; 625
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-164490 (URN)10.1016/bs.mie.2019.05.015 (DOI)000488782900019 ()31455533 (PubMedID)978-0-12-818360-1 (ISBN)978-0-12-818359-5 (ISBN)
Available from: 2019-10-25 Created: 2019-10-25 Last updated: 2019-10-25Bibliographically approved
Erttmann, S. F. & Gekara, N. O. (2019). Hydrogen peroxide release by bacteria suppresses inflammasome-dependent innate immunity. Nature Communications, 10, Article ID 3493.
Open this publication in new window or tab >>Hydrogen peroxide release by bacteria suppresses inflammasome-dependent innate immunity
2019 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 10, article id 3493Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Nature Publishing Group, 2019
Keywords
Inflammasome
National Category
Cell and Molecular Biology
Research subject
Immunology; cell research; Infectious Diseases
Identifiers
urn:nbn:se:umu:diva-162589 (URN)10.1038/s41467-019-11169-x (DOI)000478576800011 ()31375698 (PubMedID)
Available from: 2019-08-23 Created: 2019-08-23 Last updated: 2019-08-30Bibliographically approved
Gekara, N. O. & Jiang, H. (2019). The innate immune DNA sensor cGAS: A membrane, cytosolic, or nuclear protein?. Science Signaling, 12(581), Article ID eaax3521.
Open this publication in new window or tab >>The innate immune DNA sensor cGAS: A membrane, cytosolic, or nuclear protein?
2019 (English)In: Science Signaling, ISSN 1945-0877, E-ISSN 1937-9145, Vol. 12, no 581, article id eaax3521Article in journal, Editorial material (Other academic) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Washington: American Association for the Advancement of Science, 2019
National Category
Cell Biology
Identifiers
urn:nbn:se:umu:diva-159599 (URN)10.1126/scisignal.aax3521 (DOI)000467963000003 ()31088977 (PubMedID)
Available from: 2019-06-17 Created: 2019-06-17 Last updated: 2019-06-17Bibliographically approved
Panda, S., Jiang, H. & Gekara, N. O. (2019). TUBE and UbiCRest assays for elucidating polyubiquitin modifications in protein complexes. In: Sohn, J (Ed.), DNA SENSORS AND INFLAMMASOMES: (pp. 339-350). ELSEVIER ACADEMIC PRESS INC
Open this publication in new window or tab >>TUBE and UbiCRest assays for elucidating polyubiquitin modifications in protein complexes
2019 (English)In: DNA SENSORS AND INFLAMMASOMES / [ed] Sohn, J, ELSEVIER ACADEMIC PRESS INC , 2019, p. 339-350Chapter in book (Refereed)
Abstract [en]

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).

Place, publisher, year, edition, pages
ELSEVIER ACADEMIC PRESS INC, 2019
Series
Methods in Enzymology, ISSN 0076-6879 ; 625
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-164489 (URN)10.1016/bs.mie.2019.05.006 (DOI)000488782900021 ()31455535 (PubMedID)978-0-12-818360-1 (ISBN)978-0-12-818359-5 (ISBN)
Available from: 2019-10-25 Created: 2019-10-25 Last updated: 2019-10-25Bibliographically approved
Webster, S. J., Brode, S., Ellis, L., Fitzmaurice, T. J., Elder, M. J., Gekara, N. O., . . . Goodall, J. C. (2017). Detection of a microbial metabolite by STING regulates inflammasome activation in response to Chlamydia trachomatis infection. PLoS Pathogens, 13(6), Article ID e1006383.
Open this publication in new window or tab >>Detection of a microbial metabolite by STING regulates inflammasome activation in response to Chlamydia trachomatis infection
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2017 (English)In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 13, no 6, article id e1006383Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Public Library Science, 2017
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-137812 (URN)10.1371/journal.ppat.1006383 (DOI)000404511700013 ()28570638 (PubMedID)
Available from: 2017-07-26 Created: 2017-07-26 Last updated: 2018-06-09Bibliographically approved
Gekara, N. O. (2017). DNA damage-induced immune response: Micronuclei provide key platform. Journal of Cell Biology, 216(10), 2999-3001
Open this publication in new window or tab >>DNA damage-induced immune response: Micronuclei provide key platform
2017 (English)In: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 216, no 10, p. 2999-3001Article in journal, Editorial material (Other academic) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
ROCKEFELLER UNIV PRESS, 2017
National Category
Cell Biology
Identifiers
urn:nbn:se:umu:diva-140887 (URN)10.1083/jcb.201708069 (DOI)000411962700004 ()28860276 (PubMedID)
Available from: 2017-11-21 Created: 2017-11-21 Last updated: 2018-06-09Bibliographically approved
Putzova, D., Panda, S., Härtlova, A., Stulík, J. & Gekara, N. O. (2017). Subversion of innate immune responses by Francisella involves the disruption of TRAF3 and TRAF6 signalling complexes. Cellular Microbiology, 19(11), Article ID e12769.
Open this publication in new window or tab >>Subversion of innate immune responses by Francisella involves the disruption of TRAF3 and TRAF6 signalling complexes
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2017 (English)In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 19, no 11, article id e12769Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Hoboken: Wiley-Blackwell, 2017
Keywords
inflammatory responses, listeria monocytogenes, murine macrophages, tularensis lvs, cytosolic dna, in vitro, system, infection, activation, trif
National Category
Cell and Molecular Biology
Research subject
Immunology
Identifiers
urn:nbn:se:umu:diva-139636 (URN)10.1111/cmi.12769 (DOI)000412834200008 ()28745813 (PubMedID)
Funder
Swedish Research Council, 2013-8621Swedish Research Council, 2015-02857
Available from: 2017-09-19 Created: 2017-09-19 Last updated: 2019-03-15Bibliographically approved
Lindqvist, R., Mundt, F., Gilthorpe, J. D., Woelfel, S., Gekara, N. O., Kroeger, A. & Överby, A. K. (2016). Fast type I interferon response protects astrocytes from flavivirus infection and virus-induced cytopathic effects. Journal of Neuroinflammation, 13, Article ID 277.
Open this publication in new window or tab >>Fast type I interferon response protects astrocytes from flavivirus infection and virus-induced cytopathic effects
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2016 (English)In: Journal of Neuroinflammation, ISSN 1742-2094, E-ISSN 1742-2094, Vol. 13, article id 277Article in journal (Refereed) Published
Abstract [en]

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.

Keywords
Astrocytes, Interferon, TBEV, Flavivirus, Viperin
National Category
Immunology Neurosciences
Identifiers
urn:nbn:se:umu:diva-127599 (URN)10.1186/s12974-016-0748-7 (DOI)000385979600001 ()27776548 (PubMedID)
Available from: 2016-12-09 Created: 2016-11-16 Last updated: 2018-06-09Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-1269-8288

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