【病毒外文文献】2014 Active Replication of Middle East Respiratory Syndrome Coronavirus and Aberrant Induction of Inflammatory Cytokines

MAJOR ARTICLE Active Replication of Middle East Respiratory Syndrome Coronavirus and Aberrant Induction of In ammatory Cytokines and Chemokines in Human Macrophages Implications for Pathogenesis Jie Zhou 1 2 3 a Hin Chu 2 a Cun Li 2 Bosco Ho Yin Wong 2 Zhong Shan Cheng 2 Vincent Kwok Man Poon 2 Tianhao Sun 2 Candy Choi Yi Lau 2 Kenneth Kak Yuen Wong 5 Jimmy Yu Wai Chan 5 Jasper Fuk Woo Chan 1 2 3 4 Kelvin Kai Wang To 1 2 3 4 Kwok Hung Chan 2 Bo Jian Zheng 1 2 3 4 and Kwok Yung Yuen 1 2 3 4 1 State Key Laboratory of Emerging Infectious Diseases 2 Department of Microbiology 3 Research Centre of Infection and Immunology 4 Carol Yu Centre for Infection and 5 Department of Surgery University of Hong Kong Hong Kong Special Administrative Region China Middle East respiratory syndrome coronavirus MERS CoV infection caused severe pneumonia and multior gan dysfunction and had a higher crude fatality rate around 50 vs 10 than SARS coronavirus SARS CoV infection To understand the pathogenesis we studied viral replication cytokine chemokine response and antigen presentation in MERS CoV infected human monocyte derived macrophages MDMs versus SARS CoV infected MDMs Only MERS CoV can replicate in MDMs Both viruses were unable to signi cantly stim ulate the expression of antiviral cytokines interferon IFN and IFN but induced comparable levels of tumor necrosis factor and interleukin 6 Notably MERS CoV induced signi cantly higher expression levels of interleukin 12 IFN and chemokines IP 10 CXCL 10 MCP 1 CCL 2 MIP 1 CCL 3 RANTES CCL 5 and interleukin 8 than SARS CoV The expression of major histocompatibility complex class I and costimula tory molecules were signi cantly higher in MERS CoV infected MDMs than in SARS CoV infected cells MERS CoV replication was validated by immunostaining of infected MDMs and ex vivo lung tissue We con clusively showed that MERS CoV can establish a productive infection in human macrophages The aberrant induction of in ammatory cytokines chemokines could be important in the disease pathogenesis Keywords MERS CoV SARS CoV viral replication pathogenesis cytokine and chemokine response In September 2012 a novel human coronavirus HCoV EMC later renamed Middle East respiratory syndrome coronavirus MERS CoV was identi ed in 2 patients with severe pneumonia complicated with renal failure who once traveled to or resided in Saudi Arabia 1 2 Retrospective analysis of archived speci mens showed that the rst virologically con rmed cases could be traced back to early April 2012 3 As of 1 August 2013 World Health Organization has con rmed 94 cases of infection with 46 deaths and there fore an appalling fatality rate of around 50 4 Coronaviruses are the largest of all RNA viruses with positive single stranded RNA genomes of 26 32 kb Many of them are globally distributed and detectable in a wide range of animals and humans 5 They are classi ed into 4 genera alphacoronavirus betacorona virus gammacoronavirus and deltacoronavirus 6 7 HCoV OC43 lineage A betacoronavirus and HCoV 229E alphacoronavirus are known causative agents of Received 17 July 2013 accepted 27 August 2013 electronically published 24 September 2013 a J Z and H C contributed equally to the study Correspondence Kwok Yung Yuen MD Carol Yu Centre for Infection Depart ment of Microbiology University of Hong Kong Queen Mary Hospital 102 Pokfulam Rd Pokfulam Hong Kong Special Administrative Region China kyyuen hkucc hku hk The Journal of Infectious Diseases 2014 209 1331 42 The Author 2013 Published by Oxford University Press on behalf of the Infectious Diseases Society of America All rights reserved For Permissions please e mail journals permissions DOI 10 1093 infdis jit504 MERS CoV in Macrophages and Pathogenesis JID 2014 209 1 May 1331 at D H Hill Library Acquis Dept S on May 14 2014 http jid oxfordjournals org Downloaded from the common cold and rarely cause severe