Toll-Like Receptor Activation during Cutaneous Allergen Sensitization Blocks Development of Asthma through IFN-Gamma-Dependent Mechanisms

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1 ORIGINAL ARTICLE See relted ommentry on pg 874 Toll-Like Reeptor Ativtion during Cutneous Allergen Sensitiztion Bloks Development of Asthm through IFN-Gmm-Dependent Mehnisms Rit Hpkoski 1, Pii Krisol 1, Nnn Fyhrquist 1, Terhi Svinko 1, Sri Lehtimäki 1, Henrik Wolff 2, Antti Luerm 3 nd Hrri Alenius 1 Toll-like reeptors (TLRs) re pttern-reognition reeptors tht hve pivotl role s primry sensors of miroil produts nd s inititors of innte nd dptive immune responses. We investigted the role of TLR2, TLR3, nd TLR4 tivtion during utneous llergen sensitiztion in the modultion of llergi sthm. The results show tht derml exposure to TLR4 lignd lipopolyshride () or TLR2 lignd suppresses sthmti responses y reduing irwy hyperretivity, muus prodution, Th2-type inflmmtion in the lungs, nd IgE ntiodies in serum in dose-dependent mnner. In ontrst, TLR3 lignd Poly(I:C) did not protet the mie from sthmti symptoms ut redued IgE nd indued IgG2 in serum. (espeilly) nd enhned the tivtion of derml dendriti ell (DCs) y inresing the expression of CD8 nd CD86 ut deresed DC numers in drining lymph nodes t erly time points. Lter, these hnges in DCs led to n inresed numer of CD8 þ T ells nd enhned the prodution of IFN-g in ronholveolr lvge fluid. In onlusion, derml exposure to during sensitiztion modultes the sthmti response y skewing the Th1/Th2 lne towrd Th1 y stimulting the prodution of IFN-g. These findings support the hygiene hypothesis nd pinpoint the importne of derml miroiome in the development of llergy nd sthm. Journl of Investigtive Dermtology (213) 133, ; doi:1.138/jid ; pulished online 15 Novemer 212 INTRODUCTION Asthm is heterogeneous inflmmtory disese of the lungs hrterized y muus hyperseretion, irwy inflmmtion, ronhil hyperresponsiveness, nd Th2 ell infiltrtion in the lungs (Cohn et l., 24). It is estimted tht s mny s 3 million people suffer from sthm glolly, nd the numer is inresing espeilly in the Western ountries (Msoli et l., 24; von Mutius nd Verelli, 21). In ddition to multiple geneti ftors known to e involved in the pthogenesis of sthm, environmentl ftors hve lso een hypothesized to ontriute to the outome of sthm. These inlude, for exmple, different llergens, ir pollutnts, respirtory viruses, nd endotoxins (Hollowy et l., 21). 1 Unit of Immunotoxiology, Finnish Institute of Ouptionl Helth, Helsinki, Finlnd; 2 Biologil Mehnisms nd Prevention of Work-Relted Diseses, Finnish Institute of Ouptionl Helth, Helsinki, Finlnd nd 3 Deprtment of Dermtology, Allergology nd Venereology, University of Helsinki, Helsinki, Finlnd Correspondene: Hrri Alenius, Unit of Immunotoxiology, Finnish Institute of Ouptionl Helth, Topeliuksenktu 41 A, FIN-25 Helsinki, Finlnd. E-mil: Hrri.Alenius@ttl.fi Arevitions: AD, topi dermtitis; AHR, irwy hyperretivity; DC, dendriti ell; LN, lymph node;, lipopolyshride; MCh, metholine;, ovlumin;, (S)-(2,3-is(plmitoyloxy)-(2RS)-propyl)-Nplmitoyl-(R)-Cys-(S)-Ser(S)-Lys4-OH, trihydrohloride; poly(i:c), polyinosipolyytidyli id; TLR, Toll-like reeptor Reeived 7 Otoer 211; revised 2 August 212; epted 6 August 212; pulished online 15 Novemer 212 The hygiene hypothesis suggests tht exposure to miroil infetions in erly hildhood