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NLRP3 and ASC Differentially Affect the Lung Transcriptome During Pneumococcal Pneumonia

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NLRP3 and ASC Differentially Affect the Lung Transcriptome During Pneumococcal Pneumonia UvA-DARE (Digital Academic Repository) Cell-specific pattern recognition receptor signaling in antibacterial defense van Lieshout, M.H.P. Publication date 2015 Document Version Final published version Link to publication Citation for published version (APA): van Lieshout, M. H. P. (2015). Cell-specific pattern recognition receptor signaling in antibacterial defense. General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) Download date:30 Sep 2021 Chapter 7 NLRP3 and ASC differentially affect the lung transcriptome during pneumococcal pneumonia American Journal of Respiratory Cell and Molecular Biology 2014 Apr;50(4):699-712 DOI: 10.1165/rcmb.2013-0015OC Miriam H.P. van Lieshout 1,2 Brendon P. Scicluna 1,2 Sandrine Florquin 3 Tom van der Poll 1,2,4 Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands: 1Center of Infection and Immunity Amsterdam 2Center of Experimental and Molecular Medicine 3Department of Pathology 4Division of Infectious Diseases Chapter 7 Abstract Streptococcus (S.) pneumoniae is the most frequently isolated causative pathogen of community-acquired pneumonia, a leading cause of mortality worldwide. Inflammasomes are multiprotein complexes that play crucial roles in the regulation of inflammation. NLRP3 (Nod-like receptor family, pyrin domain containing 3) is a sensor that functions in a single inflammasome, whereas ASC (the adaptor apoptosis- associated speck-like protein containing a caspase activation and recruitment domain) is a common adaptor of several inflammasomes. We investigated the role of NLRP3 and ASC during S. pneumoniae pneumonia by comparing bacterial growth and spreading, and host innate immune responses in wild-type mice and mice deficient for either NLRP3 (Nlrp3-/-) or ASC (Asc-/-). Asc-/ - mice had increased bacterial dissemination and lethality compared to Nlrp3-/- mice, although the cytokine response was impaired in both mouse strains. By detailed analysis of the early inflammatory response in the lung by whole genome transcriptional profiling, we identified several mediators that were differentially expressed between Nlrp3-/- and Asc-/ - mice. Of these, interleukin-17, granulocyte-macrophage colony-stimulating factor and integrin alpha M were significantly attenuated in Asc-/- relative to Nlrp3- /- mice as well as a number of genes involved in the adaptive immune response. These differences may explain the increased susceptibility of Asc-/ - mice during S. pneumoniae infection and suggest that either ASC-dependent NLRP3-independent inflammasomes or inflammasome independent ASC functions may be involved. 124 NLRP3 and ASC differentially affect the lung transcriptome during pneumococcal pneumonia Introduction Streptococcus (S.) pneumoniae is the most frequently isolated causative pathogen in patients with community-acquired pneumonia (1, 2) and a common cause of sepsis, especially in the context of pneumonia (3). Worldwide, the mortality rate associated with pneumococcal pneumonia ranges from 6 to >40% depending on the setting of outpatients or patients in general hospital wards or intensive cares (2). This, together with the increasing incidence of antibiotic resistance in S. pneumoniae (4), emphasizes the importance of expanding our knowledge of host defense mechanisms that influence the outcome of pneumococcal pneumonia. In recent years, the importance of Nod-like receptors (NLRs) for the antimicrobial response has become apparent (5, 6). NLRs are cytosolic receptors that can be part of large multi-protein complexes called inflammasomes. Interleukin-1 beta (IL-1β), one of the most potent proinflammatory cytokines (5, 6), is at a post-transcriptional level tightly regulated by these inflammasomes, together with the proinflammatory cytokine IL-18 (5, 6). Inflammasomes recognize a diverse set of inflammation- inducing stimuli including pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) and consist of a cytosolic sensor that (together with an adaptor protein) recruits pro-caspase-1, resulting in the generation of active caspase-1, the enzyme responsible for activation of pro-IL- 1β to mature IL-1β. The sensor protein can be either a member of the NLR or the pyrin