Osterhaus Molewaterple revision rapidly to several countries in Asia North America and Europe enzyme 2 ACE2 the SARS CoV receptor Li et al 2003 Subsequently the S2 domain fuses with the cellular membrane Hamming et al 2004 Harmer et al 2002 Recently it was replication and that IFN and IFN combined have a synergistic antiviral effect against this virus Cinatl et al 2003 Morgenstern et al 2005 Sainz et al 2004 Further more we have demonstrated that pegylated IFN protected 474 Liu et al 2004 ACE2 is a component of the renin angiotensin system and mainly involved in the regulation of causing disease in almost 8000 people of whom nearly 10 died Only a few months after the first emergence of SARS a newly discovered coronavirus CoV was identified as its etiological agent Drosten et al 2003 Ksiazek et al 2003 Peiris et al 2003 SARS CoV is a positive stranded RNAvirus with a genome of about 30 kb in length that is structurally similar to that of other group 2 coronaviruses Marra et al 2003 Rota et al 2003 Snijder et al 2003 It enters the host cell by binding of the spike S protein S1 domain to angiotensin converting shown that ACE2 also acts as a receptor for human coronavirus NL63 HCoV NL63 Hofmann et al 2005 SARS CoVreplicates predominantly inthe lower respiratory tract and causes diffuse alveolar damage leading to severe respiratory distress Kuiken et al 2003 Nicholls et al 2003 Treatment of SARS patients was mainly based on the use of ribavirin and corticosteroids but the efficacy of these drugs has not been proven Several groups have examined the antiviral effect of interferons IFNs against SARS CoV In vitro studies have shown that human recombinant IFNs inhibit SARS CoV mRNA levels were also decreased after treatment with these cytokines These findings suggest that IL 4 and IFN inhibit SARS CoVreplication partly through downregulation of ACE2 2006 Elsevier Inc All rights reserved Keywords SARS CoV ACE2 Interferon gamma Interleukin 4 Introduction Severe acute respiratory syndrome SARS emerged late 2002 early 2003 in Guangdong Province China and spread heart function and blood pressure Boehm and Nabel 2002 Crackower et al 2002 It is expressed in vascular endothelia heart kidney and testis but also in epithelia of the small intestine and in alveolar epithelial cells Donoghue et al 2000 expression of angiotensin converting enzyme 2 ACE2 the SARS CoV infection In contrast to IFNs IL 4 did not show antiviral activity when administered immediately after SARS CoVinfection suggesting that IL 4 acts early during the SARS CoV replication cycle Indeed binding of recombinant SARS CoV spike protein to Vero E6 cells was diminished on cells treated with IL 4 but also on cells exposed to IFN Consistent with these observations IL 4 and IFN downregulated cell surface receptor Besides diminished ACE2 cell surface expression ACE2 Interferon and interleukin 4 downregulate coronavirus receptor ACE2 Anna de Lang Albert D M E Department of Virology Erasmus Medical Center Dr Received 18 May 2006 returned to author for Available online Abstract Interferons IFNs inhibit severe acute respiratory syndrome coronavirus In this study we demonstrate that treatment of Vero E6 cells with interleukin 4 Virology 353 2006 Corresponding author Fax 31 10 4089485 E mail address b haagmans erasmusmc nl B L Haagmans 0042 6822 see front matter 2006 Elsevier Inc All rights reserved doi 10 1016 j virol 2006 06 011 expression of the SARS in Vero E6 cells Bart L Haagmans in 50 3015 GE Rotterdam The Netherlands 12 June 2006 accepted 15 June 2006 24 July 2006 SARS CoV replication and might be valuable for SARS treatment IL 4 decreased the susceptibility of these cells to SARS CoV 481 type 1 pneumocytes from infection with SARS CoV in cynomolgous macaques Haagmans et al 2004 Usually IFNs achieve their antiviral effect through the induction of various proteins that block viral replication in the infected cell To further explore the antiviral effect of IFNs and other cytokines