R-Ras deficiency does not affect papain-induced IgE production in mice

ORIGINAL RESEARCH R-Ras de iciency does no a ec papain-induced IgE p oduc ion in mice Lau a Kummola 1 , Zsuzsanna O u ay 1 , Ma ia V€ ah€ a upa 1 , S ua P ince 1 , Hannele Uusi alo-J€ a inen 1,2 , Te o A.H. J€ a inen 1,2 , & Ilkka S. Jun ila 1,3 1 Facul y o Medicine and Li e Sciences, Uni e si y o Tampe e, Tampe e, Finland 2 Depa men s o Oph halmology and O hopaedics & T auma ology, Tampe e Uni e si y Hospi al, Tampe e, Finland 3 Fimlab Labo a o ies, Tampe e, Finland Keywo ds Alle gology, dend i ic cells, IgE, knock-ou mice, R-Ras Co espondence Ilkka S. Jun ila, School o Medicine, Uni e si y o Tampe e, Room F-354, A o 2, L€ a€ ak€ a inka u 1, 33520 Tampe e, Finland. Tel: þ358-50-327 0543; ax: þ358-3-364 0557; E-mail: [email p o ec ed] Funding in o ma ion The wo k was unded by he Sig id Juselius Founda ion, Tampe e Tube culosis Founda ion, he Academy o Finland, P€ ai ikki and Saka i Sohlbe g Founda ion, Ins umen a ium Resea ch Founda ion, Finnish Medical Founda ion, Pi kanmaa Hospi al Dis ic Resea ch Founda ion, he Finnish Cul u al Founda ion, Finnish Diabe ic Resea ch Founda ion, Finnish Eye Founda ion, Emil Aal onen Founda ion, Na ional Ins i u e o Heal h (CA125255), Na ional Science Founda ion (CBET-1403535), and Flo ida Depa men o Heal h, Bankhead Coley Cance P og am (4BB17). Recei ed: 25 Janua y 2017; Re ised: 29 Ma ch 2017; Accep ed: 13 Ap il 2017 Immuni y, In lamma ion and Disease (2017) doi: 10.1002/iid3.168 Abs ac In oduc ion: R-Ras GTPase has ecen ly been implica ed in he egula ion o immune unc ions, pa icula ly in dend i ic cell (DC) ma u a ion, immune synapse o ma ion, and subsequen T cell esponses. Me hods: He e, we in es iga ed he ole o R-Ras in alle gen-induced immune esponse ( ype 2 immune esponse) in R as de icien (R-Ras KO) and wild ype (WT) mice. Resul s: Ini ially, we ound ha he numbe o con en ional DC’s in he lymph nodes (LNs) was educed in R-Ras KO mice. The exp ession o co-s imula o y CD80 and CD86 molecules on hese cells was also educed on DC’s om he R-Ras KO mice. Howe e , he e was no di e ence in papain-induced immune esponse be ween he R-Ras WT and KO as measu ed by se um IgE le els a e he immuniza ion. In e es ingly, nei he he DC numbe no co-s imula o y molecule exp ession was di e en be ween WT and R-Ras KO animals a e he immuniza ion. Conclusions: Taken oge he , despi e ha ing educed numbe o con en ional DC’s in he R-Ras KO mice and low exp ession o CD80 on DC’s, he R-Ras KO mice a e capable o moun ing papain-induced IgE esponses compa able o ha o he WT mice. To ou knowledge, his is he i s epo add essing po en ial di e ences in in i o alle gen esponses egula ed by he R-Ras GTPase. In oduc ion R-RAS is a small GTPase, a membe o he ex ensi e Ras supe amily, and shows abou 55–60% amino acid iden i y wi h he classical RAS p o eins (H-RAS, K-RAS, N-RAS) [1, 2]. Despi e he close s uc u alsimila i y o o he membe s o he Ras amily, he unc ion o R-Ras is dis inc om o he Ras p o eins. While all o he membe s o he Ras amily may cause malignan ans o ma ion, R-Ras has e y li le o no ans o ming ac i i y. R-Ras plays a ole in se e al cellula p ocesses such as in eg in signaling [3, 