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BioMed Central Virology Journal Open Access Research Inhibition of cytokine gene expression and induction of chemokine genes in non lymphatic cells infected with SARS coronavirus Martin Spiegel and Friedemann Weber Address Abteilung Virologie Institut f r Medizinische Mikrobiologie und Hygiene Universit t Freiburg D 79008 Freiburg Germany Email Martin Spiegel martin spiegel uniklinik freiburg de Friedemann Weber friedemann weber uniklinik freiburg de Corresponding author Abstract Background SARS coronavirus SARS CoV is the etiologic agent of the severe acute respiratory syndrome SARS CoV mainly infects tissues of non lymphatic origin and the cytokine profile of those cells can determine the course of disease Here we investigated the cytokine response of two human non lymphatic cell lines Caco 2 and HEK 293 which are fully permissive for SARS CoV Results A comparison with established cytokine inducing viruses revealed that SARS CoV only weakly triggered a cytokine response In particular SARS CoV did not activate significant transcription of the interferons IFN IFN IFN 1 IFN 2 3 as well as of the interferon induced antiviral genes ISG56 and MxA the chemokine RANTES and the interleukine IL 6 Interestingly however SARS CoV strongly induced the chemokines IP 10 and IL 8 in the colon carcinoma cell line Caco 2 but not in the embryonic kidney cell line 293 Conclusion Our data indicate that SARS CoV suppresses the antiviral cytokine system of non immune cells to a large extent thus buying time for dissemination in the host However synthesis of IP 10 and IL 8 which are established markers for acute stage SARS escapes the virus induced silencing at least in some cell types Therefore the progressive infiltration of immune cells into the infected lungs observed in SARS patients could be due to the production of these chemokines by the infected tissue cells Background For most viruses the initial encounter with the host takes place in cells of non lymphatic origin The outcome of this primary infection can determine the course of disease and the cytokine response of the infected cell plays a vital part Type I interferons IFN are potent antivirally active cytokines which can be produced by most if not all body cells in response to virus infection IFNs not only trigger the synthesis of antivirally active proteins they also and chemokines activate the adaptive immune system and direct the migration of leukocytes 2 Viruses on the other hand have evolved various mechanisms to counter act the host s cytokine response 3 and their ability to induce or inhibit cytokine production in infected cells has direct consequences for the balance between host defense and virus propagation SARS coronavirus SARS CoV is the etiologic agent of Published 29 March 2006 Virology Journal2006 3 17 doi 10 1186 1743 422X 3 17 Received 28 October 2005 Accepted 29 March 2006 This article is available from 2006Spiegel and Weber licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License http creativecommons org licenses by 2 0 which permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited Page 1 of 9 page number not for citation purposes activate the innate immune system and help to shape adaptive immunity 1 Other virus induced cytokines severe acute respiratory syndrome SARS a life threaten ing new human disease which recently emerged in China Virology Journal 2006 3 17 4 7 Characteristic SARS symptoms are high fever myal gia dry cough and lymphopenia and in around 30 of cases patients also developed an atypical form of pneu monia 8 The mechanisms underlying SARS CoV mediated patho genesis remain largely unexplained Autopsies from deceased patients revealed severe damage of the lungs and lymphatic tissues accompanied by infiltrations of mono cytic cells 9 11 This may indicate that immunopatho genesis is involved in the severe outcome of the disease providing the rationale for SARS therapy with immuno suppressant corticosteroids 12 On the other hand there is evidence that cell damages could be directly caused by the virus since SARS CoV is cytolytic 13 and capable to systemically infect human hosts 14 16 In addition virus particles and signs of necrosis were found in affected tissues 11 and high viral loads are predictive of adverse clinical outcome 17 Interestingly however the acute lung injuries and respiratory failure observed in severe cases occured while viral loads were declining 16 again favouring the hypothesis of immune mediated lung dam age Virus induced cytokines not only play a significant role in host defense but also in immunopathogenesis Investiga tions of the cytokine profiles of SARS patients have shown that the proinflammatory cytokines and chemokines IL 6 IL 8 and IP 10 CXCL10 are strongly upregulated 18 both IL 8 and IP 10 were upregulated 24 whereas in other cases either only IL 8 25 26 only IP 10 27 or no cytokines were induced at all 28 IL 6 was only