fvors the development of Th1- type immunity nd protets from the lter development of the Th2-type phenotype (Strhn, 1989; Ernst nd Cormier, 2; Bh, 22; Brun-Fhrländer et l., 22; Yng nd Go, 211). Vrious Toll-like reeptors (TLRs) re importnt meditors of innte immunity tht reognize pthogen-ssoited miroil ptterns from miroil nd virl produts nd tivte distint signling pthwys. Ativtion of the innte immune system through TLRs is lso linked to the responses of dptive immunity nd thus my influene the development of llergi diseses suh s sthm (Reijmerink et l., 21; Tesse et l., 211). The skin rrier of topi individuls is often disrupted, nd severl miroil omponents nd llergens n penetrte esily through the dermis. In the present study, we investigted the role of TLR lignds in the development of sthm y mimiking the nturl route of sensitiztion from the skin to systemi nd irwy responses. The results show tht derml exposure to oth TLR4 lignd lipopolyshride () nd TLR2 lignd suppresses sthmti responses, when given t the time of sensitiztion, y reduing irwy hyperretivity (AHR), Th2-type inflmmtion, nd IgE ntiodies in dose-dependent mnner. On the other hnd, TLR3 lignd Poly(I:C) ws not le to protet the mie from sthmti prmeters in generl ut redued IgE nd indued IgG2 onentrtions in serum. The proteting effet of TLR4 lignd 964 Journl of Investigtive Dermtology (213), Volume 133 & 213 The Soiety for Investigtive Dermtology

2 R Hpkoski et l. ws found to e due to n enhned prodution of IFN-g in the irwys. RESULTS Derml exposure to TLR lignds dereses llergi inflmmtion nd AHR Mie were sensitized intrdermlly with,, nd in omintion with low (1 mg) or high (1 mg) doses of TLR lignds (TLR2), poly(i:c) (TLR3), nd (TLR4) one week for 4 weeks (Figure 1). Smples were nlyzed fter three intrnsl ovlumin () hllenges. High doses of or signifintly ttenuted irwy responsiveness to metholine (MCh), wheres Poly(I:C) hd only minor effet on AHR (Figure 1 nd Supplementry Figure S1 online). Low doses of TLR lignds hd no effet on irwy responses (dt not shown). The totl numer of ells nd inflmmtory ells in ronholveolr lvge (BAL) fluid were signifintly redued fter nd tretments, wheres Poly(I:C) hd no effet (Figure 1). The numer of eosinophils ws signifintly redued with ll three lignds (Figure 1d), wheres no signifint differenes were deteted in the numer of neutrophils or lymphoytes (dt not shown). Very high doses (1 mg) of Poly (I:C) indued the reruitment of signifintly higher numers of eosinophils nd lymphoytes in BAL fluid (Supplementry Figure S2 online). PAS þ ells were redued dose dependently with eh lignd tested, lthough the effet showed sttistil signifine only with (Figure 1e nd Supplementry Figure S3 online). In ddition, tretment signifintly deresed the numer of F4/8-positive mrophges in the lungs (Figure 1f nd Supplementry Figure S3 online). Insted, the numer of CD3 þ T ells showed no hnge with ny of the TLR lignds tested (Figure 1g nd Supplementry Figure S3 online). All TLR gonists signifintly enhned serum speifi IgG2 t high doses, nd the effet ws dose dependent (Figure 1h). In ddition, oth low nd high doses of eh TLR lignd signifintly deresed -speifi IgE (Figure 1i). TLR lignds derese Th2 ytokines in the lung nd in drining lymph nodes (LNs) Derml dministrtion of nd deresed the expression of Th2-type ytokines IL-4, IL-5, nd IL-13 s well Dy AHR BAL Lung smples Blood 