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NATURE MEDICINE VOLUME 11 NUMBER 8 AUGUST 2005 875 A crucial role of angiotensin converting enzyme 2 ACE2 in SARS coronavirus induced lung injury Keiji Kuba 1 7 Yumiko Imai 1 7 Shuan Rao 2 7 Hong Gao 3 Feng Guo 2 Bin Guan 2 Yi Huan 2 Peng Yang 2 Yanli Zhang 2 Wei Deng 3 Linlin Bao 3 Binlin Zhang 3 Guang Liu 2 Zhong Wang 4 Mark Chappell 5 Yanxin Liu 2 Dexian Zheng 2 Andreas Leibbrandt 1 Teiji Wada 1 Arthur S Slutsky 6 Depei Liu 2 Chuan Qin 3 Chengyu Jiang 2 Fig 1a and the copy numbers of SARS CoV Spike RNA were greatly reduced Fig 1b SARS CoV infection of mice is associated with the development of mild pathologi cal changes in the lungs Fig 1c d Moreover pathologic alterations in lungs were reduced in Ace2 mutant mice compared to wild type mice Fig 1c d These data provide the first genetic proof that ACE2 is indeed a crucial in vivo SARS receptor required for effective replication of infectious SARS CoV We have recently shown that the renin angiotensin system has a cru cial role in severe acute lung injury and that the SARS CoV receptor ACE2 has a protective role in acute lung failure 18 Fig 2a Notably experimental SARS CoV infections of wild type mice in vivo resulted in considerably reduced ACE2 expression in the lungs Fig 2b suggesting that reduced ACE2 expression might have a role in SARS CoV mediated severe acute lung pathologies By contrast ACE lung expression levels were not overtly changed in SARS CoV infected mice Fig 2b We therefore speculated that SARS CoV might affect lung pathologies through ACE2 To test this idea we established a defined model sys tem using recombinant SARS CoV surface Spike protein which is the essential ligand for ACE2 binding 14 This model system allowed us to avoid possible secondary effects resulting from viral replication or infec tions in vivo and to directly test whether SARS CoV Spike protein might adversely affect acute lung injury through modulation of ACE2 We first tested whether recombinant SARS CoV Spike protein Supplementary Fig 1 online binds to human as well as mouse ACE2 protein using in vitro pull down assays Our recombinant Spike Fc protein indeed pulled down both human and mouse ACE2 Fig 2c SARS CoV Spike Fc binding to human and mouse ACE2 was confirmed by FACS binding assays of Spike Fc to 293 cells overexpressing human or mouse ACE2 Fig 2d Moreover Spike Fc bound to endogenous 1 Institute of Molecular Biotechnology of the Austrian Academy of Sciences Dr Bohr gasse 7 A 1030 Vienna Austria 2 National Laboratory of Medical Molecular Biology Institute of Basic Medical Sciences 3 Institute of Laboratory Animal Sciences and 4 Peking Union Medical College Hospital Chinese Academy of Medical Sciences St Michael s Hospital 30 Bond Street Toronto M5B 1W8 Canada 7 These authors contributed equally to this work Correspondence should be addressed to C J jiang Published online 7 July 2005 doi 10 1038 nm1267 LETTERS 2005 Nature Pub lishing Gr oup http www nature com naturemedicine 876 VOLUME 11 NUMBER 8 AUGUST 2005 NATURE MEDICINE ACE2 in Vero E6 cells Fig 2e Notably binding of Spike Fc to endog enous ACE2 in Vero E6 cells resulted in downregulation of ACE2 surface expression Fig 2e and Supplementary Fig 1 online Spike Fc also decreased surface levels of human and mouse ACE2 overexpressed in 293 cells data not shown and triggered syncytia formation of mouse ACE2 transfected but not control CD4 transfected 293 cells not shown Thus analogous to other virus receptor interactions 19 SARS CoV Spike protein binding to ACE2 in cell lines or SARS CoV infections in vivo results in reduced ACE2 protein expression Because ACE2 is a crucial SARS CoV receptor Fig 1 SARS CoV Spike protein binding to ACE2 downmodulates ACE2 expression Fig 2 and loss of ACE2 expression results in severe acute respiratory failure 18 we tested whether SARS CoV Spike protein which is the crucial ACE2 binding protein 20 21 could affect the severity of acute lung injury in WT KO Uninfected