moder ately upregulated 29 or not detected at all 24 26 Thus it is still unclear whether the cytokine storm in SARS patients was directly caused by the virus i e produced by SARS CoV infected cells or whether it is a secondary effect i e the result of strong activation of the immune system With one notable exception 24 most studies investigat ing the cellular cytokine response to SARS CoV were either based on immune cells 25 27 29 or on Huh7 hepatoma cells inoculated with unphysiologically high amounts of virus 26 Thus the overall picture of the cytokine response of non immune cells which are most probably the prime targets of SARS CoV may still be incomplete To learn more about it a human cell line would be needed which on one hand can support the complete viral replication cycle but on the other hand is also able to produce cytokines which are potentially anti viral However most cell lines which are permissive for SARS CoV have lost the ability to synthesize IFNs the most potent antiviral cytokines 24 30 31 In this study we identified an IFN competent human embryonic kid ney HEK 293 cell clone which supports the growth of SARS CoV Using these cells as well as the established human colon carcinoma cell line Caco 2 24 31 we investigated the SARS CoV induced production of repre sentative cytokines chemokines and antiviral genes Our studies revealed that SARS CoV is capable to suppress the antiviral cytokine response of infected cells to a large extent Interestingly however induction of the chemok ines IP 10 and IL 8 escaped suppression by SARS CoV in Caco 2 cells but not in HEK 293s Thus SARS CoV effi ciently blocks the innate host cell defense at a very early step of infection buying time to colonize the host With the possible exception of IP 10 and IL 8 most cytokines detected in SARS patients may therefore be produced by the infiltrating immune cells and not by the resident tis sue cells These data may help to explain both the rapid rise in virus titers during the initial stage of disease caused by the suppression of antiviral cytokines as well as the progressive infiltration of immune cells into the infected lungs which could be due to the production of chemok ines by the infected tissue cells Results Growth of SARS CoV in different cell lines Vero cells which are standard for growth of SARS CoV 30 31 lack type I IFN genes 32 33 and therefore are not suitable for cytokine analyses In search of an appro priate in vitro system we tested several IFN competent Virus titersFigure 1 Virus titers Simian Vero cells white bars human Caco 2 cells grey bars and human low passage HEK 293 cells black bars were infected at a multiplicity of infection MOI of 5 infectious particles per cell Virus titers in the superna tants were determined 24 h post infection and 48 h post infection by plaque assays 1 00E 05 1 00E 06 1 00E 07 1 00E 08 1 00E 09 1 00E 10 24 h p i 48 h p i Vero CaCo2 293 log 10 PFU ml Page 2 of 9 page number not for citation purposes 23 Cell culture studies by contrast did not reveal a clear picture of SARS CoV induced cytokines In some cases human cell lines for SARS CoV growth and identified a low passage clone of HEK 293 cells 34 as being fully per Virology Journal 2006 3 17 missive Fig 1 shows that titers of HEK 293 cells and Vero cells were comparable and rather high already at 24 h post infection Caco 2 cells by contrast produce approx imately 100 fold less virus at 24 h post infection and 10 fold less at 48 h post infection Fig 1 Thus we consid CoV on the immune system independent induction of cytokines Interferon genes and their antiviral effectors To properly assess the cytokine profile of SARS CoV infec Interferon production by virus infected human cellsFigure 2 Interferon production by virus infected human cells Caco 2 cells A and HEK 293 cells B were infected with SARS CoV or the IFN inducing control viruses Bunyamwera delNSs BdelNSs Sendai virus SeV Newcastle disease virus NDV or were left uninfected mock At 8 h left panels or at 16 h right panels post infection total RNA was isolated and investi gated by RT PCR for the presence of different IFN mRNAs The cellular actin mRNA served as loading control Note that for the reliable detection of IFN in Caco 2 cells A upper right panel the infection time had to be extended to 24 h A m o c k C o V B d N S s S e V N D V S IFN O1 m o c k S C o V B d N S s S e V N D V IFN D 8 h p i 16 24 h p i IFN E IFN O2 3 8 h p i 16 h p i B m o c k C o V B d N S s S e V N D V S IFN O1 m o c k S C o V B d N S s S e V N D V IFN D IFN E IFN O2 3 J actin J actin Caco 2 293 Page 3 of 9 page number not for citation purposes ered both the Caco 2 cells and the low passage HEK 293 cells as useful systems for studying the influence of SARS tion we compared it with well characterized cytokine inducers such as Bunyamwera delNSs virus BdNSs 35 Virology Journal 2006 3 17 Sendai virus SeV and Newcastle disease virus NDV In addition we deemed it necessary to monitor