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JOURNAL OF VIROLOGY Feb 2010 p 1289 1301 Vol 84 No 3 0022 538X 10 12 00 doi 10 1128 JVI 01281 09 Copyright 2010 American Society for Microbiology All Rights Reserved Cellular Immune Responses to Severe Acute Respiratory Syndrome Coronavirus SARS CoV Infection in Senescent BALB c Mice CD4 H11001 T Cells Are Important in Control of SARS CoV Infection H17188 Jun Chen 1 Yuk Fai Lau 1 Elaine W Lamirande 1 Christopher D Paddock 2 Jeanine H Bartlett 2 Sherif R Zaki 2 and Kanta Subbarao 1 Laboratory of Infectious Diseases National Institute for Allergy and Infectious Diseases NIH Bethesda Maryland 20892 1 and Infectious Disease Pathology Activity Centers for Disease Control and Prevention Atlanta GA 30333 2 Received 22 June 2009 Accepted 27 October 2009 We characterized the cellular immune response to severe acute respiratory syndrome coronavirus SARS CoV infection in 12 to 14 month old BALB c mice a model that mimics features of the human disease Following intranasal administration the virus replicated in the lungs with peak titers on day 2 postinfection Enhanced production of cytokines tumor necrosis factor alpha TNF H9251 and interleukin 6 IL 6 and chemokines CXCL10 CCL2 CCL3 and CCL5 correlated with migration of NK cells macrophages and plasmacytoid dendritic cells pDC into the lungs By day 7 histopathologic evidence of pneumonitis was seen in the lungs when viral clearance occurred At this time a second wave of enhanced production of cytokines TNF H9251 IL 6 gamma interferon IFN H9253 IL 2 and IL 5 chemokines CXCL9 CXCL10 CCL2 CCL3 and CCL5 and receptors CXCR3 CCR2 and CCR5 was detected in the lungs associated with an influx of T lymphocytes Depletion of CD8 H11545 T cells at the time of infection did not affect viral replication or clearance However depletion of CD4 H11545 T cells resulted in an enhanced immune mediated interstitial pneumonitis and delayed clearance of SARS CoV from the lungs which was associated with reduced neutralizing antibody and cytokine production and reduced pulmonary recruitment of lymphocytes Innate defense mechanisms are able to control SARS CoV infection in the absence of CD4 H11545 and CD8 H11545 T cells and antibodies Our findings provide new insights into the pathogenesis of SARS demonstrating the important role of CD4 H11545 but not CD8 H11545 T cells in primary SARS CoV infection in this model The global outbreak of severe acute respiratory syndrome SARS in 2003 that infected more than 8 000 people in 29 countries across five continents with 774 deaths reported by the World Health Organization 54 was caused by a highly contagious coronavirus designated SARS CoV 33 The el derly were more likely to die from SARS CoV infection than younger people 7 with a case fatality rate of 50 in people older than 65 years 14 53 Disease pathogenesis in SARS is complex with multiple factors leading to severe pulmonary injury and dissemination of the virus to other organs High viral load systemic infection a cytokine storm with high levels of CXCL10 IP 10 CCL3 MIP 1H9251 and CCL2 MCP 1 massive lung infiltration by monocytes and macrophages and rapid depletion of T cells are hallmarks of SARS 5 13 15 21 28 35 The role of neutralizing antibodies Abs in protection from SARS CoV infection has been well documented Virus specific neutralizing Abs reduce viral load protect against weight loss and reduce histopathology in animal models 42 47 48 Although the role of type I interferons IFNs in the natural history of SARS is controversial 5 9 59 the innate defense system appears to be critical for controlling SARS CoV replication in mice 23 41 Mice lacking normal innate signaling due to STAT1 or MyD88 deficiency are highly sus ceptible to SARS CoV infection Virus specific T cell re sponses are present in convalescent patients with SARS 27 55 However little is known about the role of T cells in the acute phase of SARS Several mouse models have been developed for the in vivo study of