specifically against SARS CoV infection we treated Vero E6 cells with various amounts of IFN tumor necrosis factor TNF or interleukin IL 4 and infected these cells with SARS CoV Results Susceptibility of Vero E6 cells to SARS CoV or HSV infection after cytokine treatment Vero E6 cells were treated with 1 10 or 100 ng ml of IL 4 IL 10 IFN TNF or a combination of IFN and TNF and infected with SARS CoVor herpes simplex virus HSV 1 While treatment with TNF Fig 1A or IL 10 data not shown had no antiviral effect against SARS CoV or HSV 1 pretreatment with IFN Fig 1B or a combination of IFN and TNF Fig 1C dose dependently reduced the number of infected cells per well after infection with either one of these viruses as demonstrated previously Cinatl et al 2003 Feduchi et al 1989 Surprisingly recombinant human IL 4 also exerted SARS CoV antiviral activity which was not observed against HSV infection Fig 1D suggesting a virus Antiviral activity of IL 4 against SARS CoV We further evaluated the antiviral effect of IL 4 against SARS CoV using a SARS CoV specific RT PCR and plaque titration on Vero E6 cells After pretreatment of the cells with 10 ng ml IL 4 for 48 h and subsequent infection excretion of SARS CoV from the treated cells was diminished by 1 log just as SARS CoV RNA levels in these cells Figs 3A B The antiviral effect of IL 4 was abrogated after preincubation with a neutralizing IL 4 antibody Figs 3A B These results further Fig 2 Antiviral activity of IL 4 against HCoV NL63 Titration of supernatant after infection of Vero E6 cells with 10 5 TCID 50 HCoV NL63 Error bars indicate standard errors 475A de Lang et al Virology 353 2006 474 481 specific antiviral activity We also tested the antiviral activity of IL 4 against HCoV NL63 another coronavirus that also uses the ACE2 receptor to enter the cell Hofmann et al 2005 Compared to untreated control cells viral excretion from in fected Vero E6 cells was reduced by more than 1 log after IL 4 treatment Fig 2 Fig 1 Susceptibility of Vero E6 cells to SARS CoVor HSV 1 infection after treatment Triangle shaped symbols represent SARS CoVand rectangular symbols represent HSV infection while the right Y axis represents the number of infected cells per well after demonstrate that IL 4 has a modest antiviral activity against SARS CoV infection in Vero E6 cells To determine which step of the SARS CoV life cycle is affected by IL 4 conditioned medium from Vero E6 cells that were treated with IL 4 10 ng ml was removed and the cells were washed before they were infected with SARS CoV with TNF A IFN B IFN combined with TNF C or IL 4 D 1 The left Y axis indicates the number of infected cells per well after HSV SARS CoV SCV infection Error bars indicate standard errors A E6 cells 353 2006 474 481 Removal of IL 4 conditioned medium from the Vero E6 cells after 48 h of treatment still conferred protection against SARS CoV Fig 3C On the other hand when this IL 4 conditioned medium was transferred to fresh untreated Vero E6 cells and 5 Fig 3 Antiviral activity of IL 4 againstSARS CoV Plaque titration of supernatant IL 4 IL 4 treated cells that were infected with 10 5 TCID 50 SARS CoV Susceptibili conditionedmediumand subsequentwashing C and susceptibilityoffresh Vero with 10 5 TCID 50 SARS CoV D Error bars indicate standard errors 476 A de Lang et al Virology these cultures were infected 5 min later by adding 10 TCID 50 SARS CoV to the medium no reduction in the number of SARS CoV infected cells was observed Fig 3D Therefore the presence of a soluble SARS CoV infection blocking molecule released by IL 4 treated cells is unlikely Because residual IL 4 was still present in this conditioned medium not shown IL 4 probably does not inhibit replication of SARS CoV once it has entered the cell and needs to be added to the cells at an earlier time point Thus IL 4 most likely has an antiviral effect on an early step of the SARS CoV life cycle in Vero E6 cells Binding of recombinant SARS CoV spike protein to cytokine