4], ac in cy oskele on o ganiza ion [5], memb ane u les, cell sp eading [6, 7], cell adhesion [4, 8], and mig a ion [9]. Conce ning leukocy es, R as / mice show impai ed T cell a icking [10] while 1© 2017 The Au ho s. Immuni y, In lamma ion and Disease Published by John Wiley & Sons L d. This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s use, dis ibu ion and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. mu ine mac ophages exp essing cons i u i ely ac i a ed R-Ras show ele a ed phagocy o ic ac i i y [11]. R-RAS can egula e in lamma ion [12–15]. Dend i ic cells (DC’s) om R-Ras de icien animals ha e educed abili y o p ime T cell esponses due o uns able immune synapse o ma ion [14]. In expe imen al au oimmune encephalo- myeli is model (EAE), R as / mice showed a enua ed disease cou se likely due o ele a ed numbe s o pe iphe al ole ogenic and egula o y T cells (T egs) [15]. Recen ly we showed ha R-Ras is equi ed o umo de elopmen and p og ession in in lamma ion-dependen skin cance model [13]. Al hough R-Ras was p esen only in he blood essels and in skin, i was equi ed bo h o he induc ion o p o-in lamma o y cy okine p oduc ion and o he ex a a- sa ion o in lamma o y cells o he skin [13]. Gi en he key ole o DC’s in adap i e immune esponses and he ole o R-Ras in egula ing DC unc ions [14, 15] we wan ed o know whe he ype2 immune esponse is egula ed by R-Ras in i o. We ound ha despi e he ac ha un ea ed R-Ras KO had dec eased numbe o con en ional DC’s and he DC’s show educed exp ession o CD80/CD86 cos imula o y molecules, hese mice we e capable o moun ing a no mal alle gen-induced esponse a e papain immuniza ion. Me hods Mice, immuniza ions, ELISA, cell cul u e, low cy ome y R-Ras KO (¼R as G (OST24882)Lex ) mice (C57BL/6) ha e been desc ibed [16]. Animal expe imen p o ocol was app o ed by Finnish Na ional Animal Expe imen Boa d (Pe mi : ESAVI/4738/04.10.07/2014). Adminis a ion o papain was pe o med as a modi ied e sion o he p o ocol desc ibed ea lie by Sokol e al. [17]. Mice we e injec ed subcu ane- ously ei he wi h PBS alone o PBS con aining 0.5 mg papain (Me ck Millipo e, Da ms ad , Ge many) on days 0 and 14. Blood samples we e collec ed om he ail ein on day 0. Mice we e eu hanized on day 16 o day 21, and spleens and LNs we e ha es ed. Blood was collec ed by ca diac punc u e in o se um blood ubes (BD, F anklin Lakes, NJ). IgE ELISA was om eBioscience (San a Cla a, CA). Spleens and pooled LNs we e incuba ed in 75 mg/ml Libe ase DL and 10 U/ml DNase (Roche, Basel, Swi ze land) o 30 min a 378C. DC’s we e en iched wi h MACS Pan Dend i ic Cell Isola ion Ki (Mil enyi Bio ec, Be gisch Gladbach, Ge many). Al e na i ely, spleen cells we e g adi- en cen i uged wi h His opaque 1 -1077 (Sigma–Ald ich, S . Louis, MO). An ibodies we e (clones in pa en hesis): CD3-APC (145- 2C11) o -APC-eFluo 780 (17A2), CD4-FITC o -APC- eFluo 780 (bo h GK1.5), CD8-Pe CP-Cy5.5 o -APC-H7 (bo h 53–6.7), B220-Pe-Cy7 o -APC-eFluo 780 (bo h RA3- 6B2), CD11c-PE-Cy7 (N419), F4/80-APC-eFluo 780 (BM8), MHCII-FITC (M5/114.15.2), CD80-Pe CP-eFluo - (16-10A1), CD86-APC (GL1), and OX40L-PE (RM134L) (all om eBioscience). Analysis was done by FACS Can o II (BD, F anklin Lakes, NJ) and FlowJo (T ee S a , Ashland, OR). Geome ical means and s anda d e o o means a e indica ed. Real- ime PCR, s a is ical analysis Spleens we e ha es ed in o RNAla e (Qiagen, Hilden, Ge many). mRNA was ex ac ed by T izol Reagen (Li e Technologies, Ca lsbad, CA) and RNA was con e ed o cDNA by The mo Maxima Fi s S and cDNA Syn hesis Ki (Li e Technologies). FAM-labelled Taqman p obes (Applied Biosys ems, Fos e Ci y, CA) o IL-10 (Mm01288386_m1), IL-13 (Mm00434204_m1), IL-17a (Mm00439618_m1), IFN-g(Mm01168134_m1), FOXP3 (Mm00475162_m1), GATA3 (Mm00484683_m1), T-BET (Mm00450960_m1), and RORg(Mm01261022_m1) we e used wi h iQ TM Supe mix (Bio-Rad Labo a o ies Inc., He cules, CA) o qPCR. VIC-labelled Euca yo ic 18S RNA Endogenous Con ol (Applied Biosys ems) was e e ence. Resul s we e calcula ed by double del a me hod. S a is ical analysis we e done wi h P ism (G aphPad So wa e, Inc., La Jolla, CA) o R [18, 19] ( o ELISA). p- alues we e calcula ed using wo- ailed, unpai ed s uden ’s - es s. Resul s and Discussion R-Ras KO mice lacking unc ional R-Ras p o ein a e pheno ypically simila o WT coun e pa s [13, 16, 20]. Howe e , DC ma u a ion and immunological synapse o ma ion wi h T cells is impai ed in R-Ras KO mice and he capabili y o R as / DC’s o induce allogeneic o an igen-speci ic au ologous T cell p oli e a ion is e- duced [14]. As desc ibed [14], we ound ha B- and T cell numbe s in spleen o un ea ed R-Ras KO mice (n¼3) we e no mal (Fig. 1A, ga ing s a egy in Supplemen a y Fig. S1A). The less se e e EAE in R-Ras KO mice is due o a local inc ease in he numbe o T egs and ole ogenic DC’s [15]. This led us o s udy he numbe and pheno ype o DC’s in WT and R-Ras KO mice. Analysis o magne ically en iched con en ional and mig a o y DC’s om he LN (ga ing s a egy in Supplemen a y Fig. S1C) indica ed ha R-Ras KO mice had less con en ional DC’s han WT mice (Fig. 1B), (p¼0.0126), while he numbe o mig a o y DC’s was simila be ween he wo geno ypes (Fig. 1B). As p e iously shown [14], he analysis o he en iched splenic DC’s om spleen showed no di e ence be ween WT o R-Ras KO mice (Fig. 1C, ga ing s a egy in Supplemen a y Fig. S1D). We chose o exclude plasmacy oid DC’s om ou analysis, as R-Ras in alle gic- ype in lamma ion L. Kummola e al. 2© 2017 The Au ho s. Immuni y, In lamma ion and Disease Published by John Wiley & Sons L d. hey a e cu en ly conside ed less ele an o alle gic esponses: hey a e known p oduce s o IFNgupon TLR7/ TLR9 ac i a ion du ing i al in ec ions and au oimmune diso de s [21]. The s eng h o T cell ecep o (TCR)-engagemen in cy okine- ee en i onmen egula es T cell di e en ia ion. Weak TCR-signal leads o spon aneous, GATA3- and IL-2- dependen , IL-4 p oduc ion while s ong TCR signal causes, ERK-dependen , IFN-gp oduc ion [22]. The DC su ace ma ke s ha ake pa in he DC/T-cell in e ac ion include MHCII (an igen p esen a ion o TCR), CD80/CD86 (co- s imula o y molecules associa ed wi h TCR) and OX40L (enhancemen o Th2 di e en ia ion a leas in hu- mans) [23]. MHCII exp ession was iden ical in bo h con en ional and mig a o y LN DC’s be ween WT and R-Ras KO mice (Fig. 2A). CD80 exp ession, in u n, was educed in bo h con en ional (p¼0.0078) and mig a o y (p¼0.0176) subse s om R-Ras KO mice (Fig. 2A), while CD86 