31 i.n. i.d. +/ lignd i.d. +/ lignd i.d. +/ lignd i.d. +/ lignd R L (m H 2 Oml 1 s 1 ) MCh (mg/ml) Cells 1 4 per ml Totl ells μg d Cells 1 4 per ml Eosinophils μg e Cells per 2 μm of epithelium PAS + ells μg f Cells per HPF F4/8 + ells Pm 3 Cys g Cells per HPF h CD3+ ells IgG Pm 3 Cys Pm 3 Cys OD A 45 μg i OD A 45 IgE Pm 3 Cys μg Figure 1. The effets of intrderml (i.d.) dministrtion of Toll-like reeptor (TLR) lignds on the lung inflmmtory response. () The study protool. () Lung resistne (R L )nd(, d) the totl numer of ells nd eosinophils in ronholveolr lvge (BAL) fluid. (e) Periodi id-shiff (PAS) stining of muus-produing ells. Results re indited s the verge of PAS þ ells per 2 mm of ronhus surfe. Numer of immunohistohemilly stined (f) F4/8 þ ntigen-presenting ells (APCs) nd (g) CD3 þ ells in the lungs ounted s n verge from five rndomly seleted res. TLR lignds upregulted (h) ovlumin ()-speifi IgG2 nd downregulted (i) -speifi IgE ntiodies in the serum, s mesured with ELISA. Results re presented s sorne units (45 nm). P,.5; P,.1; P,.1. n ¼ 8 mie per group. AHR, irwy hyperretivity; HPF, high power field; i.n., intrnslly;, lipopolyshride; OD, optil density;, phosphte-uffered sline

3 RHpkoskiet l. s tht of pro-th2-type IL-33 in lung tissue (Figure 2 nd ). The most drsti redutions were seen with oth doses of nd with 1 mg of. The expression of IL-13 ws signifintly lower t the protein level in -stimulted LNs fter or tretment (Supplementry Figure S4 online). On the ontrry, Poly(I:C) tretment t three different doses (1, 1, nd 1 mg) showed no hnge in the expression of Th2-type ytokines in the lung (Figure 2, Supplementry Figure S2 online). When nlyzing Th1 ytokines, ws the only lignd eliiting signifint inrese in IFN-g mrna in the lung tissue (Figure 2). The expression of IL-12 showed no hnge with ny of the three TLR lignds (dt not shown). Regultory T ells re not involved in the ttenution of sthmti responses Involvement of regultory T ells (Tregs) in mediting tolerne nd suppressive funtion in sthm hs een well hrterized (Lloyd nd Hwrylowiz, 29). Mie treted with nd exhiited signifintly lower mounts of Foxp3 mrna in lung tissue when ompred with mie treted with lone, wheres other TLR lignds hd no effet (Figure 3). Expression of IL-1 ws downregulted fter derml exposure to oth doses of nd to the high dose of (Figure 3). All TLR lignds lso signifintly redued CTLA-4, negtive regultor of immune response, in the lung tissue. The most drsti redution ws oserved with (Po.1) when ompred with either (P4.1) or Poly(I:C) (Po.5) (Figure 3). Insted, smll ut signifint inrese in the expression of CD4 nd mjor histoomptiility omplex II (MHCII) on lung CD11 þ ells ws deteted (Supplementry Figure S5 online). Cutneous exposure to TLR lignds modifies the funtion of dendriti ells (DCs) during sensitiztion To understnd the erly events in derml sensitiztion, we nlyzed the funtion nd numer of DCs in drining LNs 24 hours fter dministrtion of -Alex647. Ativted DCs (CD11 þ þ ) showed 2% enhned ell dhesion nd ell prolifertion (CD4), 32 42% enhned ostimultion (CD8, CD86), nd 17% enhned ntigen presenttion (MHCII) when ompred with resting DCs (CD11 þ ) (Supplementry Figure S6 online). Among þ DCs, derml exposure to oth nd redued the totl numer of þ CD11 þ DCs in drining LNs in dosedependent mnner (Figure 4). enhned the expression of tivtion mrkers CD8 nd