treated Vero E6 cells Since IL 4 treatment reduced excretion of virus viral replication and infection only when administered before SARS CoV infection binding of the SARS CoV S protein to cytokine treated Vero E6 cells was analyzed using flow cytometry As shown in Fig 4A treatment with IL 4 reduced the ability of Vero E6 cells to bind recombinant S protein Because IFN combined with TNF also reduced suscept ibility of Vero E6 cells to SARS CoV we analyzed binding of the S protein to these cells after IFN TNF treatment and demonstrated that 10 ng ml IFN TNF reduced the binding capacity of the S protein to a background level Fig 4B These data suggest that these cytokines influence expression of the cellular receptor for SARS CoV ACE2 Effect of cytokine treatment on ACE2 expression To examine if IL 4 and IFN TNF decrease susceptibility to SARS CoVinfection through an effect on cell surface ACE2 or amountof SARS CoV RNA B presentin mock control 10 ng ml IL 4 or ty of IL 4 treated Vero E6 cells to SARS CoV infection after removal of IL 4 to SARS CoVafter receivingIL 4conditionedmediumjustbefore infection expression we analyzed ACE2 expression after cytokine treatment As shown in Fig 5A 10 ng ml IL 4 downregulated ACE2 expression and preincubation with a neutralizing IL 4 antibody abolished the IL 4 effect Fig 5B Similarly treatment of Vero E6 cells with 100 ng ml IL 13 also a Th2 cytokine downregulated ACE2 expression and inhibited SARS CoV replication data not shown Background staining was determined by incubating the cells with normal goat serum followed by the secondary antibody Fig 5 Fig 4 Binding of recombinant SARS CoV S protein to Vero E6 cells after treatment with 10 ng ml IL 4 A or a combination of 10 ng ml IFN and 10 ng ml TNF B Dotted lines represent cytokine treated cells while thick lines represent mock treated control cells The shaded areas represent back ground staining was IFN Fig 5 Influence of cytokine treatment on ACE2 expression ACE2 expression neutralizing antibody B 10 ng ml TNF C 10 ng ml IFN D or 10 ng ml A de Lang et al Virology A more pronounced decrease in ACE2 expression was observed after treatment with a combination of 10 ng ml IFN and10ng mlTNF compared to treatment with these cytokines separately Figs 5C E After removal of the cytokines ACE2 expression was restored only gradually to pretreatment levels data not shown whereas addition of fresh IFN and TNF at 48 h further decreased ACE2 expression to background level Fig 5F Downregulation of ACE2 was not caused by an overall inhibitory effect on cell surface protein expression IFN and IL 4 both upregulated MHC class I expression on Vero E6 cells Figs 5G and H When ACE2 expression on Vero E6 cells was followed in time after treatment with IL 4 a gradual decrease in ACE2 expression was observed which was maximal at 48 h after treatment Fig 6A ThedecreaseinACE2expressionovertime correlated with decreased susceptibility to SARS CoVinfection Fig 6B These experiments also demonstrate that IL 4 only displayed antiviral activity when administered at least 24 h before infection with SARS CoV Fig 6B In contrast treatment with IFN 1 h after SARS CoV infection reduced replication by at least 1 log not shown These results further substantiate our hypothesis that IL 4 inhibits SARS CoV replication through downregulation of ACE2 ACE2 mRNA levels after cytokine treatment The fact that ACE2 downregulation after IL 4 treatment is a gradual process and that ACE2 expression recovers slowly for 96 h in the presence of 10 ng ml IFN combined with 10 ng ml TNF F HLA A IFN combined with TNF H Dotted lines represent cytokine treated cells while background staining determined after treatment with 10 ng ml IL 4 A IL 4 preincubated with a combined with 10 ng ml TNF E all at 48 h Vero E6 cells were incubated 477353 2006 474 481 when cytokines are removed suggests that ACE2 is down regulated at the mRNA level Using an ACE2 specific Taqman we observed reduced ACE2 mRNA levels in Vero