Defining the Range of Pathogens Susceptible to Ifitm3 Restriction Using a Knockout Mouse Model Aaron R. Everitt1*, Simon Clare1, Jacqueline U. McDonald2, Leanne Kane1, Katherine Harcourt1, Malika Ahras1, Amar Lall3, Christine Hale1, Angela Rodgers4, Douglas B. Young4, Ashraful Haque5, Oliver Billker1, John S. Tregoning2, Gordon Dougan1, Paul Kellam1,6 1 Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United Kingdom, 2 Mucosal Infection and Immunity Group, Section of Infectious Diseases, Department of Medicine, Imperial College London, London, United Kingdom, 3 The Jenner Institute, University of Oxford, Oxford, United Kingdom, 4 Medical Research Council National Institute for Medical Research, London, United Kingdom, 5 Malaria Immunology Laboratory, Queensland Institute of Medical Research and The Australian Centre for Vaccine Development, Herston, Brisbane, Queensland, Australia, 6 Department of Infection, University College London, London, United Kingdom Abstract The interferon-inducible transmembrane (IFITM) family of proteins has been shown to restrict a broad range of viruses in vitro and in vivo by halting progress through the late endosomal pathway. Further, single nucleotide polymorphisms (SNPs) in its sequence have been linked with risk of developing severe influenza virus infections in humans. The number of viruses restricted by this host protein has continued to grow since it was first demonstrated as playing an antiviral role; all of which enter cells via the endosomal pathway. We therefore sought to test the limits of antimicrobial restriction by Ifitm3 using a knockout mouse model. We showed that Ifitm3 does not impact on the restriction or pathogenesis of bacterial (Salmonella typhimurium, Citrobacter rodentium, Mycobacterium tuberculosis) or protozoan (Plasmodium berghei) pathogens, despite in vitro evidence. However, Ifitm3 is capable of restricting respiratory syncytial virus (RSV) in vivo either through directly restricting RSV cell infection, or by exerting a previously uncharacterised function controlling disease pathogenesis. This represents the first demonstration of a virus that enters directly through the plasma membrane, without the need for the endosomal pathway, being restricted by the IFITM family; therefore further defining the role of these antiviral proteins. Citation: Everitt AR, Clare S, McDonald JU, Kane L, Harcourt K, et al. (2013) Defining the Range of Pathogens Susceptible to Ifitm3 Restriction Using a Knockout Mouse Model. PLoS ONE 8(11): e80723. doi:10.1371/journal.pone.0080723 Editor: Yasuhiro Ikeda, Mayo Clinic, United States of America Received August 1, 2013; Accepted October 16, 2013; Published November 21, 2013 Copyright: © 2013 Everitt et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Wellcome Trust. The research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement n° [115308] Biovacsafe, resources of which are composed of financial contribution from the European Union's Seventh Framework Programme (FP7/2007-2013) and EFPIA members' in kind contribution. MRC grant U117581288. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction negative bacteria, fungi, trypanosomes and surface proteins on several viruses [2]. Alternatively, other restriction factors, Intrinsic cellular defense molecules are able to detect and particularly those that target viruses, appear to have a more restrict invading pathogens at the level of the infected cell and reduced range of pathogens that they can block, as is the case constitute an initial repertoire of proteins that prevent infection. for many interferon-stimulated genes (ISGs) [3]. However, Such intrinsic defenses have the ability to detect the pathogen, restriction factors that work in defined cellular locations against and either directly block a component of the pathogen’s life a common pathogen feature of infection may also have broad cycles and / or signal to the innate and adaptive immune anti-influenza properties. system to further control the infection. In certain cases, these We and others have found that the ISG interferon-inducible intrinsic restriction factors recognise non-self pathogen- transmembrane 3 (IFITM3), initially defined as playing a assocated molecular patterns, such as lipids, proteins and developmental role in germ cell homing [4], has a profound role nucleic acids, from a broad range of pathogens through in the restriction of viruses entering the cell through the acid pathogen recognition receptors. This ability allows the host to endosomal