SARS pathogenesis However no single model accu rately reproduces all aspects of the human disease SARS CoV replicates in the upper and lower respiratory tracts of 4 to 8 week old mice and is cleared rapidly infection is associated with transient mild pneumonitis and cytokines are not detect able in the lungs 20 42 49 A SARS CoV isolate that was adapted by serial passage in mice MA 15 replicates to a higher titer and for a longer duration in the lungs than the unadapted Urbani virus and is associated with viremia and mortality in young mice 36 but the histologic changes in the lungs are caused by high titers of virus and cell death without significant infiltrates of inflammatory cells The heightened susceptibility of elderly patients to SARS led us to develop a pneumonia model in 12 to 14 month old mo BALB c mice using the Urbani virus In this model pulmonary replication of virus was associated with signs of clinical illness and his topathological evidence of disease characterized by bronchi olitis interstitial pneumonitis diffuse alveolar damage and fibrotic scarring 3 thus resembling SARS in the elderly We evaluated the host response to SARS CoV infection by exam ining the gene expression profile in the senescent mouse model and found a robust response to virus infection with an in creased expression of several immune response and cell to cell Corresponding author Mailing address Laboratory of Infectious Diseases NIAID NIH Building 33 Room 3E13C 1 33 North Drive Bethesda MD 20892 3203 Phone 301 451 3839 Fax 301 480 5719 E mail KSUBBARAO niaid nih gov These two authors contributed equally H17188 Published ahead of print on 11 November 2009 1289 on August 14 2015 by guest http jvi asm org Downloaded from signaling genes including those for tumor necrosis factor alpha TNF H9251 interleukin 6 IL 6 CCL2 CCL3 CXCL10 and IFN H9253 1 In this study we characterize the cellular immune response to SARS CoV infection in 12 to 14 mo BALB c mice in terms of the protein and gene expression of inflammatory mediators migration of inflammatory cells and virus specific T cell re sponses in the lungs during the course of disease We evaluated the role of T cells in disease pathogenesis and viral clearance by depleting T cell subsets at the time of infection and found an important role for CD4 H11001 T cells but not CD8 H11001 T cells in primary infection with SARS CoV in this model MATERIALS AND METHODS Virus SARS CoV Urbani strain a generous gift from L J Anderson and T G Ksiazek Centers for Disease Control and Prevention Atlanta GA was propagated in Vero cells with a titer of 10 6 5 50 tissue culture infective doses TCID 50 ml Vero cells were maintained in OptiPro SFM Invitrogen CA All work with infectious virus was performed inside a biosafety cabinet in a biosafety level 3 facility Mouse model of SARS The animal studies were approved by the National Institutes of Health Animal Care and Use Committee Female BALB c mice 12 to 14 mo were purchased from Taconic Germantown NY We administered 10 5 TCID 50 of SARS CoV intranasally i n to mice as previously described 42 Leibovitz 15 medium was used to mock infect control groups of mice Lungs were harvested at serial time points without perfusion for virus titration cytokine assays histopathology and flow cytometry Depletion of T cell subsets Monoclonal antibodies MAb Gk1 5 specific for mouse CD4 2 43 specific for mouse CD8 and SFR3 DR5 specific for human leukocyte antigen as an isotype control were used for in vivo depletion as described previously 17 All MAbs are rat IgG2b and were prepared as ascites fluid and delipified by the National Cell Culture Center Biovest International Mice received 1 mg ml of MAb for each dose given intraperitoneally i p 3 days before and 3 7 and 10 days after infection Passive transfer of serum Ab Postinfection hyperimmune SARS HIS anti serum was generated in BALB c mice following SARS CoV infection Mice treated with anti CD4 or anti CD4 and anti CD8 MAbs were given an i p injection