Infected S A RS spike R N A log 10 W T W T A c e 2 K O Infected 3 2 1 0 A c e 2 K O 0 1 2 3 4 Leukocyte infiltrate score W T W T A c e 2 K O A c e 2 K O Uninfected Infected WT Ace2 KO 0 2 4 6 8 10 rs l og 10 TCI D 50 g b a cd Ace2 Uninfected Infected S A R N A log 10 Uninfected Virus tit e r s l og 10 TCI D 50 g Figure 1 ACE2 is a crucial receptor for SARS CoV infections in vivo a b SARS CoV replication a and detection of SARS CoV Spike RNA b in wild type WT and Ace2 knockout mice Viral replication was determined from lung tissue at day 2 of infection Virus titers mean log 10 TCID 50 per gram lung tissue are shown for individual mice n 15 per group SARS CoV Spike RNA expression was assayed using real time RT PCR and normalized to mouse Actb Data are shown as mean s e m n 15 per group P 0 01 c Lung histopathology original magnification 200 and d lung injury scores as defined by leukocyte infiltration of control and SARS CoV infected wild type and Ace2 knockout mice Lung samples were taken on day 6 after SARS CoV infection ACE ACE2 Ang II AT1R AT2R Lung inju ry SARS CoV Spik e ACE Uninfected Infected ACE2 W T A ce 2 K O W T A c e2 K O Human ACE2 1 1 10 112 75 37 0 PMT2 Log Count Mouse ACE2 1 10 1 112 75 37 0 PMT2 Log Count C o n t r o l S p i k e F c 3 7 o C S p i k e F c 4 o C Fc Ab 1 1 10 65 32 0 PMT2 Log Count C o n t r o l S p i ke F c 3 7 o C S p i k e F c 4 o C ACE2 Ab 1 1 10 60 40 20 0 PMT2 Log Count L y s a t e C o n t r o l F c S p i k e F c mACE2 hACE2 Lung injury Spike actin c ba d e CountCount CountCount CountCount CountCount Figure 2 Downregulation of ACE2 expression by SARS CoV infection and SARS CoV Spike protein a Schematic diagram of the renin angiotensin system in acute lung failure and proposed SARS CoV action b Decreased ACE2 protein but normal ACE levels in the lungs of SARS CoV infected mice Lung homogenates were prepared from control and SARS CoV infected wild type or Ace2 knockout KO mice on day 2 and analyzed by western blot c Binding of recombinant Spike S 1190 Fc protein to human ACE2 hACE2 and mouse ACE2 mACE2 in pull down assays Spike Fc but not control Fc protein pulled down hACE2 and mACE2 from total cell extracts of A549 human alveolar epithelial cells and IMCD mouse kidney epithelial cells respectively Total lysates are shown as controls d Binding of Spike Fc protein to human and mouse ACE2 in cell culture 293 cells transfected with hACE2 or mACE2 were incubated with Spike Fc and the binding was detected by FACS blue lines Nontransfected 293 cells incubated with Spike Fc followed by Fc specific antibodies are shown as controls black line e Decreased cell surface expression of ACE2 after binding to Spike Fc protein at 37 C compared to 4 C in Vero E6 cells ACE2 surface expression was detected at 3 h of incubation with Spike Fc using an ACE2 specific monoclonal antibody Similar data were obtained using Fc specific antibody to directly detect surface bound Spike Fc and to avoid masking of the ACE2 epitope Representative FACS histograms are shown including a background control with an isotype matched antibody LETTERS 2005 Nature Pub lishing Gr oup http www nature com naturemedicine NATURE MEDICINE VOLUME 11 NUMBER 8 AUGUST 2005 877 vivo Notably treatment with Spike Fc protein worsened the lung function in wild type mice whereas control Fc protein showed no apparent effects Fig 3a Moreover Spike Fc treatment of acid challenged wild type mice augmented the pathological changes in the lung parenchyma Fig 3 b c and Supplementary Table 1 online and increased lung edemas as defined by a wet dry lung weight ratios Fig 3d We next made a Spike deletion mutant that only contained the previously mapped ACE2 binding domain amino acids 318 510 21 fused to human Fc Fig 3e and Supplementary Fig 1 online This short Spike S318 510 Fc protein containing the minimal ACE2 binding site also binds to ACE2 in cell lines using FACS assays and downmodulates the cell surface expression of ACE2 Supplementary Fig 1 online Treatment