cytokine syn thesis both at 8 h and at 16 h post infection since we pre viously found a striking difference between the early and the late host cell response to SARS CoV 36 The first set of tested cytokines comprised the classical antiviral cytokines IFN and IFN 1 and the novel interferons IFN 1 and IFN 2 3 37 To test their induc tion in cell culture we infected with 5 plaque forming units pfu of viruses per cell and analyzed cytokine mRNAs by RT PCR As it is shown in Fig 2A and 2B clear signals for all IFNs were detected after infection with the control viruses BdNSs SeV and NDV For SARS CoV by contrast only a weak signal for IFN was detected in HEK 293 cells and none for IFN or the IFN s in either cell line All preparations contained similar amounts of input RNA since the actin control mRNA was present in equal amounts Fig 2A and 2B lower panels It was of specific and sensitive markers we used the ISG56 gene which is induced both by IFNs and by virus infection 38 39 and the MxA gene which is exclusively activated by IFNs 40 As is evident from Fig 3 no significant ISG induction occurs for SARS CoV whereas the control viruses activated ISG expression Note that SeV blocks in HEK 293 cells the synthesis of IFN see Fig 2B upper right panel and of MxA Fig 3B lower right panel most probably because of its ability to inhibit IFN induced sig naling 41 Curiously this does not happen in Caco 2 cells Fig 3A lower right panel suggesting cell type spe cific differences in cytokine signaling Taken together these data demonstrate that in contrast to the other viruses tested SARS CoV suppresses the activa tion of the antiviral IFNs and the IFN induced effector genes to a large extent Induction of chemokines by infected cells Interferon stimulated genesFigure 3 Interferon stimulated genes RNA samples of Caco 2 cells A and HEK 293 cells B described in Fig 2 were investigated by RT PCR for the presence of ISG56 and MxA mRNAs As for IFN see Fig 2A for detection of MxA mRNA in Caco 2 cells an extended infection period of 24 h was necessary A lower right panel m o c k C o V B d N S s S e V N D V S m o c k S C o V B d N S s S e V N D V ISG 56 MxA 8 h p i A m o c k C o V B d N S s S e V N D V S m o c k S C o V B d N S s S e V N D V ISG 56 MxA 8 h p i 16 h p i B 16 24 h p i Caco 2 293 Page 4 of 9 page number not for citation purposes interest to see whether virus infection would lead to the upregulation of antiviral IFN stimulated genes ISGs As IP 10 and RANTES are potent chemoattractants for acti vated T cells and NK cells 2 When we infected Caco 2 Virology Journal 2006 3 17 cells significant amounts of IP 10 mRNA were synthe sized Fig 4A upper panels This strong upregulation occurred independently of the virus suggesting a general response to virus infection Although IP 10 mRNA levels induced at 16 h p i by SARS CoV are slightly lower than by the other viruses our data are in good agreement with previous studies 24 27 Induction of RANTES by con trast was restricted to the cytokine inducing viruses whereas infection with SARS CoV had no effect above background levels Fig 4A lower panels We then tested HEK 293 cells in a similar way Much to our surprise IP 10 mRNA was not detectable early after infection with SARS CoV Fig 4B upper left panel and only very weakly expressed after longer infection Fig 4B upper right panel RANTES mRNA again was not detectable for SARS CoV Fig 4B lower panels All three cytokine inducing viruses activated IP 10 and RANTES expression in HEK 293 cells as expected Fig 4B upper and lower panels cytokine response is dependent on the host cell type RANTES expression by contrast is never induced by SARS CoV although the cells respond normally to other viruses Induction of IL 6 and IL 8 The proinflammatory cytokine IL 6 and the chemokine IL 8 are strongly upregulated in SARS patients 18 19 but from cell culture studies no clear picture emerged 24 26 29 We investigated IL 6 and IL 8 production by Caco 2 and HEK 293 cells infected with SARS CoV and com pared it with the other RNA viruses As shown in Fig 5 IL 6 is induced only weakly by SARS CoV independent of the cell line used Fig 5A and 5B upper panels IL 8 by contrast is clearly induced by SARS CoV in Caco 2 cells but not in HEK 293 cells Fig 5A and 5B lower panels The control viruses invariably induced both IL 6 and IL 8 demonstrating that the cell lines are capable to produce these cytokines Thus SARS CoV strongly induces IL 8 but not IL 6 in a Chemokine productionFigure 4 Chemokine production RNA samples of Caco 2 cells A and HEK 293 cells B described in Fig 2 were assayed by RT PCR for IP 10 and RANTES mRNA levels m o c k C o V B d N S s S e V N D V S m o c k S C o V B d N S s S e V N D V IP 10 8 h p i 16 h p i RANTES A m o c k C o V B d N S s S e V N D V S m o c k S C o V B d N S s S e V N D V IP 10 8 h p i 16 h p i RANTES B Caco 2 293 Page 5 of 9 page number not for citation purposes Thus SARS CoV induces IP 10 gene expression in Caco 2 