Suppressor of cytokine signaling 1 regulates the immune response to infection by a unique inhibition of type I interferon activity

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1 5 Nture Pulishing Group Suppressor of cytokine signling 1 regultes the immune response to infection y unique inhiition of type I interferon ctivity Jennifer E Fenner 1, Royn Strr,4, Ann L Cornish,4, Jin-Guo Zhng, Donld Metclf, Roert D Schreier 3, Kthleen Sheehn 3, Dougls J Hilton, Wrren S Alexnder & Pul J Hertzog 1 Suppressor of cytokine signling 1 (SOCS1) is criticl regultor of cytokine signling nd immune responses. SOCS1-deficient mice develop severe inflmmtory disese, ut re very resistnt to virl infections. Using neutrlizing ntiody to type I interferon (IFN- nd IFN-) nd mice deficient in interferon-c or type I interferon receptor components (IFNAR1 or IFNAR), we demonstrte here tht SOCS1 deficiency mplified type I interferon ntivirl nd proinflmmtory ctions independently of interferon-c. The mechnism of the suppression of type I interferon responses y SOCS1 ws distinct from tht of other cytokines. SOCS1 ssocited with nd regulted IFNAR1- ut not IFNAR-specific signls, rogting tyrosine phosphoryltion of trnscription fctor STAT1 nd reducing the durtion of ntivirl gene expression. Thus, SOCS1 is n importnt in vivo inhiitor of type I interferon signling nd contriutes to lncing its eneficil ntivirl versus detrimentl proinflmmtory effects on innte immunity. Approprite regultion of cytokine signling is criticl not only in mintining homeostsis ut lso in the response to virl nd cteril infections nd injury. Conversely, inpproprite or unrestrined cytokine ction cn result in unwnted toxicity, leding to neurologicl dmge, growth suppression, cncer, inpproprite ctivtion of the innte nd cquired immune systems nd ultimtely chronic inflmmtory disese, utoimmunity nd sometimes deth 1. The interferons re cytokines tht were discovered y virtue of their ntivirl ctions nd were susequently demonstrted to regulte cell prolifertion nd differentition nd to ctivte most effector cells of the immune system,3. Interferons re clssified into two types sed on sequence homology, the gents tht induce their production, their cellulr origin nd their use of distinct receptor systems. Type I interferons include the vrious IFN- sutypes, IFN-, IFN-$ nd limitin, which ll ct through the receptor components designted IFN- receptor 1 (IFNAR1) nd IFNAR (refs. 36). Type I interferons re produced y vriety of cell types, including leukocytes, firolsts nd plsmcytoid dendritic cells, in response to virus nd other infectious gents 7. In contrst, the single type II interferon, IFN-g, cts through receptor components IFN-g receptor 1 nd IFN-g receptor nd is produced minly y T cells, nturl killer cells nd nturl killer T cells in response to cytokines such s interleukin 1 (IL-1) 8,9. Both clsses of interferon ctivte two Jnus kinses (Jks) tht re pressocited with prticulr receptor chin: TYK nd Jk1 for type I interferons, nd Jk1 nd Jk for type II interferons. In turn, this leds to ctivtion of complexes of signl trnsducer nd ctivtor of trnscription (STAT) fctors: interferon-stimulted gene fctor 3 (STAT1, STAT nd interferon-regultory fctor 9) for type I interferons, nd g-ctivted fctor (STAT1 homodimers) minly for IFN-g 1,11. The interferons hve properties such s potent ntivirl ctivity nd ctivtion of immune responses tht re clerly eneficil to the host. However, they lso hve undesirle side effects demonstrted y their dose-limiting toxicity in clinicl trils in humns nd neontl toxicity in mice 1,1. Therefore, lncing the potentilly eneficil nd detrimentl effects of interferons is likely to e crucil to mounting successful immune response to disese. In ddition to the cytoplsmic fctors tht drive signl trnsduction, there re negtive regultors tht inhiit or ttenute signling. These include phosphtses, protein inhiitors of ctivted STAT proteins, nd suppressor of cytokine signling (SOCS) proteins The SOCS fmily of proteins includes eight memers, designted SOCS1SOCS7 nd CIS (cytokine-inducile Src homology contining protein), which inhiit signling y inding to key phosphotyrosine residues in Jks or cytokine receptor cytoplsmic domins nd trgeting them for protesoml degrdtion 16,185. The expression of Socs1 nd Socs3 ut neither Socs nor Cish is inducile y IFN-, IFN- nd IFN-g; however, their expression is lso induced y other cytokines, including growth hormone, IL-6, 1 Centre for Functionl Genomics nd Humn Disese, Monsh Institute of Medicl Reserch, Monsh University, Clyton 3168, Austrli. The Wlter nd Eliz Hll Institute for Medicl Reserch nd The Coopertive Reserch Center For Cellulr Growth Fctors, Prkville 35, Austrli. 3 Deprtment of Pthology nd Immunology, Center for Immunology, Wshington University, St. Louis, Missouri 6311, USA. 4 Present ddresses: St Vincent s Institute for Medicl Reserch, Fitzroy 365, Austrli (R.S.), nd Deprtment of Microiology nd Immunology, University of Melourne, Melourne 31, Austrli (A.L.C.). Correspondence should e ddressed to P.J.H. (pul.hertzog@med.monsh.edu.u). Received August; ccepted 4 Octoer; pulished online 7 Novemer 5; doi:1.138/ni187 NATURE IMMUNOLOGY ADVANCE ONLINE PUBLICATION 1

