【病毒外文文献】2005 Cytokine Responses in Severe Acute Respiratory Syndrome Coronavirus-Infected Macrophages In Vitro_ Possible Relevan

JOURNAL OF VIROLOGY June 2005 p 7819 7826 Vol 79 No 12 0022 538X 05 08 00H110010 doi 10 1128 JVI 79 12 7819 7826 2005 Copyright 2005 American Society for Microbiology All Rights Reserved Cytokine Responses in Severe Acute Respiratory Syndrome Coronavirus Infected Macrophages In Vitro Possible Relevance to Pathogenesis Chung Y Cheung 1 Leo L M Poon 1 Iris H Y Ng 1 Winsie Luk 1 Sin Fun Sia 1 Mavis H S Wu 1 Kwok Hung Chan 1 Kwok Yung Yuen 1 Siamon Gordon 2 Yi Guan 1 and Joseph S M Peiris 1 Department of Microbiology The University of Hong Kong Queen Mary Hospital Hong Kong Special Administrative Region People s Republic of China 1 and Sir William Dunn School of Pathology University of Oxford Oxford United Kingdom 2 Received 19 August 2004 Accepted 9 February 2005 The pathogenesis of severe acute respiratory syndrome SARS remains unclear Macrophages are key sentinel cells in the respiratory system and it is therefore relevant to compare the responses of human macrophages to infections with the SARS coronavirus SARS CoV and other respiratory viruses Primary human monocyte derived macrophages were infected with SARS CoV in vitro Virus replication was monitored by measuring the levels of positive and negative strand RNA by immunofluorescence detection of the SARS CoV nucleoprotein and by titration of the infectious virus The gene expression profiles of macrophages infected with SARS CoV human coronavirus 229E and influenza A H1N1 virus were compared by using microarrays and real time quantitative reverse transcriptase PCR Secreted cytokines were measured with an enzyme linked immunosorbent assay SARS CoV initiated viral gene transcription and protein synthesis in macrophages but replication was abortive and no infectious virus was produced In contrast to the case with human coronavirus 229E and influenza A virus there was little or no induction of beta interferon IFN H9252 in SARS CoV infected macrophages Furthermore SARS CoV induced the expression of chemokines such as CXCL10 IFN H9253 inducible protein 10 and CCL2 monocyte chemotactic protein 1 The poor induction of IFN H9252 a key component of innate immunity and the ability of the virus to induce chemokines could explain aspects of the pathogenesis of SARS A novel coronavirus was identified as the causative agent of severe acute respiratory syndrome SARS 20 and is believed to be of zoonotic origin 10 The previously known human coronaviruses 229E HCoV 229E and OC43 have only been linked with the common cold However several animal coro naviruses result in severe animal diseases affecting the respi ratory or gastrointestinal tract or cause disseminated infec tions Compared to common respiratory viral infections SARS is unusually severe with an overall case fatality rate of about 10 The infection is not localized to the respiratory tract and the causative virus is also detected in the gastrointestinal and urinary systems 21 Contrary to what is seen with other com mon respiratory viral infections such as infections with HCoV 229E 2 and influenza A virus 13 14 the viral load in the upper respiratory tract in patients with SARS progressively increases peaking on about day 10 after the onset of symptoms 21 This suggests that the innate arm of the immune system may be unable to adequately control SARS CoV infection The appearance of antibodies in the second week of the illness appears to coincide with a falling viral load in the upper re spiratory tract Macrophages are key cells for host defense and are abun dant within all tissues of the body including the respiratory system They are potent producers of cytokines that are crucial components of innate immunity and potential mediators of immunopathology 9 Genetic resistance to strains of the coronavirus mouse hepatitis virus is associated with the ability of the virus to replicate in macrophages 1 31 On the other hand feline infectious