respiratory diseases 8 9 In contrast SARS coronavirus SARS CoV lineage B be tacoronavirus caused the severe acute respiratory syndrome SARS outbreak during 2002 2003 affecting 8000 patients in 30 countries with an overall fatality rate of 9 6 10 13 The discovery of SARS CoV aroused a growing recognition that human coronaviruses are potentially highly pathogenic The heightened awareness sparked an intense hunting for novel co ronaviruses which led to the subsequent identi cation of HCoV NL63 alphacoronavirus and HCoV HKU1 lineage A betacoronavirus that occasionally cause severe lower respira tory tract infections in young children elderly individuals and immunocompromised patients 14 15 Ten years after the SARS outbreak another novel betacoronavirus of lineage C MERS CoV might have jumped species barriers from bats to humans and caused an ongoing epidemic of severe human in fections in the Middle East 6 16 20 Most patients with MERS presented with rapidly progressive pneumonia Many of them developed multiorgan dysfunction deranged coagulation pro le and hematological changes including lymphopenia neutrophilia and thrombocytopenia 3 Although the clinical syndromes of MERS resembled those described in severe SARS MERS often had renal failure and substantially surpassed SARS in terms of crude fatality rate Because of the increasing number of person to person transmission in both nosocomial and community settings 21 22 there is a growing concern for another SARS like pandemic Among all coronaviruses that cause human infections SARS CoV is the most extensively characterized Human airway epithelial cells are the primary targets of SARS CoV 23 However virus infected macrophages contributed signi cantly to disease pathogenesis 23 24 Macrophages are the sentinel phagocytes of innate immune system which functions to contain and eliminate pathogens remove apoptotic cells and present antigens to T cells Macrophage produced cyto kines and chemokines modulate immune response eradicate invading pathogens and maintain tissue homeostasis 25 Compared with other human primary macrophages peripheral blood monocyte derived macrophages MDMs are readily available and frequently used to recapitulate the initial innate immune responses in macrophages during viral infection Before the identi cation of MERS CoV SARS CoV had been regarded as the most dangerous human coronavirus However a 5 fold higher fatality rate than that of SARS CoV highlighted the possibly higher pathogenicity of MERS CoV Recent studies demonstrated that similar to SARS CoV MERS CoV was capable of infecting and productively replicat ing in primary human airway epithelial cells and ex vivo human lung tissues Furthermore MERS CoV has a much broader tissue tropism than SARS CoV and resembled SARS CoV in its ability to suppress interferon production 26 29 The high pathogenicity of MERS CoV prompted us to explore the potential host virus interaction in macrophages the cells that were implicated in the pathogenesis of SARS 24 There fore we studied viral infection replication cellular immune re sponse and antigen presentation in MERS CoV inoculated MDMs in comparison with SARS CoV inoculated MDMs Further understanding of the pathogenesis of MERS will be im portant for optimizing treatment strategies for this highly fatal emerging infectious disease MATERIALSANDMETHODS Virus Culture and Virus Titration by a 50 Tissue Culture Infective Dose TCID 50 Assay A clinical isolate of MERS CoV was kindly provided by Fouch ier et al 2 The isolate was cultured in Vero cells ATCC with serum free minimum Dulbecco s modi ed Eagle s medium DMEM Life Technologies supplemented with 100 U mL penicillin and 100 g mL streptomycin at 37 C with 5 CO 2 SARS CoV was cultured in FRhK 4 cells ATCC with the same medium Two or 3 days after virus inoculation culture supernatants were collected and stored in 80 C freezer in ali quots For virus titration aliquots of MERS CoV and SARS CoV were applied on con uent Vero cells in 96 well plates for