and HIN domain-containging(PYHIN) family and, if the NLR sensor does not have a caspase activation and recruitment domain (CARD), ASC (the adaptor apoptosis-associated speck-like protein containing a CARD) is necessary to recruit and bind caspase-1 (6). NLRP3 (NLR family, pyrin domain containing 3) is one of the best studied members of the NLR family that can be activated by a large variety of signals, including pneumolysin, a major virulence factor of S. pneumoniae (7). Recently, the role of the NLRP3 and ASC inflammasome complexes in S. pneumoniae induced cell activation in vitro and infection in vivo was investigated (7-9). We here expanded these previous studies using a model of pneumococcal pneumonia and observed a remarkable susceptibility of especially Asc deficient (Asc-/-) mice when compared with Nlrp3-/- mice. Considering that the afore mentioned investigations on the role of the inflammasome during pneumococcal pneumonia predominantly focused on mechanistic studies in purified macrophages (7-9), we here aimed to characterize the initiation of the host response to pulmonary infection in mice deficient for inflammasome components in vivo in more detail by performing a genome-wide scan of transcriptional responses in lung tissue at an early time point after lower respiratory tract infection with S. pneumoniae. We identified a strong influence of the inflammasome components ASC and NLRP3 on the transcriptional response during pneumococcal pneumonia and in addition a differential gene expression pattern between Asc-/- and Nlrp3-/- mice. 125 Chapter 7 Methods Mice Nlrp3-/- and Asc-/- mice (10) were backcrossed 9 times to a C57Bl/6 background. Age- and sex-matched wild type (WT) C57Bl/6 mice were from Harlan (Horst, the Netherlands). Mice were infected at 9-12 weeks. All experiments were approved by the Institutional Animal Care and Use Committee of the Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands. Induction of pneumonia and tissue harvest Pneumonia was induced by intranasal inoculation with 1-2x107 CFU of S. pneumoniae D39 (serotype 2) (11). For two experiments isogenic pneumolysin- deficient D39 was used (11). Mice were followed for 14 days or euthanized at 6 or 48 hours, and bacterial outgrowth and cytokine levels were determined (11). Cytokine assays Tumor necrosis factor-α (TNF-α), Interleukin (IL)-6, IL-10 and chemokine (C-C motif) ligand 2 (CCL2) were measured by a cytometric bead assay (BD Biosciences, San Jose, CA). IL-1β, Chemokine (C-X-C motif) ligand 1 and 2 (CXCL1 and 2), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) and IL-17A (cross-reactivity with IL-17A/F) were measured by ELISA (R&D Systems, Minneapolis, MN). Myeloperoxidase (MPO) ELISA was from HyCult Biotechnology, Uden, the Netherlands. Histology Lungs were fixed in formalin and embedded in paraffin. 5 μm sections were stained with hemotoxylin and eosin. The following parameters were scored on a scale of 0 (absent) to 4 (very severe) by a pathologist blinded for experimental groups: interstitial damage, vasculitis, peri-bronchitis, oedema, thrombus formation and pleuritis. Granulocyte staining was performed (12). RNA preparation and genome-wide transcriptional profiling RNA was isolated from lung homogenates using the Nucleospin RNA II kit (Machery- Nagel, Duren, Germany). 750ng of biotinylated cRNA was hybridized onto the Illumina MouseRef-8v2 Expression BeadChip (Eindhoven, the Netherlands). The samples were scanned using the Illumina iScan array scanner(Eindhoven, the Netherlands. Preparation of cRNA, chip hybridization, washing, staining and scanning were carried out at ServiceXS (Leiden, the Netherlands). The raw scan data were read using the beadarray package (version 1.12.1) (13), available through Bioconductor (14) in the R statistical environment (version 2.13.2; R Foundation for Statistical Computing, Vienna, Austria). All non-normalized and normalized data are available at the gene expression omnibus of NCBI (GEO) with accession number GSE42464. Details on bioinformatics are available in the online supplement. 126 NLRP3 and ASC differentially affect the lung transcriptome during pneumococcal pneumonia Quantitative real time PCR Total RNA was reverse transcribed using oligo (dT) primer and Moloney murine leukemia virus reverse transcriptase (Invitrogen, Breda, the Netherlands). Quantitative PCR of 4933427D14Rik, Nlrp3, Asc, Gdpd3, Csf2, Csf3,
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