of 500 H9262l of serum on day 7 postinfection p i The control group received normal nonimmune BALB c mouse serum Harlan Indianapolis IA the experimental groups received either undiluted HIS or a 1 4 dilution in phosphate buffered saline PBS of HIS Lungs were harvested on day 9 p i Virus titration Supernatants of 10 wt vol lung homogenates were pre pared and titrated on Vero cell monolayers in 24 and 96 well plates as previously described 42 Virus titers are expressed as TCID 50 per g of tissue The lower limit of detection was 10 1 5 TCID 50 g Histopathology and immunohistochemistry IHC Mice were euthanized by cervical dislocation on days 7 9 and 12 p i The lungs were inflated with 10 formalin and embedded in paraffin wax Lung tissue sections were prepared for histopathological examination and evaluated by using conventional hematoxylin and eosin H Protein Sciences Corporation Meriden CT for 16 h with Golgi plug Golgi stop and anti CD107a b MAbs added for the last 8 h Cells were then stained with anti CD4 peridinin chlorophyll protein PerCP and PE conjugated rat anti mouse IFN H9253 MAbs clone XMG1 2 using the Cytofix Cytoperm kit for the detection of IFN H9253 positive CD4 H11001 lymphocytes The final analysis and graphical output were performed using FlowJo software Tree Star Inc Cell populations were iden tified as CD11c H11001 PDCA1 H11001 for plasmacytoid dendritic cells pDC CD3 H11002 DX5 H11001 for NK cells CD3 H11001 DX5 H11001 for NK T cells CD11b H11001 F4 80 H11001 for macrophages and CD11c H11002 Gr1 H11001 for neutrophils Neutralizing Ab assay Twofold dilutions of heat inactivated serum were tested in a microneutralization assay using 100 TCID 50 of SARS CoV as previ ously described 42 The presence of viral cytopathic effect was read on days 3 and 4 ELISA for Ab Microtiter plates were coated with 100 ng per well of recom binant truncated SARS CoV S protein Protein Sciences Corp Meriden CT Serially diluted heat inactivated mouse sera were incubated overnight Bound Abs were detected with alkaline phosphatase conjugated goat anti mouse IgG and p nitrophenyl phosphate Sigma MO substrate An optical absorbance of H110220 2 at 405 nm was considered positive Statistical analysis Statistically significant differences between groups of mice were determined by Student s t test and the Mann Whitney U test P values of H110210 05 were considered significant RESULTS SARS CoV infection is associated with pneumonitis in 12 to 14 mo BALB c mice Pulmonary viral infection was induced in senescent BALB c mice by i n administration of 10 5 TCID 50 SARS CoV High titers of virus 10 8 6 TCID 50 g were de 1290 CHEN ET AL J VIROL on August 14 2015 by guest http jvi asm org Downloaded from tected in the lungs as early as day 2 p i Titers remained high for several days H1101110 5 TCID 50 g on day 7 after which titers decreased significantly and were just above the limit of detec tion 10 1 5 TCID 50 g by day 9 p i data not shown Histologic examination of the lungs on day 2 p i showed focal perivascular infiltrates comprised predominantly of mac rophages and some lymphocytes and airway lesions character ized by bronchiolar epithelial necrosis and luminal necrotic debris data not shown On day 7 p i multifocal interstitial infiltrates comprised predominantly of mononuclear cells were identified while high titers of virus remained in the lungs Perivascular infiltrates persisted on day 9 Diffuse alveolar damage and fibrotic scarring were seen on day 9 and day 12 p i as described previously references 37 and 38 and data not shown SARS CoV infection induces a biphasic pattern of increased expression of inflammatory mediators in the lungs To identify the inflammatory mediators involved in the pathogenesis of SARS CoV induced pneumonitis we measured the protein levels of cytokines and chemokines in lung homogenates Fig 1A and B We describe events in the first 1 to 5 days p i as early and those during 6 to 13 days p i as late events and noted four patterns with