exp ession was dec eased in con en ional DC subse o R-Ras KO mice (p¼0.0241), bu no in mig a o y DC subse (Fig. 2A). OX40L exp ession was iden ical in con en ional o mig a o y DC’s be ween KO and WT mice (Fig. 2A). In spleen, MHCII exp ession was highe in he KO DC’s (p¼0.0129) while CD80 exp ession was lowe in he KO DC’s (p¼0.0207) and, CD86 o OX40L exp ession we e iden ical (Fig. 2B). To be e unde s and he ole o R-Ras in uning DC ac i a ion, we analyzed he esponse o splenic DC’s om R-Ras de icien and WT mice o IL-4, LPS, and poly(I:C) du ing o e nigh pla e cul u e, which ‘‘spon aneously’’ ac i a es DC’s [24]. Al hough LPS and poly(I:C) a e no conside ed d i ing agen s o Th2 pola iza ion, we we e in e es ed o examine whe he he e was some gene al impai men o DC esponses in R as / mice and hus included hese s imulan s in o he compa ison. Collec i ely, in splenic DC’s MHCII exp ession is highe on DC’s om R-Ras KO mice unde all es ed cul u e condi ions. The di e ences obse ed in CD80 and CD86 exp ession be ween R-Ras KO and WT cells a e o e nigh cul u e we e abolished when hecells we e u he ac i a ed wi h poly(I:C), LPS o IL-4, while OX40L was induced by poly(I:C) and LPS, bu R-Ras exp ession plays no ole in he egula ion o OX40L exp ession as no di e ence was iden i ied be ween KO and WT cells (Supplemen a y Fig. S2A–D). Taken oge he , he main di e ence in DC’s be ween WT and R-Ras KO mice is ha he e is signi ican ly less con en ional DC’s in R-Ras KO mice and hese cells exp ess less co-s imula o y molecules, pa icula ly CD80. These esul s sugges , ha an igen p esen a ion ia MHCII is no impai ed in R-Ras DC’s bu he o ma ion o immunological synapse due o dec eased CD80/CD86 exp ession is impai ed, which in u n migh esul in impai ed CD28 signalling in T-cells. Since EAE in R-Ras KO mice is less se e e han in WT mice, we wan ed o s udy i ype 2 esponses a e a ec ed in hese mice. To analyze a possible ‘‘skew’’ in he s eady-s a e Figu e 1. Immunopheno yping o un-manipula ed R-Ras KO mice. (A) Lymphocy e popula ions o “o - he-shel ”wild ype (WT, n¼3) o R-Ras deficien (R-Ras KO, n¼3) mice we e compa ed by flow cy ome y. (B and C) LNs (B) and spleens (C) we e collec ed om un ea ed wild ype (WT, n¼3) o R-Ras deficien (R-Ras KO, n¼3) mice. LNs om each mouse we e pooled sepa a ely. DC's we e en iched by magne ic sepa a ion (LN) o g adien cen i uga ion (spleens) and analyzed by flow cy ome e . LN MHCII þCD11cþ(DC all) cell g oup includes wo popula ions: con en ional (con DC) and mig a o y dend i ic cells (mig DC). (B) The amoun o all MHCII þCD11c þdend i ic cells (DC all) and hei dis ibu ion be ween con en ional (con DC) and mig a o y (mig DC) dend i ic cells. (C) The amoun o MHCII þCD11c þdend i ic cells in he spleen. Fo s a is ical analysis unpai ed wo- ailed - es s we e used. p<0.05. L. Kummola e al. R-Ras in alle gic- ype in lamma ion © 2017 The Au ho s. Immuni y, In lamma ion and Disease Published by John Wiley & Sons L d. 3 CD4 T cells in R-Ras KO mice, we s udied he mRNA exp ession o c i ical ansc ip ion ac o s ela ed o helpe T cell di e en ia ion om he o al splenocy es o WT and R-Ras KO mice, bu ound no di e ence in he mRNA exp ession o Tbe , GATA3, Foxp3, o Ro g(Fig. 2C, uppe