peritonitis is a disease caused by a coronavirus in which prior immunity or passive antibodies in crease the severity of the disease 33 In this disease macro phages are the main target cells for virus replication and antiviral antibodies enhance the replication of the virus in macrophage cultures in vitro 12 This has led to concern about whether antibody mediated enhancement of disease may be relevant to the pathogenesis of SARS It is therefore relevant to study the interaction between SARS CoV and macrophages In this study we investigate the response of human macrophages to infection with SARS CoV and compare it with the responses to HCoV 229E and influ enza A virus MATERIALS AND METHODS Viruses SARS CoV strain HK39849 was propagated in FRhK 4 cells 20 All procedures involving the use of live SARS CoV were performed in a bio safety level 3 facility The influenza A Hong Kong 54 98 H1N1 virus was prop agated in MDCK cells 5 HCoV 229E was propagated in MRC 5 cells and both the virus and cells were obtained from the American Type Culture Collec tion Manassas Va Amicon Ultra 15 centrifugal filter units Millipore Corpo Corresponding author Mailing address Department of Microbi ology University Pathology Building Queen Mary Hospital Pokfulam Road Hong Kong Special Administrative Region People s Republic of China Phone 852 28554888 Fax 852 28551241 E mail malik hkucc hku hk 7819 on June 23 2015 by UNIV OF GEORGIA http jvi asm org Downloaded from ration Bedford Mass were used for the differential filtration of SARS CoV cultures Cells Primary human monocyte derived macrophages were prepared as pre viously described and used after 14 days of differentiation in vitro in 5 autol ogous serum 5 Two days prior to the experiment the cell culture medium was changed to macrophage serum free medium Invitrogen Carlsbad Calif Infection of cells Differentiated macrophages from monocytes were seeded at 2 H11003 10 5 cells per well in 24 well tissue culture plates and were infected at a multiplicity of infection MOI of 1 to 2 After 60 min of virus adsorption at 37 C the virus inoculums were removed and the cells were washed and incubated in macrophage serum free medium supplemented with 0 6 mg liter penicillin and 60 mg liter streptomycin Samples of culture supernatants were collected and stored at H1100270 C for virus titration or cytokine analysis Other cells were seeded at 2 H11003 10 5 cells per well in 24 well tissue culture plates and were infected at an MOI of 1 to 2 After 60 min of virus adsorption at 37 C the virus inoculums were removed and infected cells were washed with warmed culture medium and incubated with the original maintenance medium Infectious viral titers in the supernatants were determined by titration at half log 10 dilutions on FRhK 4 cell monolayers in quadruplicate Cells were seeded in 96 well plates and used at about 90 confluence The 50 tissue culture infective dose was determined by the method of Reed and Muench 24 RNA extraction Total cellular RNAs were extracted with an RNeasy RNA Mini kit QIAGEN Hilden Germany with DNase digestion according to the manufacturer s instructions Extracted RNAs were stored at H1100270 C until use Microarray analysis Extracted RNAs were examined for human genome wide gene expression with a Human Genome U133A GeneChip probe array Affymetrix Inc Santa Clara Calif by the use of oligonucleotide probe sets interrogating approximately 21 000 transcripts Quality control GeneChip hy bridization and raw data analysis were performed according to the manufactur er s instructions Genome Centre University of Hong Kong Total RNAs from macrophages isolated from three experiments from separate donors were ex tracted and pooled Double stranded cDNAs were synthesized by means of the SuperScript Choice system Invitrogen and a GeneChipT7 Oligo dT promoter primer kit Affymetrix Inc The cDNAs were subjected to in vitro transcription in the presence of biotin labeled ribonucleotides by means of a BioArray High Yield RNA transcript labeling