with Spike S318 510 Fc again worsened acid induced acute lung injury in wild type mice Fig 3e Notably in vivo Spike Fc protein administration did not affect the severity of lung failure in Ace2 knockout mice Fig 3f indicating that the effect of Spike protein on acute lung injury is ACE2 specific To further clarify whether the intraperitoneally injected Spike Fc pro tein directly affected lung pathology in mice we examined the localization of the injected Spike Fc protein in lung Spike Fc was detected in lung Saline Ac Control Fc Spike F c Saline control Fc Saline Spike Fc Acid control Fc Acid Spike Fc h0123 0 50 100 150 200 Percent changes in elastance Fc 11905103181 Spike S1190 F c Spike S318 510 Fc Control Fc Spik Saline control Fc Saline Spike S318 510 Fc Acid cont Acid Spike S1190 Fc Acid Spike S318 510 Fc 0123 0 50 100 150 h Percent changes in elastance Percent changes in elastance 200 e 4 5 6 7 8 9 Wet d r y ratio Acid Spike Fc Acid Spike Fc 0 5 10 15 20 0123 h 0 50 100 150 200 250 saline control Fc Ace2 KO saline Spike Fc Ace2 KO acid control Fc Ace2 KO acid Spike Fc b Saline id a Spike id control Fc d Wet d r y ratio Wet d r y ratio Lung injury score c Ace2 KO f Figure 3 The SARS CoV Spike protein enhances the severity of acute lung injury a Lung elastance measurements after saline or acid instillation in Spike Fc protein 5 5 nmol kg or control Fc 5 5 nmol kg treated wild type mice n 5 7 per group P 0 05 for the whole time course comparing Spike Fc treated and control Fc treated wild type mice after acid injury b Lung histopathology Representative images are shown Original magnification 200 c Lung injury score Supplementary Table 1 online P 0 01 versus control Fc treated wild type d Wet to dry weight ratios of lungs as readout for pulmonary edema in control and Spike Fc treated mice in the presence or absence of acid induced lung injury P 0 05 between control and Spike Fc treated mice with acid challenge e Severe acute lung failure by Spike S318 510 Fc 5 5 nmol kg treatment in acid challenged mice The scheme upper panel shows the ACE2 binding domain of Spike S318 510 Lung elastance measurements lower panel showed that Spike S318 510 Fc induced severe acute lung failure in acid challenged wild type mice comparable to Spike S1190 Fc n 5 7 per group P 0 05 for the whole time course comparing Spike S318 510 Fc or Spike S1190 Fc treated and control Fc treated wild type mice after acid injury f Lung elastance measurements after acid instillation in Spike Fc protein S1190 5 5 nmol kg or control Fc 5 5 nmol kg treated Ace2 knockout KO mice n 5 7 for each group homogenates by western blot using human Fc specific antibody Fig 4a whereas injected control Fc was not detected In addition using immunohistochemistry we found that Spike Fc protein localized to bronchial epithelial cells inflammatory exudates and alveolar pneumocytes Fig 4b Notably Spike Fc primarily localized to severe lesions Fig 4b This localization of Spike Fc protein is similar to Spike antigen staining in SARS CoV infected mice 22 Spike Fc treatment resulted in downregulation of ACE2 protein expression in lungs of acid treated wild type mice in vivo Fig 4c consistent with ACE2 protein downre gulation in SARS CoV infected mice Fig 2a and Spike Fc protein treated cells in vitro Fig 2e These results show that the SARS CoV Spike protein can directly affect the development of severe acute lung failure through ACE2 ACE2 functions as a carboxypeptidase cleaving a single residue from angiotensin I AngI generating Ang1 9 refs 23 24 and a single residue from angiotensin II AngII to generate Ang1 7 ref 23 The ACE2 homologue ACE by contrast cleaves the decapeptide AngI into the octapeptide AngII 25 Thus ACE2 counterbalances the function of ACE and negatively regulates AngII production Fig 2a To test whether Spike Fc injections indeed affect the function of the renin angiotensin system we analyzed AngII levels in the lungs of acid and Spike Fc treated mice Acid aspiration increased AngII