panel). Also, he mRNA exp ession o IFNg, IL-13, and IL-10 was iden ical be ween WT and R-Ras KO mice (Fig. 2C lowe panel). To s udy he alle gen-induced esponse in R-Ras KO mice, we u ilized papain-immuniza ion as an in i o model. Papain-induced esponses a e media ed ia mig a o y DC’s in skin [25] and we had al eady obse ed a dec ease in CD80 exp ession in mig a o y DC’s (Fig. 2A) in R-Ras KO mice. We subjec ed R-Ras KO mice and WT animals (n¼8 o each geno ype) o papain injec ions wice: i s immuniza- ion was pe o med on day 0 and e-immuniza ion on day 14. We measu ed he se um le el o IgE on day 0 and again when he mice we e eu hanized on day 21. We ound no signi ican di e ence in he se um IgE le els be ween he wo geno ypes (Fig. 3A) 21 days a e he i s immuniza ion. Nex we wan ed o ule ou he possibili y ha he ini ia ion phase o he ype 2 esponse o papain migh be delayed due o he impai ed immune synapse o ma ion [14] in R-Ras KO mice. Fo his, we measu ed he IgE le els o papain- immunized WT and KO (n¼17 and 18, espec i ely) mice only wo days a e he second immuniza ion (i.e., 16 days a e he i s immuniza ion). Howe e , no di e ence in he se um le el o IgE was ound be ween R-Ras KO mice and WT (Fig. 3B). The concen a ion o IgE was app oxima ely 10 mg/ml on day 16, while i was 50 mg/ml on day 21, indica ing ha he alle gen esponse was s ill being induced a e day 16 (Fig. 3A and B). Nex , we de e mined he numbe o DC’s on day 16 a e he papain immuniza ion. Unlike in unmanipula ed mice, Figu e 2. Compa ison o he exp ession o dend i ic cell su ace p o eins and exp ession o T-helpe cell ansc ip ion ac o s and cy okines be ween WT and R-Ras KO mice. LNs (A) and spleens (B) we e collec ed om un ea ed wild ype (WT, n¼3) o R-Ras deficien (R-Ras KO, n¼3) mice and DC's we e en iched as in Figu e 1. (A) The exp ession o MHCII, CD80, OX40L, and CD86 su ace p o eins in con en ional (con DC) and mig a o y dend i ic cells (mig DC) om he LNs and (B) om he spleen. (C) Spleens o un-manipula ed WT and R-Ras KO mice (n¼4 o each geno ype) we e ha es ed. mRNA was isola ed om o al splenocy es and ansc ibed in o cDNA. The exp ession o Th-specific ansc ip ion ac o s and cy okines we e in es iga ed wi h qPCR. The di e ences wi hin su ace p o ein exp ession (A and B) o gene exp ession (C) alues we e s a is ically analyzed by wo- ailed unpai ed - es s. p<0.05, p<0.01. Each do ep esen s an indi idual animal. Mean alues wi h SEM a e ma ked in he sca e do plo s. R-Ras in alle gic- ype in lamma ion L. Kummola e al. 4© 2017 The Au ho s. Immuni y, In lamma ion and Disease Published by John Wiley & Sons L d. which showed a di e ence in he numbe o con en ional DC’s be ween WT and R-Ras KO animals (Fig. 1B), we ound no di e ence in hei numbe (Fig. 3C) in papain- ea ed animals. Simila ly, he numbe o splenic DC’s was alike in WT and R-Ras KO mice (Fig. 3D). We also de e mined Th2-associa ed su ace ma ke s on CD4 T cells a e papain immuniza ion. The exp ession o he cy o- kine-binding ecep o chain o IL-4 (IL-4Ra)isc i ical o CD4 T cell esponse o IL-4 and is up- egula ed on CD4 cells du ing Th2 esponse. We ound no di e ence in IL-4Raexp ession be ween WT o R-Ras KO mice (n¼5) ei he in CD4 o CD8 cells (Fig. 3E). IL-33R