CD86, wheres no differenes in MHCII were deteted with ny of the TLR lignds (Figure 4 d). Next, we onduted kineti study using derml injetions of nd with 1 mg of. After testing t ll time points (2 48 hours), the perentge of þ DCs in the drining LNs of the skin ws found to e lower in mie reeiving nd when ompred with the group given lone (Figure 4e). In ontrst, the expression levels of surfe tivtion mrkers, CD8 nd CD86, were lerly enhned in mie given nd, nd the effet lsted from 2 to 48 hours (Figure 4f nd g). No differenes were deteted in the expression of MHCII t ny time point (Figure 4h). 3, IL-4 mrna 3, IL-5 mrna 15, IL-13 mrna 2, 1, μg ,,. 6,,. 4,,. 2,,. IL-33 mrna 2, 1, μg 6, 4, 2, IFN-γ mrna 1, 5, μg μg μg Figure 2. The expression of Th1, Th2, nd proinflmmtory ytokines t the RNA level in the lung fter Toll-like reeptor (TLR) lignd exposures. () Reltive mounts of Th2-type ytokine RNAs IL-4, IL-5, nd IL-13, nd () the mount of pro-th2-type ytokine IL-33 in lung tissue. () The mount of mrna of Th1-type IFN-g in the lungs. All ytokines re determined y reverse trnsriptse PCR s reltive units (). P,.5; P,.1; P,.1. n ¼ 8 mie per group., lipopolyshride;, ovlumin;, phosphte-uffered sline. 966 Journl of Investigtive Dermtology (213), Volume 133

4 R Hpkoski et l. 2, 15, 1, 5, 8, 6, 4, 2, 5, 4, 3, 2, 1, Foxp3 mrna IL-1 mrna CTLA4 mrna IFN- is entrl to the protetive effets of Derml exposure of mie to nd or hd no effet on the numer of CD4 þ T ells in BAL fluid (Figure 5); however, slight ut insignifint enhnement in the prodution of IFN-g nd IL-13 from CD4 þ T ells in the nd group ws deteted (Figure 5 nd ). Interestingly, the numer of CD8 þ T ells nd their IFN-g prodution μg μg μg Figure 3. The effet of Toll-like reeptor (TLR) lignds on lung regultory ftors t the RNA levels fter 4 weeks of sensitiztion. The reltive expressions of () trnsription ftor Foxp3, () ytokine IL-1, nd () n inhiitory moleule CTLA-4 were determined y reverse trnsriptse PCR. P,.5;P,.1;P,.1. n ¼ 8 mie per group., lipopolyshride;, ovlumin;, phosphte-uffered sline. were signifintly enhned fter derml exposure of mie to nd (Figure 5d nd e) long with slight ut nonsignifint inrese in the totl numer of T ells in BAL fluid (Supplementry Figure S7 online). In ddition, slight inrese ws oserved in the expression of CD69 of CD8 þ Tells (Figure 5f) in BAL fluid. To ssess the role of CD8 þ T ells in mediting the effet of, we depleted the mie of CD8 þ T ells with nti-mouse CD8 ma injetions efore hllenge. Insted of reversing the protetive effet of, the depletion of CD8 þ T ells resulted in further redution in Th2-type ytokines in the lung tissue nd in the numers of eosinophils in the BAL fluid ompred with mie treted with (Supplementry Figure S8 online). To investigte the role of IFN-g in mediting the effet of, we treted the mie with nti-mouse IFN-g ma injetions efore the irwy hllenge. Neutrliztion of IFN-g resulted in signifintly redued IFN-g mrna levels in the lung tissue (Figure 6), long with inresed Th2-type ytokines IL-5 nd IL-13 (Figure 6 nd ), nd inresed numers of eosinophils in the BAL fluid (Figure 6d), ompred with mie treted with þ or with ontrol IgG. Anti- IFN-g ma tretment lso indued n influx of neutrophils (Figure 6e) nd lymphoytes in BAL fluid (Figure 6f) ut hd no effet on muus prodution or irwy retivity (dt not shown). DISCUSSION Reent PARSIFAL nd GABRIELA studies