kit Affymetrix Inc The biotin labeled cRNAs were chemically fragmented and hybridized to the GeneChip probe array Using the EukGE WS2 fluidics protocol we stained the probe array with a streptavidin R phycoerythrin conjugate Molecular Probes Eugene Oreg The image was scanned by a GeneChip scanner Affymetrix Inc at an excitation wavelength of 488 nm The amount of light emitted at 570 nm was proportional to the bound target for each probe set on the probe array The data generated were analyzed with Microarray Suite Expression Analysis software version 5 1 Affymetrix Inc Real time quantitative RT PCR for cytokine and viral gene expression Su perscript II reverse transcriptase RT Invitrogen and an oligo dT primer Invitrogen were used to convert mRNAs to cDNAs according to the manufac turer s instructions The levels of cytokine mRNA and viral RNA were measured by real time quantitative PCR using a LightCycler Roche Mannheim Ger many Each 20 H9262l reaction mixture was composed of 5 H9262l of diluted cDNA added to a master mix consisting of 2 H9262l of DNA Master SYBR green 4 mM MgCl 2 and a 0 5 H9262M concentration of each primer The primer sequences were as follows for beta interferon IFN H9252 forward 3H11032 GCC GCA TTG ACC ATC T 5H11032 and reverse 3H11032 CAC AGT GAC TGT ACT CCT 5H11032 for CXCL10 IFN H9253 inducible protein 10 IP 10 forward 3H11032 CTG ACT CTA AGT GGC ATT 5H11032 and reverse 3H11032 TGA TGG CCT TCG ATT CTG 5H11032 and for CCL2 monocyte chemotactic protein 1 MCP 1 forward 3H11032 CAT TGT GGC CAA GGA GAT CTG 5H11032 and reverse 3H11032 CTT CGG AGT TTG GGT TTG CTT 5H11032 Fluorescence readings were taken at the end of each extension cycle After a denaturation cycle at 95 C for 10 min the temperature cycling conditions for IFN H9252 and CCL2 MCP 1 were 40 cycles consisting of denaturation at 95 C for 10 s annealing at 65 C for 5 s and extension at 72 C for 11 s with acquisition at 72 C For CXCL10 IP 10 the conditions were 40 cycles consisting of denaturation at 95 C for 10 s annealing at 60 C for 5 s and extension at 72 C for 9 s with extension at 72 C After a melting curve analysis from 60 C to 95 C with fluorescence readings taken at 0 1 C s a final cooling step was carried out to reduce the rotor temperature to 40 C The primers and conditions for the SARS CoV Orf1b and nucleoprotein genes have been described previously 22 23 Positive and negative controls were included in each run and the levels of cytokine mRNA were normalized to the H9252 actin level Quantitation of cytokines Cytokines produced from macrophages were mea sured by specific enzyme linked immunosorbent assays ELISA R D Systems Minneapolis Minn according to the manufacturer s instructions Culture su pernatants were UV irradiated for 20 min to inactivate infectious viruses prior to assay in a biosafety level 3 facility Previous experiments have confirmed that cytokine levels are not affected by the dose of UV radiation used 5 RESULTS In SARS CoV infected macrophages there was an increase in the copy numbers of both the positive and negative RNA strands of the SARS CoV ORF 1b and nucleocapsid genes Fig 1A over the first few hours postinfection Viral RNA levels in macrophages peaked at modest levels at about 6 h postinfection but they continued to increase in FRhK 4 cells reaching much higher absolute levels Fig 1A and B In SARS CoV permissive cells e g FRhK 4 cells there was a marked excess of positive strand RNA for both Orf1b and the nucleoprotein Fig 1B as expected 26 In contrast compa rable amounts of positive and negative sense RNAs for both Orf1b and the nucleoprotein were detected in macrophages suggesting that macrophages do not support efficient viral rep lication SARS CoV nucleoprotein expression in SARS CoV infected macrophages was demonstrated by use of a mouse monoclonal antibody 4D11 and H1102290 of macrophages showed nucleoprotein expression when infected at an MOI of 1 to 2 Fig 2 However no infectious virus was detected in the supernatant of virus infected