is ano he cy okine ecep o chain up- egula ed in CD4 T-cells in Th2 esponses. IL-33 can egula e ‘‘e ec o cy okine,’’ such as IL-5 and IL-13, exp ession in Th2 cells in conce wi h S a 5 ac i a ing cy okines, independen ly o an igens [26]. IL-33 has been shown o be indispensable o papain- induced IgE p oduc ion in mu ine lung and geni al ac [27, 28]. We ound ha he pe cen o IL-33R posi i e cells was educed in R-Ras KO CD4 T cells (Fig. 3F, he ga ing s a egy o IL-33R is shown is supplemen a y Fig. S1B). This migh sugges ha while IL-4 media ed e en s (IgE p oduc ion, IL-4Raexp ession) a e in ac in R-Ras KO mice, IL-4 independen (bu GATA3-dependen ) e en s, such as IL-33R exp ession can be a ec ed by he dele ion o R-Ras. The equi emen o IL-4 o Th2 di e en ia ion in i o is discussed in de ail in e e ence [29]. Figu e 3. The e ec o papain-immuniza ion on WT and R-Ras-deficien mice. Wild ype (WT) o R-Ras deficien (R-Ras KO) mice we e ea ed wi h papain (500 mg/animal, subcu aneous injec ion on day 0 and day 14). Animals we e eu hanized ei he on day 21 o day 16. IgE concen a ion o blood se um was measu ed using ELISA on (A) day 21 o (B) day 16. Expe imen in A was done wice wi h ou mice o each geno ype and in B he expe imen was done ou imes wi h 4–5 mice o each geno ype. (C and D) LNs (n¼4) and spleens (n¼5) we e collec ed om papain-immunized (day 16) wild ype (WT) o R-Ras deficien (R-Ras KO) mice and dend i ic cells we e en iched as in Figu e 1. (C) The amoun o all LN MHCII þCD11c þdend i ic cells (DC all) and hei dis ibu ion be ween con en ional (con DC) and mig a o y (mig Dc). (D) The amoun o MHCII þCD11c þdend i ic cells in he spleen. (E) Exp ession o IL-4Rain CD4 and CD8 T cells o WT and R-Ras KO mice (n¼5 each geno ype) on day 16 a e papain immuniza ion. (F) Pe cen o IL-33R posi i e cells o CD4 T cells om WT and R-Ras KO mice on day 16 a e papain immuniza ion. Mean alues wi h SEM a e ma ked in he sca e do plo s. Each do ep esen s an indi idual animal. p<0.05 (unpai ed - es s). L. Kummola e al. R-Ras in alle gic- ype in lamma ion © 2017 The Au ho s. Immuni y, In lamma ion and Disease Published by John Wiley & Sons L d. 5 Then we analyzed he exp ession o DC ac i a ion ma ke s om papain-immunized mice. Unlike in he heal hy mice, he con en ional and mig a o y DC’s om he LN showed no di e ence be ween he wo geno ypes in he exp ession o MHCII, CD80, CD86, o OX40L (Fig. 4A). In splenic DC’s he exp ession o MHCII and CD80 we e simila be ween WT and R-Ras KO animals, bu CD86 exp ession was educed a e papain-immuniza ion in R-Ras KO mice (Fig. 4B). Papain has been shown o induce Th2 pola iza ion in mul iple s udies, de e mined by he IL-4 p oduc ion o T cells [17, 28, 30] and T cell ecall esponses upon in i o es imula ion wi h papain [17, 28, 31, 32]. We measu ed he mRNA exp ession o ansc ip ion ac o s associa ed wi h T cell di e en ia ion om splenocy es by qPCR a e papain-immuniza ion. The mRNA exp ession le els o Tbe , GATA3, and Foxp3 we e iden ical be ween WT and R-Ras KO mice bu o Ro g, he mas e egula o o Th17- di e en ia ion, he mRNA exp ession was signi ican ly lowe (p¼0.0492) in he R-Ras KO mice (Fig. 4C). Acco dingly, hallma k cy okine mRNA analysis om hese samples indica