show tht hildren who live on frms hve lower prevlene of sthm nd topy nd re exposed to greter vriety of environmentl miroorgnisms ompred with hildren in the referene group (Ege et l., 211). Atopi dermtitis (AD) my preede sthm during hildhood ording to the topi mrh (Spergel, 25), whih suggests tht utneous sensitiztion nd skin inflmmtion re importnt for the development of sthm lter in life. In ddition, muttions in the filggrin gene, whih hs n importnt role in skin rrier funtion, hve een shown to ssoite with sthm in AD ptients (Bthelor et l., 21). Experimentl sensitiztion is most effiient when the ntigen is dministered through skin insted of intrperitonelly (Lehto et l., 25). Miroil omponents re reognized y TLRs tht initite inflmmtory responses (Iwski nd Medzhitov, 24). In this study, we investigted the role of three TLR lignds,,, nd Poly(I:C), in the development of sthm y mimiking the nturl route of sensitiztion from skin to sthmti symptoms. We show tht derml dministrtion of TLR4 lignd during llergen sensitiztion proteted the mie from sthm nd irwy inflmmtion y reduing AHR, BAL ell numer, eosinophili, muus formtion, nd systemi IgE onentrtion. TLR2 gonist lso effetively redued Th2-type inflmmtory responses, wheres TLR3 lignd Poly(I:C) hd miniml effet on lol sthmti prmeters. Allergi sthm is usully linked to enhned Th2 responses, inluding inresed numer of eosinophils, muus overprodution, nd AHR. In ddition, Th2-type ytokines, suh s IL-4, IL-5, nd IL-13, ontriute to the indution nd mintenne of irwy inflmmtion nd sthmti response

5 % Of -Alex647+ RHpkoskiet l. Totl CD11 + ell numer CD μg + + % Of -Alex647 + DC CD μg DC CD μg d % Of -Alex647 + DC MHCII μg e 8 Reruitment of + DCs into LNs 6 CD8 + of + DCs in LNs + DCs of CD11 + ells (%) f CD8+ ells of CD11+ ells (%) Hours Hours 1 CD86 + of + DCs in LNs 15 MHCII + of + DCs in LNs ells of CD11+ gcd86+ ells (%) ells of hmhcii+ CD11+ ells (%) Hours Hours Figure 4. The reruitment nd tivtion sttus of dendriti ells (DCs) in drining lymph nodes (LNs). Ovlumin ()-Alex647 ws intrdermlly dministered lone or with 1 mg or 3 mg of Toll-like reeptor (TLR) lignds, nd the drining LNs were olleted 24 hours fter injetion. () The totl numer of þ CD11 þ DCs nd their tivtion sttus were mesured s the surfe expression of () CD8, () CD86, nd (d) mjor histoomptiility omplex II (MHCII) y flow ytometry (pool of three mie). (e h) The sme prmeters were studied in kineti study, in whih LN ells were olleted from lol drining LNs, 2, 4, 6, 9, 24, or 48 hours fter the intrderml injetion. P,.5; P,.1; P,.1.n ¼ 3 5 mie per time point., lipopolyshride. IL-5 is known to hve pivotl role s mjor mturtion nd differentition ftor for eosinophils (Tktsu et l., 29), wheres IL-13 hs een shown to diretly ontrt irwy smooth musle ells nd thus enhne the development of AHR (Kupermn et l., 22; Akri et l., 23). In our model, derml exposure to, espeilly, nd lso to, signifintly redued the prodution of IL-4, IL-5, nd IL-13 in the lungs nd tht of IL-13 in drining LNs. We therefore hypothesize tht IL-13, whih is highly produed y lung eosinophils, is relted to the signifint redution of 968 Journl of Investigtive Dermtology (213), Volume 133