macrophages for up to 7 days postinfection indicating that virus infection of these cells was abortive Table 1 Increasing the MOI infecting macrophages to 20 did not further increase viral RNA levels or lead to productive virus replication In contrast virus infected FRhK 4 cells produced infectious virus titers up to 10 5 50 tissue culture infective doses ml data not shown peaking at about 2 to 3 days postinfection There was no increase in positive or negative strand viral RNA or viral antigen expression as detected by immunofluo rescence in macrophages or FRhK 4 cells infected with UV inactivated SARS CoV data not shown In a microarray analysis of the cellular gene expression of SARS CoV infected human macrophages it was striking that in contrast to influenza A virus SARS CoV failed to induce significant IFN H9251 H9252 gene expression Table 2 These findings were confirmed by independent experiments in which the IFN H9251 and H9252 mRNA levels were determined by real time quantitative RT PCR Fig 3 and the amounts of secreted protein were assayed by ELISA data not shown Parallel experiments with influenza A virus and HCoV 299E showed that unlike SARS CoV these viruses were strong inducers of IFN H9252 It is intriguing that despite the absence of IFN H9251 H9252 induction in SARS CoV infected cells many of the IFN stim ulated gene ISG products were up regulated Table 2 RT PCRs for the interferon like cytokines interleukin 28 IL 28 and IL 29 not included in the Genechip microarray demonstrated that there was no up regulation of either of these genes by SARS CoV In contrast both HCoV 229E and influenza A virus induced the gene expression of both of these cytokines data not shown Real time quantitative RT PCR analysis was used to con firm the microarray data which showed an early induction of several chemokines such as CXCL10 IP 10 and CCL2 MCP 1 in SARS CoV infected macrophages Table 3 and Fig 4A 7820 CHEUNG ET AL J VIROL on June 23 2015 by UNIV OF GEORGIA http jvi asm org Downloaded from ELISAs for CXCL10 IP 10 and CCL2 MCP 1 in macrophage culture supernatants confirmed that SARS CoV induced CXCL10 IP 10 and CCL2 MCP 1 secretion in macrophages in the first few hours after infection Fig 4B UV irradiation of SARS CoV partially abrogated the induction of these chemo kines by 40 to 60 despite a complete loss of SARS CoV gene expression data not shown suggesting that viral repli cation was only partly necessary for the induction of these chemokine genes In order to confirm that cytokines carried over in the SARS CoV inoculum were not responsible for the induction of these chemokine genes we tested the effect of differential filtration on the virus inoculum After filtering the virus inoculum through a 0 2 H9262m filter to remove large debris we passed it FIG 1 Nonproductive replication of SARS CoV in human macrophages Differentiated primary human monocyte derived macrophages A and FRhK 4 cells B were seeded in 24 well plates 2 H11003 10 5 cells per well on glass coverslips Cells were infected at an MOI of 1 to 2 and RNAs were extracted at 3 6 and 15 h postinfection The levels of positive solid lines and negative dotted lines RNA strands of the SARS CoV Orf1b and nucleoprotein genes were determined by real time quantitative RT PCR The data show means of duplicate cultures from the same donor and are representative of three independent experiments with similar results FIG 2 Human macrophages were mock treated A or infected with SARS CoV B and C and fixed with methanol for 15 min at 15 h postinfection A mouse monoclonal antibody 4D11 specific for the SARS CoV nucleoprotein K H Chan unpublished results was tested on SARS CoV infected and uninfected macrophages A and C A mouse monoclonal antibody against influenza A virus hemagglutinin was used as a control B All three cell smears were stained with a secondary fluorescein isothiocyanate conjugated anti mouse antibody Zymed Laboratories San Francisco Calif and Evans blue was used as a counterstain VOL 79 2005 CYTOKINE