ed no di e ence be ween IFN-g, IL-13, o IL-10 exp ession (Fig. 4C). The no ion ha RORg- ansc ip ion ac o was exp essed a educed le els in R-Ras KO mice compa ed o WT is logical as IL-17 p oduc ion is dec eased in CNS o R as / mice [15] unde going EAE o ca cinogen induced umo de elopmen [13]. RORg, on he o he hand, plays a c i ical ole in he de elopmen o IL-17 p oducing CD4þT cells [33]. Also, a ecen s udy showed ha Figu e 4. The exp ession o dend i ic cell su ace ma ke s and mRNA exp ession o T-helpe cell ansc ip ion ac o s and cy okines a e papain- immuniza ion in WT and R-Ras KO mice. (A) LNs we e collec ed om papain-immunized (day 16) wild ype (WT, n¼4) o R-Ras deficien (R-Ras KO, n¼4) mice and dend i ic cells we e en iched by magne ic sepa a ion. The exp ession o di e en su ace p o eins (MHCII, CD80, OX40L, and CD86) was in es iga ed di ec ly a e eu hanizing he mice. (B) Spleens we e collec ed om papain-immunized (day 16) wild ype (WT, n¼5) o R-Ras deficien (R-Ras KO, n¼5) mice and dend i ic cells we e en iched by g adien cen i uga ion. The exp ession o di e en su ace p o eins (MHCII, CD80, and CD86) was analyzed wi h flow cy ome e . The di e ences wi hin su ace p o ein exp ession we e s a is ically analyzed (unpai ed wo- ailed - es s. p<0.05). (C) Spleens we e ha es ed om day 16 papain-immunized wild ype (WT, n¼5) and RAS-deficien (R-Ras KO, n¼5) mice and p epa ed as in Figu e 2. qPCR analysis o T cell ansc ip ion ac o s and cy okines was pe o med. Each do ep esen s an indi idual animal. Mean alues wi h SEM a e ma ked in he sca e do plo s. The di e ences wi hin gene exp ession alues we e s a is ically analyzed by wo- ailed unpai ed - es s. p<0.05 R-Ras in alle gic- ype in lamma ion L. Kummola e al. 6© 2017 The Au ho s. Immuni y, In lamma ion and Disease Published by John Wiley & Sons L d. epicu aneous papain adminis a ion was able o induce he di e en ia ion o alle gen-speci ic Th17 cells [31], sugges - ing ha papain induces no only Th2 bu also Th17 esponses. To ou knowledge, his is he i s s udy ha add esses he e ec o R-Ras on alle gen-induced immune esponse. Al hough we could no demons a e any ole o R-Ras in egula ing he immune esponse owa d papain- immuniza ion, u u e wo k u ilizing IL-4-dependen , and IL-4-independen Th2 pola izing models (such as T ichu us mu is and Nippos ongylus b asiliensis in ec- ions) could speci y a mo e de ined ole o R-Ras in immune esponse. Au ho s’Con ibu ions T.J., I.J., L.K., Z.O., S.P., M.V., and H.U-J. designed he esea ch. L.K., Z.O., and M.V. pe o med he expe imen s. L.K., Z.O., M.V., S.P., H.U-J., T.J., and I.J analyzed he da a. H.U.J. and M.V. con ibu ed he geno yped mice li e ma es. L.K., Z.O., T.J., and I.J. w o e he manusc ip . Acknowledgmen s We hank Ma ianne Ka lsbe g o p ac ical suppo and P o esso E kki Ruoslah i (San o d-Bu nham-P ebys Medi- cal Disco e y Ins i u e, La Jolla, CA, USA) o p o iding he R-Ras knockou mice o he s udy and o his commen s on he manusc ip . We also hank D . Masanobu Koma su (San o d-Bu nham-P ebys Medical Disco e y Ins i u e a Lake Nona, O lando, FLA, USA) o cons uc i e commen s. The wo k was unded by he Sig id Juselius Founda ion (IJ, TJ), Tampe e Tube culosis Founda ion (IJ), he Academy o