2 5 Nture Pulishing Group IL-3, IL-13, grnulocyte-mcrophge colonystimulting fctor, leukemi inhiitory fctor, erythropoietin, IL-1 nd leptin. Likewise, when constitutively overexpressed, SOCS1 nd SOCS3 inhiit signling in response to cytokines such s interferons nd other cytokines cting through the Jk-STAT pthwy 15,17,18,68.Thein vivo function of SOCS1 hs een explored through the genertion of mice with null lleles of Socs1. SOCS1-deficient mice die during the neontl period with severe heptotoxicity, multiorgn inflmmtion nd errnt hemtopoiesis 9,3. This phenotype requires IFN-g, s the genertion of mice deficient in oth SOCS1 nd IFN-g or the injection of Socs1 / mice with neutrlizing ntiody to IFN-g rescues the lethl neontl Socs1 / phenotype 31. Susequent studies hve estlished tht mice lcking SOCS1 show oth incresed production of nd heightened sensitivity to IFN-g, which contriute to the perintl lethlity 3,33. Those studies hve een instrumentl in demonstrting the dire consequences of unchecked IFN-g signling. However, the lethl IFN-g-medited phenotype of SOCS1-deficient mice my e msking the in vivo importnce of SOCS1 in regulting the responses to other cytokines, prticulrly the type I interferons. In this study, we demonstrte tht mice with trgeted muttion in Socs1 hd heightened ntivirl responses, resulting in decresed virl lod nd incresed survivl fter infection, trits tht re indictive of mplified protective effects of interferons. Using Socs1 / neontl mice, neutrlizing ntiody to type I interferon nd crosses to Ifng / mice or Ifnr1 / mice, we show tht these eneficil effects were consequence of mplifiction of type I interferon ntivirl ctions independently of IFN-g. TheSocs1 / Ifnr1 / mice lso survived eyond wening nd did not demonstrte the dire inflmmtion ssocited with SOCS1 deficiency. Thus, SOCS1 lso cts to suppress the proinflmmtory effects of type I interferons. Unlike the production of IFN-g, the production of type I interferons ws not suppressed y SOCS1; however, cells lcking SOCS1 showed prolonged STAT1 phosphoryltion nd ntivirl interferon-stimulted gene stimultion in response to type I interferon. Comprison of the result of crosses of Socs1 / mice with Ifnr / or Ifnr1 / mice demonstrted unique specificity of SOCS1 ction on signling through only the IFNAR1 chin of the receptor. Consistent with tht oservtion, we demonstrte here y coimmunoprecipittion tht SOCS1 ssocited with IFNAR1. Thus, SOCS1 uses unique nd very specific mechnism to regulte type I interferon signling vi IFNAR1 nd contriutes in vivo to lncing its vrious effects on innte immunity. Therefore, SOCS1 modultion my e useful djunct to selective ntivirl therpy. RESULTS Amplified ntivirl responses in Socs1 / neontl mice Socs1 / neontl mice survive sustntilly longer thn their wildtype nd heterozygous littermtes fter infection with Semliki Forest virus (SFV); however the mechnisms of enhnced virl resistnce hve remin unexplored 31. To ddress tht issue, we mesured virl lods in the serum, spleens, kidneys, livers, thymuses nd lungs of Socs1 / nd wild-type neontl mice 48 h fter infection with SFV. Virl titers in the serum nd in orgns including the lung, thymus, Virl lod (titer) Kidney Liver Lung Socs1 +/+ nd Socs1 +/ Socs1 / Serum Spleen Thymus Socs1 +/+ nd Socs1 +/ Socs1 / Figure 1 Virl repliction nd interferon secretion from SFV-infected neontl mice with trgeted muttion of Socs1. Neontl mice from Socs1 +/ mtings were injected with SFV t 1 TCID 5 etween 5 nd 1 d of ge. Neontes were monitored for 48 h, t which time they were killed nd orgns nd serum were collected. () Virl titers, mesured y CPE ssy nd expressed s men log titer ± s.e.m., P o.5,, P o.1, nd, P o.1, Socs1 / neontl mice (n ¼ 9) versus their wild-type nd heterozygous littermtes (n ¼ 4; Mnn-Whitney rnk sum tests). () Serum interferon, mesured y CPE-reduction ssy. Ech point represents n individul mouse; horizontl lines indicte men concentrtions., P o.5, Socs1 / neontl mice (n ¼ 9) versus their wildtype nd heterozygous littermtes (n ¼ 31; Mnn-Whitney rnk sum test). spleen, kidney nd liver in the Socs1 / mice were.11% those in wild-type nd heterozygous littermtes (Fig. 1). The reduced virl lod in Socs1 / orgns suggested tht the improved survivl of the SFV infected Socs1 / mice resulted from n incresed ility to resolve the virl infection. SOCS1 does not ffect IFN- or IFN- production The reduced virl lod in Socs1 / mice could hve een due to either incresed production of interferons fter virl infection or incresed ntivirl response of cells to similr mounts of interferon. In untreted helthy mice, oth type I nd II interferon is generlly undetectle in serum. However, fter virl chllenge, expression of type I interferons is induced rpidly, resulting in redily detectle mounts of these cytokines 34. We mesured interferon ntivirl ctivity y cytopthic effect (CPE)reduction iossy of serum smples from SFVinfected neontl mice. After virl infection, interferon ws detected in serum from 77% (4 of 31) wild-type nd Socs1 +/ mice, with concentrtions rnging from out 1 to 1, IU/ml with men of 3 IU/ml. In contrst, interferon ctivity ws detected in the serum of only 33% (three of nine) Socs1 / mice, with men concentrtion of 3 IU/ml, rnging from to 3 IU/ml (Fig. 1). Thus, the serum interferon concentrtions in Socs1 / mice fter virl infection were significntly lower thn those in wild-type mice (Po.5). Therefore, the incresed resistnce of Socs1 / mice to SFV chllenge proly resulted from n incresed sensitivity to interferons. Virl resistnce in Socs1 / mice is IFN-c independent Becuse the incresed virl resistnce of Socs1 / mice ws not due to incresed interferon production fter virl infection, we next investigted the regultion y SOCS1 of ntivirl ctions of interferons. Although SOCS1 hs een shown y overexpression studies in vitro to suppress the ntivirl effects of oth type I nd II interferons 35,the in vivo effects hve not een chrcterized. Becuse SOCS1 regultes other responses to IFN-g in vivo, ffecting moleculr signling, hemtopoiesis, inflmmtory nd immune responses 3638, we ddressed whether the incresed ntivirl response in Socs1 / mice could e ttriuted to n incresed sensitivity to IFN-g 11,39,4. We first exmined whether removl of endogenous IFN-g ffected the outcome of SFV infection. We infected Ifng / nd wild-type neontl Interferon (IU/ml) ADVANCE ONLINE PUBLICATION NATURE IMMUNOLOGY