pathway [5,6], including influenza and dengue detect bacterial, viral, fungal and protozoan pathogens [1]. For viruses [7]. Since the discovery of IFITM3’s antiviral role, the example, Toll-like receptor 4 alone is able to detect Gram- number of viruses restricted by the IFITM family has expanded PLOS ONE | www.plosone.org 1 November 2013 | Volume 8 | Issue 11 | e80723 Pathogens Restricted by Ifitm3 considerably [5,7-14]. This has led to the generation of Ifitm3-/- mice were phenotyped through pipelines at the hypotheses about how the IFITM family achieves restriction; Wellcome Trust Sanger Institute as described previously namely through preventing the fusion of viral and cellular [23,24]. membranes [15,16]. Recently, the role of IFITM3 has been expanded by the Immunohistochemistry discovery of nonenveloped reoviruses’ restriction [9]. This has 5-µm sections of paraffin-embedded tissue were incubated important implications, as nonenveloped viruses do not rely on with anti-Ifitm3 antibody (Abcam), which was subsequently membrane fusion to gain release from late endosomes. bound to a secondary horse radish peroxidase-conjugated anti- Instead, it is hypothesised that these viruses may physically rabbit antibody (Dako). Sections were counterstained with disrupt the endosomal membrane through their surface hematoxylin (Sigma-Aldrich) and were assessed for expression proteins [17,18]. This therefore potentially broadens the actions by microscopy. of IFITM3 beyond enveloped viruses and may also include other non-viral pathogens. The role of IFITM3 in vivo shows it is crucial restriction factor Salmonella Typhimurium challenge for preventing the onset of severe influenza viral infections in a Groups of 8 Ifitm3-/- and 8 C57BL/6J mice were challenged knockout mouse model [6]. Further, the overrepresentation of a intravenously with 5 x 105 colony forming units (cfu) Salmonella single nucleotide polymorphism (SNP), rs12252 C allele in the Typhimurium M525 containing TetC, (Fragment C of tetanus human IFITM3 gene in two cohorts of patients hospitalised with toxin, to act as an antigen for subsequent antibody influenza virus during the 2009 H1N1 pandemic shows that the quantification), and followed for 28 days. On day 14 post- rs12252 CC genotype confers an 4-5 fold increased risk infection (pi), four from each group of mice are culled and developing a severe influenza virus infection [6,19]. organs (spleen, liver and caecum) removed. A small piece of Here we sought therefore to expand and define the role of the spleen and liver was fixed in 4% formalin and then later Ifitm3 in pathogen restriction by assessing the susceptibility of processed to paraffin blocks as a biobank of infected tissue for Ifitm3-deficient (Ifitm3-/-) mice to bacteria (Salmonella histological study if interesting. The rest of the organs were Typhimurium, Citrobacter rodentium, Mycobacterium weighed then homogenized, serially diluted and plated to tuberculosis), a parasite (Plasmodium berghei) and a virus determine viable bacterial load. At day 28pi, the remaining four (respiratory syncytial virus, RSV) to determine the specificity of mice are culled following a terminal bleed and the organs this crucial antimicrobial protein. We show that Ifitm3 is removed and processed as above. The blood was allowed to specifically an antiviral protein; yielding no significant clot then centrifuged when the serum was removed and used phenotype in mice when challenged with bacteria and to detect TetC antigen specific antibodies by enzyme-linked protozoa, despite studies implicating the IFITM family in immuosorbent assay (ELISA). Mice were weighed and restriction of these pathogens [20,21]. monitored daily for signs of clinical illness. We also show a novel role for Ifitm3 in vivo in restriction of RSV: a virus that does not enter cells through the endosomal Citrobacter rodentium challenge pathway, adding further to the role of IFITM3 as a central -/- antiviral restriction factor that targets cellular entry. Groups of 8 Ifitm3 and 8 C57BL6j mice were orally infected by gavage with 1 x 109 cfu of and followed for 28 days. Every Methods 2-3 days faeces from infected mice are collected. These were then weighed and homogenized in 1 ml per 100 mg of faeces. Ethics statement This was then serially diluted and plated to determine viable bacterial load. On day 14pi four mice per group were culled All animal experiments were conducted under Prof. Gordon and organs (spleen, liver, caecal contents, caecum, 6cm of Dougan’s project