6 R Hpkoski et l. 8, CD4 + 3, CD4 + IFN-γ + 8, CD4 + IL-13 + Numer of CD4 + TCR + ells 6, 4, 2, Numer of IFN-γ produing CD4 + T ells 2, 1, Numer of IL-13 produing CD4 + T ells 6, 4, 2, d Numer of CD8 + T ells 4, 3, 2, 1, CD8 + e Numer of IFN-γ produing CD8 + T ells 6, 4, 2, CD8 + IFN-γ + f CD69 + of CD8 ells (%) CD69 + Figure 5. T-ell phenotyping of ronholveolr lvge (BAL) fluid ells y FACS. BAL fluids were olleted fter 4 weeks of sensitiztion with ovlumin () or nd Toll-like reeptor (TLR) lignds. The () totl numer of CD4 þ T ells nd their () IFN-g nd () IL-13 prodution were mesured y flow ytometry. (d) The numer of CD8 þ T ells nd their (e) IFN-g prodution s well s their tivtion sttus were determined y the expression of (f) CD69. P,.5;P,.1; P,.1. n ¼ 3, eh representing pool of BAL fluid from three mie., lipopolyshride;, phosphte-uffered sline. 4, 3, 2, IFN-γ mrna 1, 8, 6, 4, IL-5 mrna 12, 9, 6, IL-13 mrna 1, 2, 3, d Cells 1 5 per ml Eosinophils e Cells 1 5 per ml Neutrophils f Cells 1 5 per ml Lymphoytes Figure 6. The effet of IFN-g neutrliztion on Th1/Th2-type ytokines nd irwy inflmmtion. Mie were sensitized s shown in Figure 1 y ovlumin () with 1 mg of. On dy 27, the mie were treted intrperitonelly (i.p.) with 2 mg nti-mouseifn-g ma or ontrol, nd intrnslly (i.n). on dys 28, 29, nd 3 together with. The reltive mount of () Th1-type ytokine IFN-g nd Th2-type () IL-5 nd () IL-13 mrnas ws mesured t the RNA level in lung tissue y reverse trnsriptse PCR on dy 31. The mount of (d) eosinophils, (e) neutrophils, nd (f) lymphoytes in ronholveolr lvge fluids ounted from My-Grünwld Giems (MGG)-stined slides. P,.5; P,.1; P,.1. n ¼ 8 mie per group., lipopolyshride;, phosphteuffered sline. eosinophils y or tretment. This result is supported y deresed muus prodution in onert with redued prodution of IL-4 nd IL-5. Further nlysis showed tht expression levels of regultory trnsription ftor Foxp3, ytokine IL-1, nd inhiitory mrker CTLA-4 were deresed espeilly fter tretment. Therefore, our findings strongly 969

7 RHpkoskiet l. suggest tht the inhiition of llergi responses ould not e result of n indued mount or funtion of regultory T ells ut insted euse of deresed mount of eosinophils in the lungs. Endotoxin tolerne (ET) or TLR tolerne is phenomenon, where repeted systemi pplition of or other TLR lignds n indue stte of immune unresponsiveness (Brod et l., 27; Bisws nd Lopez-Collzo, 29). Mtsushit et l. (21) suggest tht the downregulted expression of MHC lss II nd ostimultory moleules, CD86 nd CD4, on DCs nd monoytes is good iomrker for ET in vivo. However, we sw smll ut signifint inrese insted of redution in the expression of CD4 nd MHCII on lung CD11 þ nd CD11 þ ells fter 4 weeks of sensitiztion nd eliittion. This dvotes tht TLR tolerne does not explin the deresed sthm symptoms in this model. TLR lignds re potent tivtors of DCs resulting in the initition of dptive immunity. In our study, derml dministrtion of TLR lignds during llergen sensitiztion deresed the numer of ntigen-ering DCs in LNs ut enhned their tivtion sttus. DCs tht engulfed or nd hd similr kinetis, nd they trveled into drining LNs peking y 4 hours fter ntigen dministrtion. However, their numer ws deresed in the -treted groups t ll time points tested, mye euse of deresed intrinsi migrtory ility s desried y Grnui et l. (1999). In ddition, is shown to elerte the mturtion nd redistriution of DCs followed y mrked derese in DC numers, unless signl is reeived from Ag-speifi T ells (De Smedt et l., 1996, 1998). DCs tht survived through the nd tretment in our study