INDUCTION IN SARS CoV INFECTED MACROPHAGES 7821 on June 23 2015 by UNIV OF GEORGIA http jvi asm org Downloaded from through filters with a 30 kDa cutoff Titration of the filtrate on FRhK 4 cells and real time quantitative RT PCR with viral RNA indicated that most of the SARS CoV was retained by the 30 kDa cutoff filter Table 4 In human macrophages the retained material eluted from the filter induced the gene ex pression of CXCL10 IP 10 and CCL2 MCP 1 to similar levels as those induced by the initial virus inoculum An assay of the filtrate was negative for SARS CoV and did not up regulate CXCL10 IP 10 and CCL2 MCP 1 gene expression in human macrophages DISCUSSION SARS CoV infections of macrophages lead to the initiation of viral gene transcription and viral protein synthesis No in fectious virus was produced and hence SARS CoV infections of macrophages appeared to be abortive Table 1 The copy number of Orf1b or nucleoprotein RNA was found to increase with time reaching a plateau at 6 h postinfection with the ratio of positive to negative strand RNA being about 1 The viral nucleoprotein was expressed in H1102290 of infected macro phages Fig 1 and 2 In contrast the amounts of positive sense RNA for Orf1b and the nucleoprotein progressively in creased in infected FRhK 4 cells MOI 1 to 2 These results confirmed that virus gene transcription and translation were TABLE 1 Infectious virus yields from macrophages infected with SARS CoV HCoV 229E or influenza A H1N1 virus a Virus Yield log 10 TCID 50 ml at indicated time postinfection 3 h 1 day 2 days 3 days 7 days SARS CoV 1 1 H110211 H110211 H110211 HCoV 229E 1 H1102113H110211 H110211 Influenza A H1N1 virus 2 3 5 6 H110211 a Differentiated human monocyte derived macrophages were infected with SARS CoV HCoV 229E and influenza A HK 54 98 H1N1 virus and culture supernatants were titrated on monolayers of FRhK 4 SARS CoV MRC 5 HCoV 229E and MDCK H1N1 cells The data shown are averages from three independent experiments corrected to 1 significant figure TABLE 2 Microarray analysis of IFN related genes a Gene product Log 2 fold change compared to mock treated cells SARS CoV Influenza A virus Beta interferon NC 7 1 Alpha interferon 1 2 4 5 8 10 and 21 b NC NC Omega interferon NC NC Interferon stimulated gene 20 kDa 6 8 NC Interferon induced protein with tetratricopeptide repeats 1 4 9 3 5 Alpha interferon inducible protein clone IFI 15K 3 8 2 5 Interferon inducible guanylate binding protein 1 67 kDa 3 7 2 6 Interferon induced protein with tetratricopeptide repeats 4 3 7 2 6 Interferon induced transmembrane protein 1 9 27 3 4 NC Myxovirus influenza virus resistance 1 interferon inducible protein p78 mouse 3 1 1 Interferon induced protein 44 2 7 NC Interferon induced protein 35 1 9 NC Retinoic acid and interferon inducible protein 58 kDa 1 9 3 3 Alpha interferon inducible protein 27 1 7 NC Interferon induced transmembrane protein 3 1 8U 1 7 NC Protein kinase interferon inducible double stranded RNA dependent 1 6 NC Alpha interferon inducible protein clone IFI 6 16 1 5 NC Interferon induced transmembrane protein 2 1 8D 1 2 NC Gamma interferon inducible protein 16 1 1 NC Interferon stimulated transcription factor 3 gamma 48 kDa 1 1 NC Interferon inducible guanylate binding protein 2 1 NC a Macrophages were infected at an MOI between 1 and 2 and RNAs were extracted at 3 h postinfection RNAs were pooled from three different donors and gene expression was analyzed with an Affymetrix U133A Genechip microarray Many of the IFN stimulated genes but not IFN H9251 H9252 were up regulated in SARS CoV infected macrophages and are shown relative to influenza A H1N1 virus infection A significant up regulation in gene expression is indicated by an increase of twofold or more and was accompanied by a positive signal intensity call by the Microarray Suite Expression Analysis software version 5 Affymetrix Inc Only genes that have at least twofold change compared to mock treated cells are shown NC no change compared with mock treated ma