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of March 14 2015 This information is current as Control of Murine Coronavirus Infection Macrophages and Dendritic Cells Secures Type I IFN Mediated Protection of Volker Thiel and Burkhard Ludewig Martin K nig Boris Reizis Constantino L pez Mac as Luisa Cervantes Barrag n Ulrich Kalinke Roland Z st http www jimmunol org content 182 2 1099 doi 10 4049 jimmunol 182 2 1099 2009 182 1099 1106 J Immunol References http www jimmunol org content 182 2 1099 full ref list 1 23 of which you can access for free at cites 43 articlesThis article Subscriptions http jimmunol org subscriptions is online at The Journal of ImmunologyInformation about subscribing to Permissions http www aai org ji copyright html Submit copyright permission requests at Email Alerts http jimmunol org cgi alerts etoc Receive free email alerts when new articles cite this article Sign up at Print ISSN 0022 1767 Online ISSN 1550 6606 Immunologists Inc All rights reserved Copyright 2009 by The American Association of 9650 Rockville Pike Bethesda MD 20814 3994 The American Association of Immunologists Inc is published twice each month byThe Journal of Immunology at North Carolina State University Libraries on March 14 2015 http www jimmunol org Downloaded from at North Carolina State University Libraries on March 14 2015 http www jimmunol org Downloaded from Type I IFN Mediated Protection of Macrophages and Dendritic Cells Secures Control of Murine Coronavirus Infection 1 Luisa Cervantes Barraga n Ulrich Kalinke Roland Zu st Martin K nig Boris Reizis Constantino Lo pez Mac as Volker Thiel and Burkhard Ludewig 2 The swift production of type I IFNs is one of the fundamental aspects of innate immune responses against viruses Plasmacytoid dendritic cell derived type I IFNs are of prime importance for the initial control of highly cytopathic viruses such as the mouse hepatitis virus MHV The aim of this study was to determine the major target cell populations of this first wave of type I IFNs Generation of bone marrow chimeric mice expressing the type I IFN receptor IFNAR on either hemopoietic or non bone marrow derived cells revealed that the early control of MHV depended mainly on IFNAR expression on hemopoietic cells To establish which cell population responds most efficiently to type I IFNs mice conditionally deficient for the IFNAR on different leukocyte subsets were infected with MHV This genetic analysis revealed that IFNAR expression on LysM H11545 macrophages and CD11c H11545 dendritic cells was most important for the early containment of MHV within secondary lymphoid organs and to prevent lethal liver disease This study identifies type I IFN mediated cross talk between plasmacytoid dendritic cells on one side and macrophages and conventional dendritic cells on the other as an essential cellular pathway for the control of fatal cytopathic virus infection The Journal of Immunology 2009 182 1099 1106 F or the control of fast replicating cytopathic virus infec tions the immune system must act rapidly to control viral replication and dissemination before tissue damage and inflammation endanger survival of the host Secretion of type I IFNs is an essential component of the innate immune response against viruses These soluble factors induce an array of intracel lular effectors including protein kinase R 2H11032 5H11032 oligoadenylate synthetases and Mx proteins which halt viral replication 1 Fur thermore type 1 IFNs exert proapoptotic activities that control viral spread by eliminating infected cells 2 and they deliver im munomodulatory stimuli that affect cell migration 3 4 cross presentation 5 8 B cell responses and Ig isotype switch 9 11 CD4 H11001 T cell activation 12 13 or CTL expansion 14 15 How ever chronic activation of the type I IFN system can be detrimen tal for the host because autoimmune responses might be aggra vated 16 17 The fact that almost all cells are able to produce type I IFNs under certain conditions and also respond to it led to the initial idea of a general antiviral state However several in vitro stud ies have provided insight into the subtle differences of cell pop ulation specific effects of type I IFNs which depend largely on the constitutive vs inducible expression of STAT proteins and IFN regulatory factors and the state of cellular differentiation 18 19 It therefore appears that there is a cell type specific context dependent differential requirement of type I IFN re sponsiveness that secures optimal protection against viral in fection while reducing potential immunopathological side ef fects of these potent cytokines The murine hepatitis virus MHV 3 A59 is a group II corona virus that causes hepatitis and demyelinating encephalomyelitis in mice This natural mouse pathogen is one of the most extensively studied coronaviruses 20 A strong CTL response mediates clear ance of the virus between days 6 and 8 postinfection 21 22 and neutralizing Abs appear to be required to prevent re emergence