TCID50 assay Brie y serial 10 fold dilutions of each virus were inoculated in a Vero cell monolayer in sextuplets and cul tured in penicillin streptomycin supplemented DMEM The plates were observed for cytopathic effect for 4 5 days Viral titer was calculated with the Reed and M nch end point method One TCID 50 is interpreted as the amount of virus that causes cytopathic effect in 50 of inoculated wells MDM Culture and Virus Infection Healthy adult blood samples were collected from Hong Kong Red Cross Blood Transfusion Service according to a protocol approved by the Institutional Review Board of the University of Hong Kong Monocyte preparation and differentiation were performed according to a well established protocol described previously 30 The purity of the macrophages assessed by ow cytometry analysis of CD14 and CD68 was 93 on average 31 For viral infection MDMs in 24 well plates were inoculat ed with MERS CoV or SARS CoV at 2 TCID 50 cell or were mock inoculated for 1 hour at 37 C Supernatants and cell lysates were harvested 0 5 10 24 48 and 72 hour s after in fection TCID 50 assay was performed on cell free supernatants for viral titration Cell lysates were extracted for RNA to detect viral RNA and cellular messenger RNA mRNA For immunostaining MDMs were seeded in 24 well plates on glass coverslips The cells were inoculated with MERS CoV at 2 TCID 50 cell Twelve and 48 hours after infection the cells were xed with 4 paraformaldehyde and immunos tained 1332 JID 2014 209 1 May Zhou et al at D H Hill Library Acquis Dept S on May 14 2014 http jid oxfordjournals org Downloaded from Quanti cation of Viral and Cellular RNATranscript by Reverse Transcription Quantitative Polymerase Chain Reaction RT qPCR Cellular RNA extraction and RT qPCR were performed as de scribed previously 32 Viral RNA in the supernatant was ex tracted with the PureLink Viral RNA DNA mini kit Life Technologies Virus speci c primer for MERS CoV or SARS CoV was used in reverse transcription to generate complemen tary DNAs cDNAs for the viruses and oligo dT for cellular cDNAs Speci c primers used in the qPCR analysis were listed in Supplementary Table 1 Cellular gene expression results were normalized to GAPDH and presented as the fold change in gene expression of virus infected MDMs relative to that of mock infected cells Ex Vivo Lung Tissue Culture and Virus Infection Experiment The ex vivo lung tissue culture and virus infection experi ment was approved by the Institutional Review Board of the University of Hong Kong Hospital Authority Hong Kong West Cluster Fresh normal lung tissue was obtained from a patient undergoing resection of lung tumor Lung tissue was cut into 2 mm 3 cubes and subsequently infected by a MERS CoV inoculum of 1 10 7 TCID 50 mL or were mock infected for 1 hour at 37 C After inoculation tissue cubes were maintained in DMEM F12 medium supplemented with 10 human serum and penicillin streptomycin before xation and cryosectioning as described previously 32 Immuno uorescence Staining MERS CoV was detected using our previously described guinea pig anti nucleocapsid protein NP antibody 29 for 1 hour at room temperature followed by uorescein isothiocyanate conjugated rabbit anti guinea pig immunoglobulin G IgG Life Technologies or Alexa 594 goat anti guinea pig IgG Abcam as the secondary antibody In tissue slides macrophages were labeled with mouse anti CD68 antibody KP1 Abcam followed by Alexa 488 goat anti mouse IgG Abcam All primary and secondary antibodies were diluted at 1 200 Finally slides were mountedwithProLongGoldantifadereagent LifeTechnologies and examined with a Carl Zeiss LSM 710 microscope Statistical Analysis Experimental results represented mean and standard errors of the mean from at least 3 different donors Statistical compari son between the groups was performed by the Student t test using GraphPad Prism 6 Differences were considered statisti cally signi cant when the P value was 05 RESULTS Viral Infectivity and Replication of MERS CoV in MDMs To