early late biphasic or no increases respec tively First a modest increase in the levels of IFN H9251 and IFN H9252 was detected in the lungs early on day 3 following SARS CoV infection but no statistical difference was found compared to mock infected control samples P H11022 0 05 Sec ond a marked increase in inflammatory chemokines CCL5 RANTES and CXCL9 MIG accompanied by an increase of inflammatory cytokines IFN H9253 IL 2 and IL 5 was detected in the lungs predominantly at day 7 the late stage of infection P H11021 0 05 Third a biphasic pattern of significantly increased production of proinflammatory cytokines TNF H9251 and IL 6 and chemokines CCL2 MCP 1 CCL3 MIP 1H9251 and CXCL10 IP 10 was seen both early and late following infection P H11021 0 05 The first wave of these cytokines and chemokines was detected as early as day 2 p i High levels of these cytokines and che mokines remained on day 3 and decreased dramatically on day 5 p i By day 7 when pneumonitis was observed we detected a second peak of production of these proteins Fourth IL 4 IL 10 and IL 12 were not detectable throughout the course of disease data not shown Consistent with the findings of chemokine protein levels a biphasic pattern of increased expression of chemokine mRNA was observed in the lungs early and late in infection Fig 1C An increase in mRNA transcripts for CCL2 MCP 1 CCL3 MIP 1H9251 and CXCL10 IP 10 was detected in the lung early on days 2 and 3 p i when peak viral replication occurred Com pared to those in mock infected animals these mRNA tran scripts in SARS CoV infected lungs significantly increased at day 2 p i declined thereafter and then showed a moderate increase on day 7 In contrast mRNA transcripts for CCL5 RANTES and CXCL9 MIG increased predominantly late in infection on days 5 and 7 when histopathologic evidence of pneumonitis was seen in the infected lungs An increase in mRNA transcripts for CCR2 CCR5 and CXCR3 which are receptors for the upregulated chemokines CCR2 for CCL2 MCP 1 CCR5 for CCL3 MIP 1a and CCL5 RANTES and CXCR3 for CXCL9 MIG and CXCL10 IP 10 was also found in the lungs after infection with SARS CoV Fig 1C Compared to those in mock infected lungs a 4 fold increase in CCR2 mRNA and a H110229 fold increase in CCR5 or CXCR3 mRNA were detected around days 7 to 9 p i The profile of increased expression of these receptor transcripts correlated closely with the upregulation of their ligands in the SARS CoV infected lung in the late phase of infection SARS CoV infection induces two waves of recruitment of inflammatory cells into the lungs To analyze the cellular re sponses to SARS CoV infection we evaluated cell migration into the lungs FACS analyses revealed a local accumulation of leukocytes in the lungs following infection Fig 2A and B We observed a significant increase in the number of CD45 H11001 leu kocytes in the lungs of SARS CoV infected mice 7 days p i P H11005 0 0015 The number of CD45 H11001 leukocytes slightly in creased as early as day 2 p i reaching a peak on day 7 and progressively declining by days 9 and 12 p i Three patterns of cell migration were detected in the lungs following SARS CoV infection First plasmacytoid DC pDC migrated into the lungs at the early phase of infection The number of pDC peaked on day 2 P H11005 0 001 followed by a sharp decline by day 7 and day 9 p i P H11021 0 001 Second the pulmonary migration of inflammatory cells such as NK T cells NK cells macrophages and CD4 H11001 T cells began on day 2 increased rapidly to reach a peak on day 7 and or 9 P H11021 0 01 and declined thereafter Third neutrophils and CD8 H11001 T cells mi grated into the lungs at the late phase of infection A large number of neutrophils were detected in the lungs of mock infected senescent mice and these numbers increased mod estly on day 7 p i P H11021 0 05 The number of CD8 H11001 T cells increased sharply on days 7 and 9 P H11021 0 05 remaining at a high level or declining by day 12 p i The two waves of inflam matory cells