Finland, P€ ai ikki and Saka i Sohlbe g Founda ion (TJ), Ins umen a ium Resea ch Founda ion (TJ), Finnish Medi- cal Founda ion (TJ, IJ), Pi kanmaa Hospi al Dis ic Resea ch Founda ion (TJ), he Finnish Cul u al Founda ion (TJ), Finnish Diabe ic Resea ch Founda ion (TJ), Finnish Eye Founda ion (TJ), Emil Aal onen Founda ion (LK), Na ional Ins i u e o Heal h CA125255 (TJ), Na ional Science Founda ion CBET-1403535 (TJ), and Flo ida Depa men o Heal h, Bankhead Coley Cance P og am 4BB17 (TJ). Con lic o In e es The au ho s decla e no con lic o in e es . Re e ences 1. Lowe, D. G., D. J. Capon, E. Delwa , A. Y. Sakaguchi, S. L. Naylo , and D. V. Goeddel. 1987. S uc u e o he human and mu ine R- as genes, no el genes closely ela ed o as p o o- oncogenes. Cell 48(1):137–146. 2.Eh ha d ,A.,G.R.Eh ha d ,X.Guo,andJ.W. Sch ade . 2002. 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Cua, and D. R. Li man. 2006. The o phan nuclea ecep o RORgamma di ec s he di e en ia- ion p og am o p oin lamma o y IL-17þT helpe cells. Cell 126(6):1121–1133. SUPPORTING INFORMATION Addi ional suppo ing in o ma ion may be ound in he online e sion o his a icle a he publishe ’s web-si e. Figu e S1. Ga ing s a egies used in low cy ome y. (A) Spleens we e ha es ed om eu hanized mice. Single cell suspension was c ea ed as desc ibed in Ma e ials and Me hods and ed blood cells lysed be o e s aining he cells o su ace ma ke s and applying hem on o he low cy ome e . Cell deb is was excluded and emaining cells we e s udied o CD3 and B220 exp ession, and T cells we e ga ed as CD3 þ popula ion (T cell) and B cells as B220 þ g oup (B cell). T cells we e u he analyzed o CD4 and CD8 su ace ma ke exp ession, and CD4 þ CD8  and CD8 þ CD4  subpopula ion we e iden i ied as CD4þT cells (CD4þ)andCD8þT cells (CD8þ), espec i ely. (B) S a egy o IL-33R ga ing in CD4 cells. (C) Fo LN DC’s, single cell suspensions o LN’s we e p epa ed as in Figu e 1. CD11c þ MHCII þ dend i ic cells in ol e wo subpopula- ions: con en ional dend i ic cells (con DC) show highe CD11c and lowe MHCII exp ession while mig a o y dend i ic cells exp ess less CD11c bu mo e MHCII. (D) R-Ras in alle gic- ype in lamma ion L. Kummola e al. 8© 2017 The Au ho s. Immuni y, In lamma ion and Disease Published by John Wiley & Sons L d. Fo splenic DC’s, spleens we e p epa ed as desc ibed in Ma e ials and Me hods. Cell deb is was excluded and cells we e in es iga ed o he exp ession o CD3 (T cell ma ke ), B220 (B cell ma ke ) and F480 (mac ophage ma ke ). CD3  B220  F480  cells (lin.neg.) we e hen s udied u he o he exp ession o CD11c and MHCII. The igu e shows he ga ing o one ep esen a i e sample o each case. Figu e S2. Exp ession o DC su ace ma ke s in WT and R-Ras KO splenic DC’s in esponse o a ious s imulan s. Spleens we e p epa ed as desc ibed in ma e ials and me hods om WT (N¼3) an R-Ras KO (n¼3) mice. The cells we e ei he s ained di ec ly (ex i o) o a e o/n cul u e wi h medium alone o wi h poly(I:C), LPS, o IL-4 as indica ed. The exp ession o MHCII (A), CD80 (B), OX40L (C), and CD86 (D) was measu ed wi h low cy ome e . L. Kummola e al. R-Ras in alle gic- ype in lamma ion © 2017 The Au ho s. Immuni y, In lamma ion and Disease Published by John Wiley & Sons L d. 9