understand the ability of MERS CoV to infect human macrophages we inoculated MDMs with MERS CoV and SARS CoV in parallel and observed the outcome of infection by examining levels of viral gene in the cell lysate and culture super natant As shown in Figure 1 the level of MERS CoV RNA increased from 5 hours after infection in both cell lysate Figure 1A andsupernatant Figure1B of MDMs Despite the variation among different donors a 2 4 log increase in viral RNA level was consistently detected MERS CoV infection and replication in MDMs was con rmed with TCID 50 assays in which increasing titers of infectious virus were observed after inoculation On the contrary when the same dose of SARS CoV was inoculated onto MDMs no sign of viral replication were observed because the viral RNA levels gradually decreased in the cell lysate and culture supernatant Infectious viruses detected in the supernatants of SARS CoV infected MDMs remained at low levels indicating an abortive infection Replication of MERS CoV in MDMs was further veri ed by immuno uorescence study As shown in Figure 2 strong uo rescence signals for MERS CoV NP were observed in the cyto plasm of virus inoculated MDMs Twelve hours after infection most of the infected cells displayed puncta like positivity in the cytoplasm whereas 48 hours after infection there were more cells displaying homogeneous immunoreactivity to NP throughout the cytoplasm Moreover there were more NP pos itive cells 48 hours after infection than 12 hours after infection data not shown compatible with a productive infection and replication Mock infected MDMs did not display any immu noreactivity to NP Moreover replacing anti NP sera with pre immune sera did not yield any positive signal in infected MDMs data not shown Collectively we demonstrated the in fection and replication capability of MERS CoV in MDMs We next assessed the cell viability of MERS CoV infected SARS CoV infected and mock infected MDMs by calculating the numbers of viable cells 48 hours after infection and com paring these data with values obtained right before infection It was shown that approximately 87 of mock infected MDMs remained viable 48 hours after infection whereas the percent age of viable cells among MERS CoV infected MDMs was sig ni cantly reduced to 64 Figure 3 In contrast compared with mock infection SARS CoV appeared to have a protective effect from cell death The difference however was not statisti cally signi cant Our result suggested that MERS CoV induced signi cantly higher cytotoxicity than SARS CoV in infected MDMs Innate Immune Response in MERS CoV Infected MDMs To evaluate the response of MDMs to active MERS CoV repli cation we assessed the mRNA expression levels of a series of antiviral cytokines proin ammatory cytokines and chemo kines in MERS CoV infected SARS CoV infected and mock infected MDMs The expression of antiviral cytokines type I interferon interferon IFN and IFN type II interferon IFN proin ammatory cytokines tumor necrosis factor TNF interleukin 6 IL 6 and interleukin 12 IL 12 and MERS CoV in Macrophages and Pathogenesis JID 2014 209 1 May 1333 at D H Hill Library Acquis Dept S on May 14 2014 http jid oxfordjournals org Downloaded from chemokines IP 10 CXCL 10 MCP 1 CCL 2 MIP 1 CCL 3 RANTES CCL 5 and interleukin 8 IL 8 was examined by RT qPCR As shown in Figure 4 both MERS CoV infected and SARS CoV infected MDMs displayed fairly weak and comparable expression levels of IFN and IFN whereas these IFNs were highly induced in in uenza A H5N1 infected MDMs data not shown However IFN expression was highly induced by both viruses IFN induction was more prominent in MERS CoV inoculated MDMs than in SARS CoV inoculated cells TNF and IL 6 were comparably induced in both MERS CoV infected and SARS CoV infected cells IL 12 was more signi cantly induced by MERS CoV than SARS CoV Consistent with the high induction of IFN IP 10 IFN inducible protein 10 which is secreted by a variety of cells in response to INF was highly upregulated by both