E6 cells after 48 h of IL 4 or IFN TNF treatment Fig 7 This implicates that downregulation of ACE2 by these cytokines is regulated at transcription level Discussion It is well established that IFNs inhibit replication of many different viruses including SARS CoV Cinatl et al 2003 Haagmans et al 2004 Morgenstern et al 2005 Sainz et al 2004 When IFNs bind to their receptors downstream signaling from these receptors can block one or more steps in the virus life cycle IL 4 on the other hand does not induce these proteins and may even antagonize the protective effects of IFN Lohoff et al 1990 In general the Th2 derived cytokine IL 4 is not considered to display antiviral activity In fact only a few studies have demonstrated IL 4 mediated inhibition of viral replication Goletti et al 2002 Lin et al 2003 Furthermore expression of IL 4 by recombinant ectromelia virus or vaccinia virus exacerbated infection in vivo and the administration of recombinant IL 4 delayed virus clearance in influenza virus infected mice Jackson et al 2001 Moran et al 1996 Sharma et al 1996 In this study we demonstrate that treatment of Vero E6 cells with IL 4 decreased susceptibility of these cells to SARS CoV infection by 10 to 100 fold Interestingly ACE2 expression on the cell surface was downregulated after B C expression on Vero E6 cells after treatment with 10 ng ml IL 4 G or thick lines represent mock treated control cells The shaded areas represent treatment with IL 4 or IFN Therefore we postulate that IL 4 and IFN may decrease susceptibility to SARS CoV infection partially through modulation of ACE2 cell surface expression Cytokine mediated downregulation of viral receptors has been reported earlier in the case of the coxsackievirus adenovirus receptor which can be downregulated synergistically by IFN and TNF in vitro Vincent et al 2004 In this study we show that the antiviral activity of IL 4 was not observed when IL 4 was added after SARS CoV infection This suggests that IL 4 inhibits entry of the virus possibly through an effect on the viral receptor ACE2 In line with these observations IL 4doesnothaveanantiviral effectagainstHSV but does show antiviral activity against HCoV NL63 which also utilizes ACE2 as a receptor Subsequent experiments re vealed that ACE2 expression indeed is downregulated by IL 4 Confirmation of ACE2 s involvement as an intermediate of the IL 4 antiviral activity is hard to proof because of its essential role in SARS CoV infection ACE2 is a component of the renin angiotensin system and enzymatically cleaves angiotensin I into angiotensin 1 9 and angiotensin II into angiotensin 1 7 Donoghue et al 2000 Vickers et al 2002 Several studies have reported regulation of ACE2 expression most likely related to its enzymatic activity ACE2 protein expression is downregulated in the kidneys of hand also downregulated ACE2 suggesting the involvement of the IL 4 type II receptor in the IL 4 induced antiviral activity However this hypothesis could not be further substantiated because antibodies against the IL 4 type II receptor tested were not reactive on non human primate Vero E6 cells Forced downregulation of ACE2 expression by cytokines may reveal a novel antiviral strategy against SARS CoV infection Potentially addition of cytokines able to down regulate ACE2 expression could further improve the efficacy of IFN based anti SARS CoV therapy However downregula tion of ACE2 may have a potential negative effects on SARS pathogenesis since it was recently reported that ACE2 has a protective role in acute lung failure in mice Imai et al 2005 Therefore it will be necessary to explore the consequences of ACE2 downregulation on SARS pathogenesis The most important finding of this study is the observation 478 A de Lang et al Virology Fig 6 ACE2 expression and SARS CoV susceptibility at different time points after IL 4 treatment ACE2 expression on Vero E6 cells was monitored at different time points after IL 4 treatment relative to control cells A