levels in the lungs of wild type mice Notably we observed a further significant increase in AngII levels in the lung tissue of mice treated with Spike Fc Fig 4d To confirm whether Spike Fc promotes lung disease pathogenesis through increased AngII production and functional alterations of the renin angiotensin system we blocked the AngII receptor type 1 AT1R 26 with a specific inhibitor The AT1R is the crucial receptor that mediates AngII induced vascular permeability and severe acute lung injury 18 LETTERS 2005 Nature Pub lishing Gr oup http www nature com naturemedicine 878 VOLUME 11 NUMBER 8 AUGUST 2005 NATURE MEDICINE Inhibition of the AT1R indeed attenuated acute severe lung injury in Spike Fc treated mice Fig 4e Inhibition of the AT1R also attenuated pulmonary edema Fig 4f Taken together our data show that SARS CoV Spike can exaggerate acute lung failure through deregulation of the renin angiotensin system Moreover SARS CoV Spike mediated lung failure can be rescued by inhibition of AT1R It has been estimated that the Spanish flu virus that killed more than 20 million people at the beginning of the twentieth century was lethal to around 0 5 of infected people 9 10 whereas the lethality of SARS CoV infections reached 10 even with modern intensive care treatment 1 3 Given this very high lethality of SARS and the enormous economic and social impact of the worldwide SARS outbreak elucidation of the disease pathogenesis is crucial for future treatment in case of renewed outbreaks Moreover a recent outbreak of avian influenza A H5N1 in humans resulted in up to 70 lethality from acute respiratory failure 12 Before the discovery of SARS CoV two coronaviruses HCoV 229E and HCoV OC43 were known to infect humans but they caused only self limiting upper respiratory tract infections 30 of the common colds and had never been reported to cause severe illness 27 The molecular determi nants that may account for the dramatic differences in pathogenesis between these human coronaviruses and SARS CoV were unknown Our data provide a molecular explanation for the severe lung failure and lethality associated with SARS we hypothesize that infections with SARS CoV result in ACE2 downregulation through binding of SARS CoV Spike protein to ACE2 Given that ACE2 is a key negative regulatory factor for severity of lung edema and acute lung failure SARS CoV Spike protein mediated ACE2 downregulation then contributes to the severity of lung pathologies This scenario would explain how this family member of the relatively harmless coronaviruses has turned into a lethal virus Notably recent data in a small cohort of individuals with SARS suggested that an insertion deletion ACE polymorphism that affects ACE function correlates with disease severity 28 implying that our findings are indeed relevant for humans Our data provide a molecular link between SARS pathogenesis and the role of the renin angiotensin system in lung failure Recombinant ACE2 protein therefore could not only be a treatment to block spreading of SARS CoV but modulation of the renin angiotensin system could also be used to protect individuals with SARS and possibly individuals infected with other viruses such as avian influenza A strains from devel oping acute severe lung failure and acute respiratory distress syndrome METHODS For details of Methods see Supplementary Methods online In vivo SARS infections The SARS CoV Beijing strain PUMC01 isolate used in this study was provided by Z Wang and Y Liu Chinese Academy of Medical Sciences Chinese National Human Genome Center 29 All mouse studies were approved by the Ministry of Health Science and Technology division of the People s Republic of China We intranasally inoculated mice with 100 l virus 10 5 23 TCID 50 At day 2 mice were killed and the lungs were removed for fur ther analyses We assessed lung injury scores as described previously 30 SARS CoV Spike protein binding experiments We cloned the coding sequence of SARS CoV Spike protein amino acids 1 