of persistent CNS infection 23 24 Nonetheless before effective adaptive immune responses are elicited type I IFN mediated in nate immune responses are essential for the survival of the host in the early phase of infection The first wave of type I IFNs is pro duced almost exclusively by plasmacytoid dendritic cells pDC leading to containment of the virus and prevention of disease 25 Thus MHV infection represents a well suited model to investigate whether a particular hierarchy exists in the dependency on pDC derived type I IFNs which secure control of cytopathic viral in fection and protect the host from severe disease In this study we have used type I IFNR deficient ifnar H11002 H11002 bone marrow chimeric mice and conditionally gene targeted mice with cell type specific IF NAR deletion to elucidate whether type I IFN signaling is required on all nucleated cells We found that during MHV infection the presence of the IFNAR on LysM H11001 macrophages and CD11c H11001 con ventional dendritic cells cDC is of utmost importance whereas type I IFN responsiveness of other MHV target cells such as B cells appeared not to be critical for the control of the virus Overall our results indicate that cells from the hemopoietic system and in particular macrophages and cDCs are the prime target cells for type 1 IFNs during murine coronavirus infection Research Department Kantonal Hospital St Gallen St Gallen Switzerland Un idad de Investigacio nMe dica en Inmunoqu mica Hospital de Especialidades Centro Me dico Nacional Siglo XXI IMSS Mexico City Mexico Department of Immu nology Paul Ehrlich Institut Langen Germany and Department of Microbiology Columbia University Medical Center New York NY 10032 Received for publication May 20 2008 Accepted for publication November 12 2008 The costs of publication of this article were defrayed in part by the payment of page charges This article must therefore be hereby marked advertisement in accordance with 18 U S C Section 1734 solely to indicate this fact 1 This project received financial support from the Swiss National Science Foundation Deutsche Forschungsgemeinschaft and the National Institutes of Health Grant AI067804 to B R 2 Address correspondence and reprint requests to Dr Burkhard Ludewig Research Department Kantonsspital St Gallen 9007 St Gallen Switzerland E mail address burkhard ludewig kssg ch 3 Abbreviations used in this paper MHV mouse hepatitis virus pDC plasmacytoid dendritic cell cDC conventional dendritic cell IFNAR type I IFNR ALT alanine 2 oxoglutarate aminotransferase Copyright 2009 by The American Association of Immunologists Inc 0022 1767 09 2 00 The Journal of Immunology www jimmunol org at North Carolina State University Libraries on March 14 2015 http www jimmunol org Downloaded from Materials and Methods Mice and viruses C57BL 6 B6 mice were obtained from Charles River Laboratories Type I IFNR deficient mice ifnar H11002 H11002 Ref 26 on the B6 background were kindly provided by Dr Martin Bachmann Cytos Schlieren Switzerland and bred in our facilities R26 EYFP H11001 H11002 mice 27 were kindly provided by Dr Ari Waisman University of Mainz Mainz Germany R26 EYFP H11001 H11002 mice and mice expressing a loxP flanked ifnar1 ifnar1 fl fl 4 were bred with mice that express Cre recombinase specifically in B cells CD19 Cre T cells CD4 Cre T and B cells CD19 CreCD4 Cre mac rophages LysM Cre Ref 28 or CD11c H11001 dendritic cells CD11c Cre Ref 29 For the generation of bone marrow chimeric mice recipients were lethally irradiated with 900 rad from a linear accelerator Clinic of Radio Oncology Kantonal Hospital St Gallen St Gallen Switzerland and injected i v 1 day later with 2 H11003 10 7 of the indicated donor bone marrow cells Chimeric mice were maintained on antibiotic water contain ing sulfadoxin and trimethoprim Borgal Veterinaria for the following 3 wk Mice were used for experiments 8 10 wk after bone marrow recon stitution The degree of chimerism induced using this protocol has been routinely evaluated by reconstituting B6 mice expressing the congenic marker Thy1 2 with bone marrow cells derived from B6 Thy1 1 mice Chi merism in these control animals was always H1102297 MHV A59 was gen erated from a molecularly cloned cDNA 30 based on the Albany strain of MHV A59 and propagated on L929 cells GFP recombinant MHV was generated as previously described 31 Experiments were performed in accordance with Swiss Kantonal and Federal legislations Virus infections determination of virus titers liver enzyme values liver histology and IFN H9251 Mice were injected i p with 50 PFU of MHV A59 representing a low dose infection with maximal liver disease around day 5 comparable with the kinetics of systemic infection as described previously 25 To achieve maximal target cell infection in B6 mice and minimal infection associated death in ifnar H11002 H11002 mice a dose of 5 H11003 10 3 PFU GFP recombinant MHV 31 was used Intranasal infection was done with 5 H11003 10 4 PFU of MHV A59 because