Susceptibility of Vero E6 cells to SARS CoV was monitored at different time pointsbeforeandaftertreatmentwith10ng mlIL 4 B Shownisthenumberof SARS CoV infected cells per well hypertensive or diabetic rats Crackower et al 2002 Tikellis et al 2003 whereas ACE2 upregulation has been demonstrated after blocking of the angiotensin II receptors in the kidneys of rats during pregnancy and in the human failing heart Brosnihan et al 2003 Crackower et al 2002 Goulter et al 2004 Ishiyama et al 2004 Tikellis et al 2003 Furthermore it was recently shown that ACE2 is downregulated in the lungs of mice after acute lung injury including SARS CoV infection Kuba et al 2005 Most probably ACE2 can be regulated by several different factors and expression of this protein is dynamic on various cell types Recently it was reported that ACE2 expression is dependent on the differentiation state of epithelia ACE2 was poorly expressed on undifferentiated airway epithelial cells while it was abundantly expressed on well differentiated cells Jia et al 2005 It will be of interest to study the effectof IL 4 andIFN on ACE2expression onthese cells The ACE2 downregulation was cytokine specific while IFN and IL 4 both downregulated ACE2 expression IFN a cytokine that inhibits SARS CoV replication in vivo and in vitro did not affect ACE2 expression on Vero E6 cells data not shown Treatment of Vero E6 cells with IL 13 on the other Fig 7 ACE2 mRNA levels in Vero E6 cells after treatment with 10 ng ml IL 4 or a combination of 10 ng ml IFN and 10 ng ml TNF compared to ACE2 mRNA levels in untreated control cells ACE2 mRNA levels in untreated control cell and in IL 4 treated cells are represented as the fold change in gene expression relative to the IFN TNF treated cells which were used as the calibrator GAPDH was used as an endogenous control 353 2006 474 481 that cytokines modulate ACE2 expression The physiological relevance of the IL 4 induced antiviral activity may be limited because this cytokine is probably not produced in large quantities during natural SARS CoV infection Huang et al 2005 Wong et al 2004 However vaccination strategies utilizing inactivated SARS CoVor subunits in the presence of specific alum based adjuvants may skew immune response to TH2 or TH0 allowing the production of IL 4 upon restimula tion Spruth et al 2006 The relevance of ACE2 expression modulation by IFN in the pathogenesis of SARS and NL 63 CoV needs further investigation Materials and methods Cells and cytokine treatment Vero E6 cells ATCC were grown in Dulbecco s modified Eagle smedium DMEM supplemented with10 fetalbovine serum FBS sodium bicarbonate and 20 mM HEPES buffer Unless stated otherwise recombinant human r hu IL 4 BD Pharmingen r hu IFN r hu TNF r hu IL 13 r hu IL 10 Peprotech Inc and r hu IFN Roche were added to cell cultures ataconcentrationof10ng ml 48hbeforeinfection As a specificity control r hu IL 4 was preincubated with a neutralizing IL 4 antibody Ebiosciences for 30 min at 37 C before addition to the cells All treatments were done in quadruplets 96 well experiments or triplicate 6 well and 24 well experiments Infection of Vero E6 cells and immunohistochemistry Vero E6 cells grown in 96 well plates were infected with 10 5 50 tissue culture infective doses TCID 50 SARS CoV HKU 39849 or HSV 1 by adding the virus directly to Vero E6 cell cultures After 16 h cells were fixed with 10 neutral buffered formalin and treated with 70 ethanol 10 min RT SARS CoV infected cells were visualized using purified human IgG from a convalescent SARS patient CSL followed by staining with an antibody to human IgG linked to horseradish peroxidase Amersham Biosciences HSV infected cells were stained using a rabbit anti HSV 1 antiserum followed by staining with an antibody to rabbit IgG linked to horseradish peroxidase DAKO In the case of the HCoV NL63 infection Vero E6 cells cultured in the presence or absence of IL 4 were infected with 10 4 TCID 50 HCoV NL63 The inoculum was removed after