viruses and was more signi cantly induced in MERS CoV infected cells Figure 5 Chemokines were extensively studied and implicated in the immunopathogenicity of SARS CoV 33 34 Our data demonstrated that MERS CoV induced signi cantly higher levels of MCP 1 MIP 1 and IL 8 when compared with SARS CoV Figure 5 A trend of higher RANTES expression was documented in MERS CoV inoculated MDMs Notably all of these chemokines as well as IP 10 exhibited a sus tained induction curve from an early time point 5 hours after infection to a late time point 72 hours after infection in MERS CoV inoculated cells The expression patterns of many of these cytokines chemokines in SARS CoV infected Figure 1 Middle East respiratory syndrome coronavirus MERS CoV replication kinetics in human monocyte derived macrophages MDMs compared with SARS coronavirus SARS CoV MERS CoV or SARS CoV was inoculated onto MDMs at 2 tissue culture infective doses TCID 50 per cell At the indi cated hours after infection cell lysate A and culture supernatant B were collected to detect the levels of positive strand viral RNA by reverse transcrip tion quantitative polymerase chain reaction Viral titration of the culture supernatants was performed with a TCID 50 assay C The results of 3 representative donors are demonstrated 1334 JID 2014 209 1 May Zhou et al at D H Hill Library Acquis Dept S on May 14 2014 http jid oxfordjournals org Downloaded from MDMs in this study were in agreement with those in a previ ous report 35 Collectively we found that similar to SARS CoV MERS CoV was unable to induce effective antiviral IFN response IFN and IFN Both viruses similarly up regulated the expression of TNF and IL 6 but MERS CoV induced more IFN expression Remarkably MERS CoV profoundly and sustainably induced immune cell recruiting chemokines and cytokines suchasIP 10 CXCL 10 MCP 1 CCL 2 MIP 1 CCL 3 RANTES CCL 5 IL 8 and IL 12 in MDMs Antigen Presenting Function of MERS CoV Infected MDMs Apart from dendritic cells macrophages are the foremost antigen presenting cells in the host immune system Viral anti gens displayed on infected cells via major histocompatibility complex MHC class I molecules can be recognized by speci c cytotoxic T cells that trigger the elimination of infected cells and thereby abrogate further viral replication and dissemi nation On the other hand macrophages present viral antigens coupled with MHC class II molecules to helper T cells and B cells to elicit the cellular and humoral immune responses The effective host immune response is an important determinant for disease presentation and outcome of viral infection A number of molecules for antigen processing PSMB8 and CD74 antigen expression and stability in MHC class I complex B2M and costimulation and activation CD40 CD80 and CD86 together with MHC class I HLA A and HLA B and MHC class II HLA DMB and HLA DPB1 mole cules were examined upon inoculation with MERS CoV SARS CoV or mock control We demonstrated that SARS CoV largely failed to stimulate the expression of MHC class I and MHC class II molecules in inoculated MDMs However Figure 2 Immuno uorescence staining of Middle East respiratory syndrome coronavirus MERS CoV nucleocapsid protein NP in MERS CoV inoculated human monocyte derived macrophages MDMs MDMs seeded on glass coverslips were inoculated with MERS CoV at 2 tissue culture infective doses per cell or subjected to mock infection Twelve hours A and 48 hours B after inoculation cells were xed blocked and permeabilized incubated with anti NP primary antibody for 1 hour and stained with uorescein isothiocyanate conjugated secondary antibody for 1 hour The slides were mounted with DAPI containing mounting buffer and examined with a Carl Zeiss LSM 710 microscope C Findings for mock inoculated MDMs that underwent the same procedure as that described for MERS CoV infected MDMs MERS CoV in Macrophages and Pathogenesis JID 2014 209 1 May 1335 at D H Hill Library Acquis Dept S on May 14 2014 http jid oxfordjournals org Downloaded from MERS CoV