at this dose 100 of the mice were reproducibly infected and the virus did not spread systemically in B6 mice Mice were sacrificed at the indicated time points and organs were stored at H1100270 C until further analysis or disrupted for FACS analysis Blood was incubated at room temperature to coagulate and then centrifuged and serum was used for alanine 2 oxoglutarate aminotransferase ALT measurements using a Hitachi 747 autoanalyzer Virus titers in organs were deter mined from frozen organs after weighing and homogenization Viral titers were determined by standard plaque assay using L929 cells Liv ers were fixed in 4 formalin and embedded in paraffin Sections were stained with H and F4 80 PE CD11c PE and anti MHV N Alexa 647 N556 kindly provided by Dr Stuart Sidell Department of Cellular and Molecular Medicine University of Bristol Bristol U K Images were acquired using a Leica DMRA microscope and processed using Adobe Photoshop Adobe Systems Cell culture of primary cells and in vitro infections Bone marrow derived cDCs or pDCs were generated as described 25 with either GM CSF containing supernatant from the cell line X63 GM CSF kindly provided by Dr Antonius Rolink University of Basel Basel Switzerland or Flt3 L R pore size 0 4 H9262m was used to prevent cell cell contact between pDCs and macrophages cDCs Statistical analysis Statistical analyses were performed with Graphpad Prism 5 0 using either a nonpaired two tailed Student t test or one way ANOVA with Bonferroni posttest comparing the samples with their corresponding control group Survival curves were generated using the Kaplan Meier method and the significance of differences was calculated by the log rank test Statistical significance was defined as a value of p H11021 0 05 Results Early control of MHV depends on type I IFN responsiveness of hemopoietic cells To better define the cellular targets for the activity of type I IFNs bone marrow chimeras were generated using ifnar H11002 H11002 or B6 mice The chimeric mice that expressed the IFNAR on either hemopoi etic or nonhemopoietic cells were infected i p with 50 PFU of MHV A59 Because ifnar H11002 H11002 mice succumb to MHV infection rapidly 25 mice were sacrificed after 48 h and IFN H9251 produc tion severity of liver disease and viral titers in spleens livers and lungs were determined As shown in Fig 1A neither the lack of the IFNAR on hemopoietic nor that on nonhemopoietic cells pre cluded production of IFN H9251 Furthermore induction of IFN H9252 was not influenced by the absence of IFNAR on different cell subsets data not shown The lack of the IFNAR on radio resistant pa renchymal cells B63ifnar H11002 H11002 did not lead to significantly ele vated liver enzyme values whereas the absence of the IFNAR on bone marrow derived cells ifnar H11002 H11002 3B6 resulted in severe liver disease Fig 1C Moreover viral titers in livers spleens and lungs Fig 1B from these mice were significantly higher than in mice that expressed the IFNAR only on hemopoietic cells Most importantly the expression of IFNAR on the hemopoietic cells B63ifnar H11002 H11002 secured significantly longer survival of the mice Fig 1D These results indicate a clear hierarchy in the impor tance of the IFNAR expressed on hemopoietic vs nonhemopoietic cells the presence of the IFNAR on hemopoietic cells appears to be important to contain the virus within secondary lymphoid or gans and thereby contributes critically to the prevention of disease Target cells of MHV within the bone marrow derived cell compartment It is likely that those cells that are most easily infected by a cyto pathic virus and therefore rapidly lost during the infection are most dependent on the protection provided by the type I IFN system Working along this assumption we first determined which cell 1100 TARGET CELLS OF pDC DERIVED IFN at North Carolina State University Libraries on March 14 2015 http www jimmunol org Downloaded from populations within the hemopoietic compartment support MHV infection In a first set of experiments splenocytes from B6 or ifnar H11002 H11002 mice were infected in vitro with GFP recombinant MHV at a MOI of 1 After 12 h MHV replication in macrophages F4 80 H11001 CD11b H11001 neutrophils Ly6G H11001 CD11b H11001 cDCs CD11c H11001 B220 H11002 B cells CD19 H11001 CD4 H11001 T cells CD3 H11001 CD4 H11001 and CD8 H11001 T cells CD3 H11001 CD8 H11001 was determined by flow cytometry Fig 2A This analysis revealed that primary macrophages cDCs neutrophils and B cells could be infected with MHV and that the lack of IFNAR on these cells slightly increased their susceptibility To confirm whether this target cell tropism of MHV for particular leukocyte subsets can be reproduced in vivo B6 and ifnar H11002 H11002 mice were infected with 5 H11003 10 3 PFU of GFP recombinant MHV i p and the different spleen cell populations were probed for GFP ex pression 36 h postinfection using flow cytometric analysis as de scribed previously We could not detect GFP positive