recruited into the lungs at the early and late phases of infection correlated well with the biphasic expression of chemokines and the receptors in the lungs of SARS CoV infected mice Fig 1B and C SARS CoV infection induces an enhanced virus specific T cell response in the lungs To determine the function of T cells in response to SARS CoV infection we measured the number and frequency of IFN H9253 producing CD8 H11001 and CD4 H11001 T cells iso lated from the lungs of mock and SARS CoV infected mice on day 7 p i by intracellular staining with and without exogenous stimulation with phorbol myristate acetate PMA ionomycin or viral peptides Fig 2C and D FACS analysis revealed an in creased frequency of CD8 H11001 IFN H9253 H11001 and CD4 H11001 IFN H9253 H11001 cells seen in the lungs of SARS CoV infected mice following exogenous stimulation with SARS CoV S protein and to a lesser extent nucleocapsid N proteins and PMA ionomycin Compared to that in mock infected mice the number of CD8 H11001 IFN H9253 H11001 cells increased from 1 6 H11003 10 4 to 4 3 H11003 10 4 cells lung in SARS CoV infected mice following stimulation with PMA ionomycin and from0to1H11003 10 5 cells lung when stimulated with a pool of overlapping peptides from the SARS CoV S protein The num ber of CD4 H11001 IFN H9253 H11001 cells increased from 1 1 H11003 10 4 cells lung in mock infected mice to 3 7 H1100310 4 cells lung in SARS CoV infected mice when stimulated with PMA ionomycin and from 0 to 1 9 H11003 10 5 cells lung when stimulated with SARS CoV S peptides Stim ulation with a pool of overlapping peptides from the SARS CoV N protein did not significantly increase IFN H9253 production by T cells This result indicates that antigen specific T cell responses occurred to the SARS CoV S protein but not to the N protein VOL 84 2010 PATHOGENESIS OF SARS CoV INFECTION IN SENESCENT MICE 1291 on August 14 2015 by guest http jvi asm org Downloaded from FIG 1 Kinetics of inflammatory mediators in the lungs following SARS CoV infection Protein levels of cytokines A and chemokines B and fold increase in gene transcripts of chemokines and the receptors C were measured in the lungs at the indicated time points following i n inoculation of 10 5 TCID 50 SARS CoV Data are shown as mean H11006 standard error of the mean SEM for four mice at each time point and the units are reported next to the name of the protein P H11021 0 05 for the comparison between mock and SARS infected groups by Student s t test 1292 on August 14 2015 by guest http jvi asm org Downloaded from CD4 H11001 T cells exhibited a higher IFN H9253 response to S peptides than CD8 H11001 T cells Taking the above data together our studies showed an en hanced virus specific T cell response in the lungs that coincided with the development of pneumonitis and was accompanied by viral clearance see Fig 7 and data not shown These data indi cate that T cells may play an important role in SARS CoV infec tion FIG 2 Presence of inflammatory cells in the lungs of mice following SARS CoV infection A FACS analysis of inflammatory cells CD11c H11001 PDCA1 H11001 for pDC CD3 H11002 DX5 H11001 for NK CD3 H11001 DX5 H11001 for NKT CD11b H11001 F4 80 H11001 for macrophages and CD11c H11002 Gr1 H11001 for neutrophils in mock infected and SARS CoV infected lungs Cells for analysis are gated on a CD45 H11001 population B Total number of inflammatory cells isolated from the lungs on the indicated day p i Data are the means H11006 SEMs for three to five mice analyzed at each time point Results from one representative experiment out of three are shown P H11021 0 05 P H11021 0 01 for the comparison between mock and SARS CoV infected groups by Student s t test C FACS analysis of IFN H9253 producing CD8 H11001 and CD4 H11001 T cells isolated from the lungs of SARS CoV infected mice on day 7 p i with or without ex vivo stimulation with PMA ionomycin or SARS CoV S or N peptides Cells for analysis are gated on a CD3 H11001 population D Total number of C