3 c Survivl (%) Ifng/ Time fter inocultion (d) 5 Survivl (%) Time fter infection (d) Virl lod (titer) Kidney Lung Spleen 5 Nture Pulishing Group Figure IFN-g responses do not ffect the hyper-resistnce of Socs1 / mice to virl infection. () Resistnce to infection with SFV t 1 TCID 5 in wildtype (; n ¼ 13) nd Ifng / (n ¼ 16) neontl mice etween 4 nd 11 d of ge. P 4.5, wild-type versus Ifng / (log-rnk test). () Socs1 / Ifng / neontl mice show more resistnce in response to SFV thn do wild-type neontl mice. Survivl plot reports resistnce to infection with SFV t 1 TCID 5 in wild-type (n ¼ 4) nd Socs1 / Ifng / (n ¼ 6) neontl mice etween 5 nd 1 d of ge., P o.1 (log-rnk test). In,, the helth of neontl mice ws monitored nd ll ftlities were recorded. (c) Virl titers of homogentes of kidneys, lungs nd spleens of in Socs1 / Ifng / (n ¼ 6) nd wild-type (n ¼ 4) neontl mice injected with SFV t 3 TCID 5. Neontl mice were monitored for 48 h, t which time they were killed nd orgns were collected. Virl titers were mesured y CPE ssy nd re expressed s men log titer ± s.e.m., P o.5, nd, P o.1, titers of wild-type mice versus Socs1 / Ifng / mice. (Mnn-Whitney rnk sum tests.) Dt re representtive of one experiment. mice with SFV t 1 the hlf-mximl tissue culture infectious dose (TCID 5 ; the concentrtion of virus tht will kill 5% of cells) nd mesured resistnce to the virus s survivl fter infection. There ws no significnt difference in the survivl of wild-type neontl mice nd Ifng / mice (P 4.5). All mice of oth genotypes were ded y 4.5 d fter infection (Fig. ), indicting tht IFN-g does not hve sustntil involvement in the resistnce of mice to SFV infection. The survivl curve of infected Socs1 / mice closely follows tht of untreted Socs1 / mice 31, suggesting tht these mice my e lmost completely resistnt to the SFV infection nd were in fct succuming to the inflmmtory disese ssocited with SOCS1 deficiency. To overcome the prolem of the inflmmtion-medited deth of Socs1 / mice efore wening, we took dvntge of Socs1 / Ifng / mice, which fil to produce IFN-g, re helthy nd survive to dulthood 31. Socs1 / Ifng / neontl mice seemed lmost completely resistnt to infection with SFV, with 84% of infected neontl mice (five of six) surviving pst d. In contrst, none of the infected wild-type mice survived eyond 7 d of infection (zero of four; P o.1; Fig. ). Similr to the titers of Socs1 / mice, virl titers in the orgns of SFVinfected Socs1 / Ifng / mice were pproximtely 1% those of wildtype control mice (Fig. c). We noted similr trends in experiments using higher dose of virus (3 TCID 5 ; dt not shown). These results suggest tht the doule-knockout mice were le to cler the virus from most orgns more efficiently thn wild-type control mice. Becuse these trends were similr to tht demonstrted for Socs1 / mice, the suppression of ntivirl effects nd enhnced resistnce to infection y SOCS1 is clerly independent of IFN-g. Type I IFN medites SFV resistnce in Socs1 / Ifng / mice The results presented ove suggested tht type I interferons medited the heightened ntivirl response noted in oth the Socs1 / nd the Socs1 / Ifng / neontl mice. To estlish directly the involvement of type I interferons, we injected 1, IU of neutrlizing ntiody to type I IFN (which recognizes oth IFN- nd IFN-) into the peritonel cvities of 5- to 1-dy-old Socs1 / Ifng / neontl mice 6 h efore SFV infection. We then monitored the survivl of mice for 1 d s descried ove. In the sence of ntiody tretment, the survivl of Socs1 / Ifng / mice ws 1% fter 3 d, 75% fter 4 d nd 55% fter 1 d of virl infection. Pretretment of Socs1 / Ifng / mice with neutrlizing ntiody to type I IFN reversed the reltive resistnce of these mice to virl infection nd resulted in incresed sensitivity to SFV, with ll mice ded y 4 d, similr to wild-type mice (Fig. 3). To demonstrte y n independent method tht SOCS1 regultes type I interferons, we generted mice lcking oth SOCS1 nd IFNAR1 nd ssessed the survivl of 5- to 1-dy-old mice in response to SFV. Ifnr1 / mice re reported to hve reduced type I interferon signling nd to e highly susceptile to virl infection 41,4. Socs1 / Ifng / mice infected in this experiment demonstrted 1% survivl t 4 d, 9% survivl t 5 d nd 5% survivl t 1 d fter infection. When we crossed Socs1 / mice with Ifnr1 / mice, however, the offspring were more sensitive to infection, with 65% survivl t 4 nd 5 d fter infection nd % survivl t 6 d fter infection (P o.1; Fig. 3). These dt collectively suggest tht SOCS1 regultion of type I ut not type II interferon ntivirl ction hs profound effect on virl repliction (y up to 1,-fold) nd survivl fter infection. Mechnism of SOCS1 suppression of type I IFN signling To exmine the mechnism of SOCS1 regultion of type I interferon responses in vivo independently of IFN-g, we first exmined the durtion of tyrosine phosphoryltion of STAT1, primry meditor of interferon-induced ntivirl ctivity 1. Tretment of one mrrow mcrophges (BMMs) from Ifng / mice with IFN-1 resulted in trnsient increse in tyrosine phosphoryltion of STAT1, which egn to decrese y 6 min, ws rely detectle fter 8 min (Fig. 4) nd ws not detectle fter 1 