1 190 from Urbani strain or a Spike sequence that only contains the previously mapped 21 ACE2 binding domain amino acids 318 510 to generate a fusion protein with the Fc portion Spik e IgG C o n t r o l F c S p i k e F c Bronchi Control Fc Spike Fc 0 0 5 1 0 1 5 2 0 A n g II f m o l mg prot Saline Spike Fc vehicl Saline Spike Fc AT1 inhibito Acid Spike Fc vehicle Acid Spike Fc AT Acid control Fc vehi 012 0 50 100 150 h Percent changes in 200 4 5 6 7 8 9 Wet d r y ratio Acid Spike Fc AT1 inhibitor Acid Spike Fc C o n t r o l F c S p i ke F c ACE2 actin a Bronchiole Alveoli Control Fc Spike Fc d A n g II f m o l mg protien fe Saline Spike Fc vehicle 1 inhibitor 1 inhibitor hicle 3 Percent changes in elastance Wet d r y ratio c b Figure 4 SARS CoV Spike mediates lung injury through modulation of the renin angiotensin system a b Localization of intraperitoneally injected Spike S 1190 Fc in lung tissue a Spike Fc was detected by pull down assay with Protein G Sepharose and western blot with human Fc specific antibody Mouse IgG is shown as loading control b Lung immunohistochemistry to detect Spike S 1190 Fc or control Fc protein using a human Fc specific antibody Spike S 1190 Fc localizes to bronchial epithelial cells left original magnification 100 inflammatory exudates cells middle original magnification 200 and alveolar pneumocytes right original magnification 200 c Decreased ACE2 protein expression in the lungs of Spike S 1190 Fc treated mice Lung homogenates were prepared from control Fc and Spike S 1190 Fc treated wild type mice and analyzed by western blot with ACE2 specific antibody d AngII peptide levels in lungs of Spike S1190 Fc protein or control Fc treated wild type mice after saline or acid aspiration AngII levels were determined at 3 h by enzyme immunoassay Bars mean s e m P 0 05 comparing Spike S1190 Fc and control Fc treated wild type mice after acid injury e Lung elastance measurements in acid plus Spike S1190 Fc challenged wild type mice treated with the AT1R inhibitor losartan 15 mg kg n 4 6 per group P 0 05 comparing losartan treated Spike S1190 Fc challenged mice with vehicle treated Spike S1190 Fc challenged mice f Wet to dry weight ratios of lungs of acid and Spike S1190 challenged mice in the presence or absence of losartan 15 mg kg n 4 6 mice per group P 0 05 comparing losartan treated wild type with vehicle treated wild type mice at 3 h after acid injury LETTERS 2005 Nature Pub lishing Gr oup http www nature com naturemedicine NATURE MEDICINE VOLUME 11 NUMBER 8 AUGUST 2005 879 of human IgG1 We purified Spike Fc protein from transfected CHO cells using affinity chromatography For in vitro binding assays we used cell lysates from A549 human alveolar epithelial cells or IMCD mouse kidney epithelial cells and we pulled down Spike Fc or control human IgG Fc protein using Protein G Sepharose followed by western blot For flow cytometry we detached Vero E6 cells using a 2 mM mixture of EDTA and PBS and incubated them with Spike Fc or control human IgG Fc protein at 4 C or 37 C for 3 h We then incubated cells with ACE2 specific antibodies or a FITC conjugated human IgG specific antibody Recombinant Spike Fc in vivo challenge in mice We used the mouse model of acid aspiration induced acute lung injury 18 for all Spike Fc in vivo experiments Mice received Spike S1190 Fc Spike S318 510 Fc or control Fc 5 5 nmol kg each intraperitoneally three times at 30 min before and at 1 and 2 h after dur ing acid treatment For AT1 inhibition of Spike Fc mediated acute lung injury we treated the Spike S1190 Fc challenged mice with the AT1 inhibitor losartan 15 mg kg Lung injury scores were assessed as described previously 30 Statistical analyses All data are shown as mean s e m Measurements at single time points were analyzed by unpaired t test virus titers SARS CoV Spike RNA levels ANOVA with two tailed t test percent changes in elastance wet dry ratio AngII levels or Kruskal Wallis test histological scores Time courses were analyzed by re