cells in the different splenocyte fractions derived from infected B6 mice Fig 2B top row suggesting that the intact type I IFN system in these mice had efficiently blocked viral replication below the level of detection Indeed macrophages cDCs B cells and neutrophils from infected ifnar H11002 H11002 mice showed significant GFP expression Fig 2B bottom row Other leukocyte populations such as CD4 H11001 and CD8 H11001 T lymphocytes Fig 2B and NK cells not shown did not exhibit significant GFP expression Furthermore B6 and ifnar H11002 H11002 mice were infected with 5 H11003 10 3 PFU of MHV and fluorescence microscopic analysis was performed using anti MHV nucleoprotein Ab to identify infected cells in situ Whereas MHV infected F4 80 H11001 cells in the red pulp Fig 2C and CD11c H11001 in the white pulp Fig 2D could be readily detected in spleens of ifnar H11002 H11002 mice colocalization of the MHV nucleoprotein with the B cell marker B220 Fig 2 C and D and with the neutrophil marker Ly6G not shown was rare As expected only very few MHV infected cells were found in B6 mice not shown thus con firming the high susceptibility of cDCs and macrophages to MHV infection in the absence of a functional type I IFN system Requirement of IFNAR expression on different leukocyte populations To assess the differential requirement of type I IFN responsiveness of the MHV target populations we used a set of conditionally gene targeted mice Crossing of mice with a loxP flanked ifnar1 ifnar1 fl fl with mice that express the Cre recombinase in a cell type specific manner resulted in deletion of the IFNAR in T cells CD4 Cre H11001 H11002 ifnar fl fl 4 in B cells CD19 Cre H11001 H11002 ifnar1 fl fl 4 in T and B cells CD4 Cre H11001 H11002 CD19 Cre H11001 H11002 ifnar1 fl fl in macrophages neutrophils and some dendritic cells LysM Cre H11001 H11002 ifnar1 fl fl Ref 28 and spe cifically in CD11c H11001 cDCs CD11c Cre H11001 H11002 ifnar1 fl fl Ref 29 These mice were infected with MHV and survival was monitored for 2 wk As shown in Fig 3A the expression of the IFNAR on the surface of LysM H11001 or CD11c H11001 cells was essential for survival since LysM Cre H11001 H11002 ifnar1 fl fl and CD11c Cre H11001 H11002 ifnar1 fl fl mice succumbed to the infection LysM Cre H11001 H11002 ifnar1 fl fl developed a more severe phenotype with lethal disease after 4 days of infection Likewise LysM Cre H11001 H11002 ifnar1 fl fl mice showed the most severe liver pathology with sig nificantly elevated ALT values as early as day 2 postinfection Fig 3B and a massive damage of liver tissue Fig 3C Because neu trophils can be infected with MHV in vivo Fig 2B we determined next whether the presence of neutrophils in LysM Cre H11001 H11002 ifnar1 fl fl FIGURE 1 Type I IFN responsiveness of bone marrow derived cells is essential for early control of MHV infection Bone marrow chimeric mice B63ifnar H11002 H11002 ifnar H11002 H11002 3B6 B63B6 ifnar H11002 H11002 3ifnar H11002 H11002 were infected i p with 50 PFU of MHV A59 After 48 h IFN H9251 concentration in serum and spleens A viral titers in livers spleens and lungs B ALT values in serum C were determined Results represent means H11006 SD of five to six mice per group D Survival of bone marrow chimeric mice Health status was monitored twice daily and moribund animals were euthanized n H11005 5 6 mice per group Statistical analysis was performed using one way ANOVA with Bonferroni posttest H11569H11569H11569 p H11021 0 001 H11569H11569 p H11021 0 01 H11569 p H11021 0 05 n s p H11022 0 05 n d Not detected Survival curves were generated using the Kaplan Meier method and the significance of differences was calculated by the log rank test Statistical significance was defined as H11569H11569H11569 p H11021 0 001 H11569H11569 p H11021 0 01 H11569 p H11021 0 05 n s p H11022 0 05 1101The Journal of Immunology at North Carolina State University Libraries on March 14 2015 http www jimmunol org Downloaded from could affect viral distribution and virus mediated disease To this end neutrophils were depleted in LysM Cre H11001 H11002 ifnar1 fl fl and B6 mice using the NIMP R14 Ab 32 NIMP R14 mediated deple tion of neutrophils in B6 mice had no significant effect on MHV replication in the major target organs data not shown Likewise MHV replication and infection associated hepatitis was not af fected by the absence of neutrophils in LysM Cre H11001 H11002 ifnar1 fl fl mice Fig 3D indicating that in these mice it is the absence of the IFNAR on macrophages not on neutrophils that determines the high susceptibility to MHV infection CD11c Cre H11001 H11002 ifnar1 fl fl FIGURE 2 MHV target cells in vitro and in vivo A 10 6 splenocytes or low density cell enriched fractions for cDC analysis from ifnar H11002 H11002 or B6 mice were infected with GFP recombinant MHV MOI 1 Cells were harvested 12 h later and stained for the indicated surface molecules macrophages F4 80 H11001 CD11b H11001 neutrophils Ly6G H11001 CD11b H11001 cDCs B220 H11002 CD11c