min (dt not shown). In contrst, BMMs deficient in SOCS1 reproducily demonstrted prolonged STAT1 tyrosine phosphoryltion tht ws still strongly detectle 8 min (Fig. 4) nd 1 min (dt not shown) fter tretment. Notly, STAT1 serine phosphoryltion ws unffected y the presence of SOCS1 (dt not shown), consistent with inctivtion of tyrosine kinse ssocited with IFNAR, such s Jk, y SOCS1. To determine whether the ctions of SOCS1 ffected interferonstimulted ntivirl gene ctivity, we next monitored the ctivity of type I interferoninducile, ntivirl enzyme, -5 -oligodenylte synthetse ( -5 -OAS), which is regulted y STAT1 vi IFNARmedited signling 4. This ssy hs the dvntge of eing le to ccurtely quntify sl s well s inducile enzyme ctivity. The sl -5 -OAS ctivity in Socs1 / cells ws not significntly different from tht of wild-type cells (75. ± 43.9 nd ± 35.7 mmol/mg protein in wild-type (n ¼ 3) nd Socs1 / (n ¼ 5) mouse emryo firolsts, respectively; P o.5). After stimultion with IFN-4, the NATURE IMMUNOLOGY ADVANCE ONLINE PUBLICATION 3

4 5 Nture Pulishing Group Survivl (%) Survivl (%) Time fter infection (d) Time fter infection (d) Anti-IFN-αβ No ntiody ctivity in wild-type mouse emryo firolsts peked fter 7 h nd therefter decresed stedily to pproximtely 4% of the mximum vlue y 144 h (Fig. 4). In contrst, lthough -5 -OAS ctivity reched similr pek in Socs1 / mouse emryo firolsts fter 48 h, the ctivity ws mintined t this pek for the entire time course (P o.5; Fig. 4) without the decrese noted in wild-type cells. These results show tht lthough SOCS1 hd no pprent effect on the mplitude of the IFN- induction of this interferon-stimulted gene, it ws crucil in its temporl regultion, cting vi IFNAR on receptorproximl signling events tht regulte the durtion of susequent interferon-stimulted gene ctivtion. Becuse type I interferons trnsduce signls through the IFNAR1 nd IFNAR components ech with specific interction domins for signl-trnsducing proteins, we compred nd chrcterized mice deficient in SOCS1 nd either IFNAR1 or IFNAR to ctegorize the SOCS1 effects on IFNAR signling. The survivl of uninfected Ifnr1 / Socs1 / mice ws different from tht of Socs1 / mice, with no deths t 3 weeks of ge, when ll Socs1 / mice hd died (Fig. 5). Histologicl exmintion of orgns from the Ifnr1 / Socs1 / mice showed tht these mice did not hve the fmilir inflmmtory syndrome of the Socs1 / mice (dt not shown), demonstrting Ifnr1 / Socs1 / Figure 3 Type I interferon medites resistnce to SFV in Socs1 / Ifng / mice. () Survivl fter SFV infection in Socs1 / Ifng / neontl mice is due to the type I interferon ntivirl response. Socs1 / Ifng / neontl mice etween 5 nd 1 d of ge were divided into two groups, control (n ¼ 11)ndtest(n ¼ 9). The test group ws given 1, IU sheep nti-type I IFN (nti-ifn- /); the control group ws injected with PBS. After 6 h, oth groups were infected with SFV t 1 TCID 5 nd were monitored. As reference, wild-type neontl mice (; n ¼ 5) of the sme ge were infected with SFV t 1 TCID 5 nd were monitored., P o.1, Socs1 / Ifng / mice with nti-type I IFN versus no ntiody (log-rnk test). () Deletion of Ifnr1 in Socs1 / neontl mice decreses their resistnce to virl infection. Survivl plot mesures the resistnce to infection with SFV t 3 TCID 5 in Ifnr1 / Socs1 / (n ¼ 3) nd Socs1 / Ifng / (n ¼ 8) neontl mice etween 5 nd 1 d of ge. The helth of neontl mice ws monitored nd ftlities were recorded., P o.1 (log-rnk test). the importnt contriution to this phenotype of signling through IFNAR1. In contrst, Ifnr / Socs1 / mice were ll ded y 3 weeks of ge, similr to the originl Socs1 / mice (Fig. 5). Becuse this phenotype ws unexpectedly different from the survivl pttern of Ifnr1 / Socs1 / mice, it demonstrtes tht signls trnsduced through the IFNAR receptor component re not influenced y SOCS1. These results represent n importnt dvnce in elucidting the mechnism of SOCS1 ction in vivo. In regulting the type I interferon system, SOCS1 intercts with only one receptor chin, IFNAR1 nd/or its ssocited signl-trnsducing proteins. Becuse the result reported ove constituted compelling genetic evidence for n ssocition of SOCS1 with IFNAR1, we next sought to demonstrte this ssocition directly. First, we coimmunoprecipitted IFNAR1 nd Flg-tgged SOCS1 overexpressed in 93T cells (Fig. 5). Notly, we then demonstrted ssocition of endogenous SOCS1 with IFNAR1 y coimmunoprecipittion from IFN-1-treted BMM lystes using monoclonl ntiody to immunoprecipitte IFNAR1 nd monoclonl ntiody to SOCS1 to detect this protein y immunolot (Fig. 5c). Thus, our results demonstrte tht SOCS1 intercts endogenously with IFNAR1 fter stimultion nd tht this interction is independent of Jk1. DISCUSSION In this study we hve demonstrted tht SOCS1 is crucil in ttenuting type I interferon signling in vivo nd hence in limiting the host response to virl infection. Mice nd cells lcking SOCS1 responded to type I interferons for longer to limit virl repliction nd to cler virus more efficiently nd, in doing so, llowed survivl of n otherwise lethl infection. Unlike the sitution with IFN-g, in which SOCS1 deficiency led to oth incresed production nd Figure 4 Mechnism of SOCS1 suppression of type I interferon ntivirl signling. () Durtion of STAT1 tyrosine phosphoryltion in BMMs, nlyzed y immunolot of cell lystes. BMMs from Ifng / nd Socs1 / Ifng / mice were pulse-treted for min with 1, IU/ml of IFN-1, then cell lystes were prepred t -minute intervls up to 8 min fter tretment. Immunolots used n ntiody specific for tyrosine-phosphorylted STAT1 (STAT1-P(Tyr)) or for STAT1; -tuulin, loding control. () Durtion of -5 -OAS stimultion in response to IFN-4 is prolonged in SOCS1- kd , Socs1 / Ifng /, Time (min) STAT1-P(Tyr) Time fter tretment (h) deficient mice. Wild-type (; n ¼ 3) nd Socs1 / (n ¼ 5) mouse emryo firolst cell lines t dy 13 were individully pulse-treted for 4 h with 1, IU/ml of IFN-4. Cells were collected every 4 h until 144 h fter initil tretment nd were lysed t density of cells/ml of lysis uffer; lystes were nlyzed y enzyme ssy mesuring -5 -OAS ctivity in rdioctive redout., P o.5 (Mnn-Whitney rnk sum tests). β-tuulin Totl STAT1 β-tuulin -5 -OAS ctivity (mmol/µg lyste) 15, 1, 5, ADVANCE ONLINE PUBLICATION NATURE IMMUNOLOGY