cells but not in HEK 293 cells again suggesting that the cell type dependent manner This may suggest that the IL 8 detected in SARS patients 18 19 is directly synthesized Virology Journal 2006 3 17 by infected resident cells whereas IL 6 is more likely a sec ondary response mediated by infiltrating immune cells Taken together our data demonstrate that SARS CoV in general is a weak inducer of cytokines and antiviral genes in non lymphatic cells Chemokines like IP 10 and IL 8 however can be directly upregulated in SARS CoV in a cell type dependent manner Discussion The activation of immune relevant cytokines and host cell genes by SARS CoV in cells and patients was the subject of several previous investigations 18 21 22 24 29 42 44 However most of the cell culture studies were either based on immune cells which do not represent the major ity of infected cells 25 27 29 or on Huh7 hepatoma cells which needed to be infected with 100 pfu per cell i e with unphysiologically high amounts of virus 26 More over Huh7 cells are known to be deficient in the antiviral virus infected cells or whether it was mediated by the acti vated immune system Furthermore it was not systemati cally investigated how the cytokine induction by SARS CoV compares to other viruses Here we have used three control viruses and two different cells lines to elucidate and compare the induction of cytokines by SARS CoV Our results demonstrate that SARS CoV does not induce significant amounts of IFNs antiviral genes RANTES and IL 6 In agreement with this finding SARS CoV infected macrophages and dendritic cells lack IFN induction 27 29 IP 10 and IL 8 however can be activated by SARS CoV This suggests that these chemokines which are reliable markers of acute stage SARS 18 20 21 23 are not only produced in response to IFN after activation of the immune system as suggested but may also be directly secreted by infected tissue cells An upregulation of either IP 10 and or IL 8 was observed in several studies using SARS CoV infected Caco 2 cells 24 macrophages 27 peripheral blood mononuclear cells 25 and dendritic Interleukin productionFigure 5 Interleukin production RNA samples of Caco 2 cells A and HEK 293 cells B described in Fig 2 were investigated by RT PCR for the presence of IL 6 and IL 8 mRNAs m o c k C o V B d N S s S e V N D V S m o c k S C o V B d N S s S e V N D V IL 6 IL 8 8 h p i 16 h p i A m o c k C o V B d N S s S e V N D V S m o c k S C o V B d N S s S e V N D V IL 6 IL 8 8 h p i 16 h p i B Caco 2 293 Page 6 of 9 page number not for citation purposes cytokine response 45 Thus it was not entirely clear whether the patients cytokine response was caused by cells 29 Using HEK 293 cells by contrast we found that SARS CoV is able to downregulate also IP 10 and IL 8 pro Virology Journal 2006 3 17 duction Similarly recent studies showed that peripheral blood monocytes from SARS patients do not produce any cytokines 28 Thus chemokine induction by SARS CoV appears to be highly cell type specific With the exception of IP 10 and IL 8 SARS CoV is capable to suppress the production of a wide range of cytokines This is in agreement with our previous finding that SARS CoV inhibits the crucial cytokine transcription factor IRF 3 36 providing a possible mechanism for the high potential of this pathogen to suppress the host response Of note SARS CoV is highly sensitive to the antiviral action of IFNs both in vivo and in vitro 46 51 thus explaining why the virus needs to suppress IFN induction in advance Conclusion In the initial phase of SARS the virus grows exponentially and spreads to different organs including the lungs 8 14 Our data may explain this rapid and efficient dis semination of SARS CoV By slowing down expression of IFNs and their antiviral genes in the infected tissue cells the virus buys time during the initial critical phase of infection in order to grow unhindered in the host At the same time however the virus induced chemokines IP 10 and IL 8 attract immune cells Possibly this mixture of high level virus replication followed by the invasion of activated immune cells results in a strong inflammatory response leading to a cytokine storm and the severe and potentially fatal respiratory distress which is the hallmark of full blown SARS Methods Cells and viruses Simian VeroE6 cells human Caco 2 cells and human embryonic kidney HEK 293 cells were maintained and grown as described 24 36 The low passage HEK 293 cell clone 34 was purchased from Microbix Biosystems Toronto Canada All experiments were performed with HEK 293 cells between passage 38 and 48 The FFM 1 iso late of SARS CoV was kindly provided by Stephan Becker University of Marburg Germany Bunyamwera delNSs virus 35 Sendai virus and Newcastle disease virus were used as controls Plaque assays Virus plaque assays were performed as described previ ously 50 Briefly Vero cell monolayers were infected with dilutions of supernatants from infected cells over laid with soft agar and