5 5 Nture Pulishing Group c Survivl (%) kd kd Ifng / Age (weeks) Time (min) SOCS1 IFNAR1 heightened sensitivity, only the ltter ws ffected with respect to type I interferons. The difference in the effect of SOCS1 on the production of the two types of interferon could hve een due to differences in the mechnism of their induction. One prominent mens of induction of IFN-g is y IL-1, cytokine known to e regulted y SOCS1 (ref. 43). Conversely, type I interferons re ctivted directly y virl ctivtion of interferon-regultory fctors 4,44, pthwy tht is not known to e sensitive to SOCS1. We used severl independent methods to demonstrte tht SOCS1 regultion of type I interferon ctivity ws responsile for the resistnce to virl infection independently of IFN-g. Those methods included the use of Socs1 / mice in comintion with Ifng / mice, Ifnr1 / mice nd injection of neutrlizing ntiody to type I IFN. Those results hve provided n independent demonstrtion tht SOCS1 inhiition of type I interferon responses in vivo modulted resistnce or sensitivity to virl infection. Notly, in the sence of exogenous virl infection, these Ifnr1 / Socs1 / mice survived eyond 3 weeks of ge nd did not die from the inflmmtion tht chrcterized the Socs1 / mice, which ll died efore 3 weeks of ge 9. Tht result demonstrtes the proinflmmtory potency of type I interferons, which is incresingly eing recognized s n importnt meditor of inflmmtion nd product of selective Toll-like receptor ctivtion 4,44,45. Furthermore, tht pthwy my contriute to the function of SOCS1 in regulting Toll-like receptor signling 45. Type I interferons contriute to the IFN-g response, possily y recruitment of IFNAR1 into the IFN-g receptor signling complex 46. It is therefore possile tht the rogtion of inflmmtion in Ifnr1 / Socs1 / mice reflected (prtil) reduction in IFN-g signling, leit through type I interferon production nd ction vi IFNAR1. We consider this possiility unlikely, ecuse deficiency in either IFNAR chin reduced IFN-g signling (P.J.H. et. l., unpulished oservtions) ut only one ffects survivl from the inflmmtory response (discussed elow). To determine the selectivity of SOCS1 regultion of interferon signling, we lso crossed the Socs1 / mice with Ifnr / mice tht we hve generted nd chrcterized (P.J.H. et l., unpulished Ifnr1 / Socs1 / Ifnr / Socs1 / Socs1 / IFNAR1 SOCS1 Jk1 SOCS1 IFNAR Figure 5 IFNAR1 nd SOCS1 show direct interction. () Survivl of uninfected Socs1 / mice (n ¼ 8), Ifnr1 / Socs1 / mice (n ¼ 14) nd Ifnr / Socs1 / mice (n ¼ 7) versus wild-type control mice (n ¼ 6). () Immunolot of cells overexpressing IFNAR1 nd SOCS1. Cells (93T) were infected with vrious constructs, pef-bos-flg-jk1, pef-bos-flg- SOCS1 nd pef-bos-ifnar1; cell lystes were immunoprecipitted with monoclonl ntiody to IFNAR1 followed y immunolot with iotinylted nti-socs1 nd horserdish peroxidseconjugted nti-flg. (c) Immunolot of BMM lystes from Ifng / nd Socs1 / Ifng / mice treted with 1, IU/ml of IFN-1, showing n interction etween IFNAR1 nd SOCS1. Lystes were immunopreciptted with monoclonl ntiody to IFNAR1 followed y immunolot with iotinylted monoclonl ntiody to SOCS1. Dt re representtive of three experiments. dt). This cross did not rescue the inflmmtory phenotype of the Socs1 / mice, s Ifnr / Socs1 / mice ll died efore 3 weeks of ge, like the Socs1 / mice. Tht result demonstrtes tht the mechnism wherey SOCS1 regultes type I interferon responses in vivo is vi ction selectively on IFNAR1 or its ssocited signling molecules such s the TYK kinse 11 nd does not ffect IFNAR- medited signling. Notly, we demonstrted the mechnism of selective ction of SOCS1 on IFNAR1 y coimmunoprecipittion of endogenous IFNAR1 with SOCS1 from IFN-1-treted BMMs nd of IFNAR1 nd SOCS1 proteins overexpressed in 93T cells. We hve further chrcterized the moleculr mechnisms responsile for the mplified interferon responses noted in the Socs1 / neontl mice s resulting from sustined response to type I interferons. Tyrosine phosphoryltion of STAT1, proly y Jk fmily memer such s TYK, ws prolonged, wheres serine phosphoryltion ws unffected. Also, the interferon-inducile -5 -OAS ntivirl enzyme ctivity in cells isolted from Socs1 / mice produced signl tht incresed to the sme intensity s in wild-type mice ut ws sustined for longer period. Despite the impression tht SOCS1 hs reltively specific function in vivo in suppressing IFN-g-medited neontl deth, it in fct hs multiple functions in vivo. When exmined more closely (in the cse of IL-1, IL-, IL-4, IL-7 nd IL-15), SOCS1 hs more sutle ut nevertheless importnt functions in regulting other spects of the immune response 43,47,48. The loss of IFN-g regultion hs n erly nd profound effect on the survivl of young nimls, nd we hve now demonstrted tht type I interferon signls contriute to tht phenotype. We hve further demonstrted here tht fter chllenge with virus, n essentil function of SOCS1 on regulting type I interferon responses in vivo is uncovered. Tht result my extend eyond the ntivirl ctions of these interferons to cteril infections, s reports hve indicted tht SOCS1-deficient mice re hyporesponsive to chllenge with lipopolyscchride. Notly, type I interferon signls re importnt in Toll-like receptor 4medited lipopolyscchride responses 4,44,45. Becuse type I interferons regulte oth innte immunity (dendritic cells, mcrophges nd Toll-like receptor medited responses) nd cquired immunity (memory T cells nd CD8 + T cells) nd re reltively more specific in ction thn re other cytokines, regultion of SOCS1 could e considered s n djunct to type I interferons in the clinicl context nd could provide therpeutic trget in type I interferonsensitive diseses. METHODS Mice. Socs1 / nd Socs1 +/ mice nd their equivlent wild-type mice were generted nd mintined on mixed 19/Sv nd C57BL/6 ckground 9 nd were housed in clen ut not specific pthogenfree conditions efore virl infection experiments. These mice were genotyped y Southern lot nlysis of EcoRI-digested genomic DNA nd hyridiztion with 1.5-kilose (1.5-k) NATURE IMMUNOLOGY ADVANCE ONLINE PUBLICATION 5

6 5 Nture Pulishing Group EcoRIHindIII frgment of mouse Socs1 (ref. 9). This frgment hyridized to 5.3-k nd for the wild-type llele nd n 8.-k nd for the knockout llele. Socs1 / Ifng / mice, Ifng / mice nd their equivlent C57BL/6 wild-type mice were mintined s seprte colonies 31. Ifnr1 /, Ifnr1 / Socs1 / nd wild-type control mice were generted y intercrossing of Ifnr1 +/ Socs1 +/ mice housed in conventionl conditions nd genotyped y Southern lot nlysis of EcoRI-digested genomic DNA. Hyridiztion with 1.7-k BmHI ScI frgment of mouse Ifnr1 (ref. 4) identified 3.8-k nd for the wildtype llele nd 4.8-k nd for the knockout llele. The Socs1 genotype of these mice ws determined s descried ove. Ifnr / mice were generted y trgeting of exon 4 of Ifnr, resulting in null muttion wherey oth the trnsmemrne nd solule isoforms of IFNAR re not expressed (P.J.H. et l., dt not shown). Ifnr /, Ifnr / Socs1 / nd wild-type control mice were generted y intercrossing of Ifnr +/ Socs1 +/ mice housed in conventionl conditions nd genotyped y PCR. All mice were ge- nd sex-mtched for experiments. Animl experimenttions were in complince with guidelines set y the institutionl ethics committee (Monsh Medicl Centre AEC-A, Monsh University, Clyton, Austrli). Virl infection of neontl mice. Pups etween 5 nd 1 d of ge were injected intrperitonelly with 5 ml SFV t 1, 3 nd 1 TCID 5, determined y CPE iossy of mouse L99 cell cultures 44. One of the experiments using Socs1 / Ifng / nd equivlent control mice involved preinjection of neutrlizing ntiody (1, IU) specific to type I interferons (sheep ntiody to mouse IFN- nd IFN-; Reserch Dignostics) 6 h efore virl infection. The neutrliztion ctivity of this ntiody ginst virus-induced type I interferons ws verified in the CPE-reduction iossy descried elow. Experiments with Socs1 / nd Ifnr1 / Socs1 / used littermte controls nd were conducted y reserchers linded to experimentl conditions, with genotypes eing determined fter completion of the experiments. Experiments with Socs1 / Ifng / mice nd their equivlent controls used offspring from seprte colonies s descried ove. Mice were monitored t 3- to 6-hour intervls nd resistnce ws recorded. In seprte series of experiments, mice were killed 48 h fter infection nd lood, spleens, kidneys, livers, thymuses, lungs nd rins were collected. The lood ws left to cogulte, then ws centrifuged, nd serum ws collected nd ws stored t 4 1C efore ssy. The orgns were snp-frozen in liquid nitrogen nd were stored t 8 1C. Virus titers. Ech frozen orgn ws weighed nd then ws trnsferred to 1 ml of cold PBS on ice. Smples were sonicted for three 5-second pulses on ice with n Ultrsonics Homogenizer set t n mplitude of 4, ensuring the smple remined cold. Orgn homogentes were centrifuged t 9,g for 3 min t 4 1C. Superntnts were serilly titrted in semi-log steps in duplicte cross monolyers of L99 cells plted in 96-well microtiter pltes t density of cells/ml in RPMI medium supplemented with 3% (vol/vol) FBS nd.5% (vol/vol) penicillin nd streptomycin. Virus ws titrted onto pltes s positive control nd, s negtive control, cells were left in culture for the durtion of the ssy without dditions. Pltes were incuted for 3 d t 37 1C in 5% CO. After tht time, pltes were viewed under microscope nd ech well ws ssigned score for CPE ccording to the following scle: 4, pproximtely 1% deth; 3, 75% deth;, 5% deth; 1, 5% deth;, % deth. Virl titers were then determined s the dilution t which 5% deth occurred nd re expressed s log 1 titers or s fold dilution. Comprisons of virl titers for ech smple group were then ssessed y sttisticl nlysis y the Mnn-Whitney rnk sum test. Serum interferon ssys. Serum smples from neontl mice were diluted in semi-log 1 steps in duplicte into 96-well flt-ottom tissue culture pltes contining L99 cell monolyers, s descried ove. Medi ws removed 4 h lter nd SFV ws dded t concentrtion of 1 TCID 5 in fresh RPMI medium. Pltes were susequently incuted for 3 d t 37 1C in5%co, fter which pltes were ssigned scores for CPE. This ssy mesures the ctivity of ll type I nd type II interferons. The interferon ctivity in IU/ml ws clculted y comprison of the titer of the smple with lortory IFN- stndrd clirted to the Ntionl Institutes of Helth reference stndrd G STAT1 phosphoryltion. BMMs were isolted from mice nd cultured s descried 49. Cells were plted t density of cells/plte nd were cultured for further 4 h without colony-stimulting fctor 1. Cells were treted with 1, IU/ml of IFN-1 for -minute pulse, then were collected t, 4, 6 nd 8 min fter initil tretment. Cells were collected into oiling lysis uffer (66 mm Tris-HCl, ph 7.4, 1 mm phenylmethylsulfonyl fluoride (PMSF), 1 mm NF, 1 mm sodium vndte nd % SDS, plus one protese inhiitor tlet (Roche) per 1 ml uffer, with resulting lyste concentrtions determined y the Lowry ssy. Protein (5 mg) ws seprted y 8% SDS-PAGE, followed y immunolot for phosphorylted STAT1 (Tyr; Cell Signling), totl STAT1 (Cell Signling) nd -tuulin (Chemicon). -5 -OAS ssys. Wild-type, Socs1 +/ nd Socs1 / mouse primry emryo firolsts were generted s descried 5. Cells were treted with 1, IU/ml of mouse IFN-4 (otined from D. Gewert, BioLuncher, Cmridge) for 4-hour pulse nd were wshed, then were incuted for further 48, 7, 96, 1 or 144 h. Cell lystes were prepred nd -5 -OAS ctivity ws determined s descried 4. Protein concentrtion ws determined y the Brdford ssy. Enzyme ctivity ws clculted s millimoles phosphte incorported per microgrm protein t ech time point 4. Trnsient trnsfection. The 93T cells were mintined in DMEM contining 1% FBS. Cells expressing the PEF-BOS-Flg-Jk1+pEF-BOS-Flg-SOCS1, pef-bos-ifnar1+pef-bos-flg-socs1 nd pef-bos-flag-jk1+pef- BOS-IFNAR1+pEF-BOS-Flg-SOCS1 constructs were generted using the fugene6 trnsfection regent (Roche) ccording to the mnufcturer s instructions nd were collected 48 h lter. Immunoprecipittion nd immunolot nlysis. BMMs were isolted from mice nd cultured s descried 49. Cells were plted t density of cells/ plte nd were cultured for further 4 h with the ddition of colonystimulting fctor 1. Cells were pretreted for 4 min with 1 mm MG13 (Cliochem), then were treted for further 6 min with 1, IU/ml of IFN1. Cells were collected in ice-cold lysis uffer (.5% Nonidet-P4, 5 mm Tris- HCl, ph 7.4, 15 mm NCl, 1 mm sodium vndte, 1 mm PMSF nd 1 mm NF, plus one protese inhiitor tlet (Roche) per 5 ml uffer). The resulting lystes were preclered y the ddition of 5% protein ASephrose slurry, then were immunoprecipitted with monoclonl ntiody to mouse IFN-R1 (ref. 51). Protein A eds were incuted with the immunoprecipitting lystes for h, fter which they were wshed nd were oiled in reducing or nonreducing smple uffer. Proteins were seprted y 8% SDS-PAGE, followed y immunolot for IFNAR1, or were seprted y 15% SDS-PAGE, followed y immunolot with monoclonl ntiody to SOCS1 (otined from J. Zhng, Wlter nd Eliz Hll Institute of Medicl Reserch, Melourne, Austrli). Trnsfected cells were collected in lysis uffer (1% Triton X-1, 5 mm Tris-HCl, ph 7.4, 15 mm NCl, 1 mm EDTA, mm sodium vndte, 1 mm NF nd 1 mm PMSF plus one protese inhiitor tlet (Roche) per 5 ml uffer). The resulting lystes were immunoprecipitted with monoclonl ntiody to IFNAR1. Protein A eds were incuted with the immunoprecipitting lystes for h, fter which they were wshed nd oiled in reducing smple uffer. Proteins were seprted y 1% SDS-PAGE (precst gels; Novex), followed y immunolot for Flg (rt monoclonl ntiody to Flg (9H1; ref. 5) is n in-house ntiody of the Wlter nd Eliz Hll Institute of Medicl Reserch, Melourne, Austrli) or IFNAR1. ACKNOWLEDGMENTS Supported y the Ntionl Helth nd Medicl Reserch Council of Austrli. COMPETING INTERESTS STATEMENT The uthors declre tht they hve no competing finncil interests. Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions/ 1. Guttermn, J.U. Cytokine therpeutics: lessons from interferon-. Proc. Ntl. Acd. Sci. USA 91, (1994).. Issc, A. & Lindemnn, J. Virus interference I: the interferons. Proc. Royl Soc. Lond. B 147, 5867 (1957). 6 ADVANCE ONLINE PUBLICATION NATURE IMMUNOLOGY

7 5 Nture Pulishing Group 3. Pestk, S., Lnger, J.A., Zoon, K.C. & Smuel, C.E. Interferons nd their ctions. Annu. Rev. Biochem. 56, (1987). 4. Hertzog, P.J. et l. A gene on humn chromosome 1 locted in the region 1q. to q.3 encodes fctor necessry for signl trnsduction nd ntivirl response to type I interferons. J. Biol. Chem. 69, (1994). 5. Weissmnn, C. & Weer, H. The interferon genes. Prog. Nucleic Acid Res. Mol. Biol. 33, 513 (1986). 6. Oritni, K., Kincde, P.W., Zhng, C., Tomiym, Y. & Mtsuzum, Y. Type I interferons nd limitin: comprison of structures, receptors nd functions. Cytokine Growth Fctor Rev. 1, (1). 7. Le Bon, A. et l. Type I interferons potently enhnce humorl immunity nd cn promote isotype switching y stimulting dendritic cells in vivo. Immunity 14, (1). 8. Bch, E.A., Aguet, M. & Schreier, R.D. The IFN-g receptor: prdigm for cytokine receptor signling. Annu. Rev. Immunol. 15, (1997). 9. Godfrey, D.I., Hmmond, K.J., Poulton, L.D., Smythe, M.J. & Bxter, A.G. NKT cells: fcts, functions nd fllcies. Immunol. Tody 1, (). 1. Drnell, J.E., Kerr, I.M. & Strk, G.R. Jk-STAT pthwys nd trnscriptionl ctivtion in response to IFNs nd other extrcellulr signling proteins. Science 64, (1994). 11. Strk, G.R., Kerr, I.M., Willims, B.R., Silvermn, R.H. & Schreier, R.D. How cells respond to interferons. Annu. Rev. Biochem. 67, 764 (1998). 1. Gresser, I. How does interferon inhiit tumour growth? Phil. Trns. R. Soc. Lond. B 99, 6976 (198). 13. Neel, B.G. Role of phosphtses in lymphocyte ctivtion. Curr. Opin. Immunol. 9, 454 (1997). 14. Irie-Sski, J. et l. CD45 is JAK phosphtse nd negtively regultes cytokine receptor signling. Nture 49, (1). 15.Strr, R. et l. A fmily of cytokine-inducile inhiitors of signling. Nture 387, (1997). 16. Endo, T.A. et l. A new protein contining n SH domin tht inhiits JAK kinses. Nture 387, 9194 (1997). 17. Nk, T. et l. Structure nd function of new STAT-induced STAT inhiitor. Nture 387, 9499 (1997). 18. Yoshimur, A. et l. A novel cytokine-inducile gene CIS encodes n SH-contining protein tht inds to tyrosine-phosphorylted interleukin-3 nd erythropoietin receptors. EMBO J. 14, (1995). 19. Hilton, D.J. et l. Twenty proteins contining C-terminl SOCS ox form five structurl clsses. Proc. Ntl. Acd. Sci. USA 95, (1998).. Nicholson, S.E. et l. Muttionl nlyses of the SOCS proteins suggest dul domin requirement ut distinct mechnisms for inhiition of LIF nd IL-6 signl trnsduction. EMBO J. 18, (1999). 1. Sski, A. et l. Cytokine-inducile SH protein-3 (CIS3/SOCS3) inhiits Jnus tyrosine kinse y inding through the N-terminl kinse inhiitory region s well s SH domin. Genes Cells 4, (1999).. Zhng, J.-G. et l. The conserved SOCS ox motif in suppressors of cytokine signling inds to elongins B nd C nd my couple ound proteins to protesoml degrdtion. Proc. Ntl. Acd. Sci. USA 96, 7176 (1999). 3. Nicholson, S.E. et l. Suppressor of cytokine signling-3 preferentilly inds to the SHP--inding site on the shred cytokine receptor suunit gp13. Proc. Ntl. Acd. Sci. USA 97, (). 4. Ysukw, H. et l. The JAK inding protein JAB inhiits Jnus tyrosine kinse ctivity through inding in the ctivtion loop. EMBO J. 18, (1999). 5. Kile, B.T. et l. The SOCS ox: tle of destruction nd degrdtion. Trends Biochem. Sci. 7, 3541 (). 6.Adms, T.E. et l. Growth hormone preferentilly induces the rpid, trnsient expression of SOCS3, novel inhiitor of cytokine signling. J. Biol. Chem. 73, (1998). 7. Skmoto, H. et l. A Jnus kinse inhiitor, JAB, is n interferon-g inducile gene nd confers resistnce to interferons. Blood 9, (1998). 8. Bjorek, C., Elmquist, J.K., Frntz, J.D., Shoelson, S.E. & Flier, J.S. Identifiction of SOCS3 s potentil meditor of centrl leptin resistnce. Mol. Cell 1, (1998). 9. Strr, R. et l. Liver degenertion nd lymphoid deficiencies in mice lcking suppressor of cytokine signling-1. Proc. Ntl. Acd. Sci. USA 95, (1998). 3. Nk, T. et l. Accelerted poptosis of lymphocytes y ugmented induction of Bx in SSI-1 (STAT-induced STAT inhiitor-1) deficient mice. Proc. Ntl. Acd. Sci. USA 95, (1998). 31. Alexnder, W.S. et l. SOCS 1 is criticl inhiitor of interferon g signling nd prevents the potentilly ftl neontl ctions of this cytokine. Cell 98, 1 (1999). 3. Mrine, J.C. et l. SOCS1 deficiency cuses lymphocyte-dependent perintl lethlity. Cell 98, (1999). 33. Brysh, M. et l. Suppressor of cytokine signling 1 ttenutes the durtion of interferon gmm signl trnsduction in vitro nd in vivo. J. Biol. Chem. 76, 8689 (1). 34. Hertzog, P.J., Emery, P., Cheethm, B.F., Mcky, I.R. & Linnne, A.W. Interferons in rheumtoid rthritis: Altertions in production nd response relted to disese ctivity. Clin. Immunol. Immunopthol. 48, 191 (1988). 35. Song, M.M. & Shui, K. The suppressor of cytokine signling (SOCS) 1 nd SOCS3 ut not SOCS proteins inhiit interferon-medited ntivirl nd ntiprolifertive ctivities. J. Biol. Chem. 73, (1998). 36. Kuo, M., Hnd, T. & Yoshimur, A. Suppressors of cytokine signling nd immunity. Nt. Immunol. 4, (3). 37. Bullen, D.V., Drwiche, R., Metclf, D., Hndmn, E. & Alexnder, W.S. Neutrlistion of interferon-g in neontl Socs1 / mice prevents ftty degenertion of the liver ut not susequent ftl inflmmtory disese. Immunology 14, 998 (1). 38. Metclf, D., Mifsud, S., Di Rgo, L. & Alexnder, W.S. The lethl effects of trnsplnttion of Socs1 / one mrrow cells into irrdited dult syngeneic recipients. Proc. Ntl. Acd. Sci. USA 1, (3). 39. Dlton, D.K. et l. Multiple defects of immune cell function in mice with disrupted interferon-g genes. Science 59, (1993). 4. Biron, C.A. Cytokines in the genertion of immune responses to, nd resolution of, virus infection. Curr. Opin. Immunol. 6, (1994). 41. Müller, U. et l. Functionl role of type I nd type II interferons in ntivirl defense. Science 64, (1994). 4. Hwng, S.Y. et l. A null muttion in the gene encoding type I interferon receptor component elimintes ntiprolifertive nd ntivirl responses to interferons nd nd lters mcrophge responses. Proc. Ntl. Acd. Sci. USA 9, (1995). 43. Eyles, J.L., Metclf, D., Grusy, M.J., Hilton, D.J. & Strr, R. Negtive regultion of interleukin-1 signling y suppressor of cytokine signling 1. J. Biol. Chem. 77, (). 44. Hmilton, J.A., Whitty, G.A., Kol, I. & Hertzog, P.J. Endogenous IFN/ suppresses colony-stimulting fctor (CSF)-1-stimulted mcrophge DNA synthesis nd medites inhiitory effects of lipopolyscchride nd TNF. J. Immunol. 156, (1996). 45. Hertzog, P.J., O Neill, L.A. & Hmilton, J.A. The interferon in TLR signling: more thn just ntivirl. Trends Immunol. 4, (3). 46. Tkok, A. et l. Cross tlk etween interferon-g nd / signling components in cveolr memrne domins. Science 88, (). 47. Cornish, A.L. et l. Suppressor of cytokine signling-1 regultes signling in response to interleukin- nd other gc-dependent cytokines in peripherl T cells. J. Biol. Chem. 78, (3). 48. Chong, M.M. et l. Suppressor of cytokine signling-1 is criticl regultor of interleukin-7-dependnt CD8 + T cell differentition. Immunity 18, (3). 49. Sweet, M.J. et l. A novel pthwy regulting lipopolyscchride-induced shock y ST/T1 vi inhiition of Toll-like receptor 4 expression. J. Immunol. 166, (1). 5. Hertzog, P.J. Isoltion of emryonic firolsts nd their use in the in vitro chrcteriztion of gene function. Methods Mol. Biol. 158, 515 (1). 51. Dunn, G.P. et l. A criticl function for type I interferons in cncer immunoediting. Nt. Immunol. 6, 779 (5). 5. Wilson-Annon, J. et l. Propoptotic BH3-only proteins trigger memrne integrtion of prosurvivl Bcl-w nd neutrlize its ctivity. J. Cell. Biol. 16, (3). NATURE IMMUNOLOGY ADVANCE ONLINE PUBLICATION 7