mintined high ntigen presenttion pity (MHCII), nd tivtion y highly inresed expression of CD8 nd less inresed expression of CD86. CD86 is linked to Th2 development, wheres CD8 might e more Th1-type or neutrl ostimultory moleule (Kuhroo et l., 1995; Moser nd Murphy, 2). Exposure to household endotoxins during hildhood is inversely relted to llergen sensitiztion (Gered et l., 2) nd to the development of llergi sthm (Brun- Fhrländer et l., 22). The mehnisms tht drive immunity fter endotoxin or exposure re multiftoril nd prtly unknown. n support the development nd funtion of the mjor T-ell susets depending on the sitution, whih hs led to re-evlution of its effets on dptive immunity (MAleer nd Vell, 21). In our study, when ompring the nd group with the group fter 4 weeks of sensitiztion, the numer of CD8 þ T ells in BAL fluid ws seen to hve inresed signifintly, wheres the numer of CD4 þ T ells hd remined unhnged. T ells in - nd -treted mie produed signifintly more IFN-g euse of inresed numers of CD8 þ T ells, nd these ells were lso more tive s indited y the expression of CD69. However, in vivo depletion of CD8 þ T ells did not reverse the protetive effet of ut insted led to further deresed numers of eosinophils nd redution of Th2-type ytokines in the lung, likely onsequene of depleting the entire omprtment of CD8 þ T ells, inluding Th2-like CD8 þ T ells, whih re known to medite irwy inflmmtion nd AHR (Miyhr et l., 24; Stok et l., 24). Nevertheless, in vivo neutrliztion of IFN-g demonstrted ler dependene of the protetive effet of on IFN-g. To the est of our knowledge, no other report hs studied the erly events of diretly in vivo in similr study settings. In one study, low-dose -pulsed one mrrow derived DCs used mssive eosinophili nd enhned Th2 ytokine prodution in mie, wheres high-dose -indued Th17- type immune responses nd -free did not indue llergi symptoms t ll (Peters et l., 21). Depending on the lol miroenvironment, T ells might e polrized from Th2 to Th1, Th17, or to Treg ells. Tretment with TLR gonists during the hllenge efore or fter llergen exposure hs produed quite different outomes (Duehs et l., 211). We hve previously shown enhned Th1 polriztion fter derml TLR9 lignd CpG exposure during llergen sensitiztion. (Hpkoski et l., 211). Here we show tht identil exposure regimes of different TLR lignds exhiit vrious effets on the Th2 inflmmtory response. TLR2 nd TLR4 gonists indued protetive effets, wheres TLR3 gonist poly I:C hd no effet t low onentrtions nd ugmented the Th2-type response t high onentrtions. The different outomes might e the result of the distint signling pthwys used y the TLRs, with TLR3 utilizing exlusively the TRIF-dependent pthwy nd TLR2 nd TLR4 signl utilizing the MyD88-dependent pthwys s well (Akir et l., 26). The vrious responses medited y TLRs likely evolved to fine-tune dptive immunity, nd different dose, time, nd dministrtion routes eliit qulittively distint signls through TLRs, influening T-ell polriztion. Aording to the hygiene hypothesis, the inrese in topy nd sthm is relted to improved hygiene sttus nd redued Th1-type immune responses in the industrilized world (Yng nd Go, 211). Endotoxins nd other teril derivtives present in the environment re suggested to hve role in the proess y ffeting DC mturtion nd T-ell polriztion y fvoring the development of protetive immunity. Our results show tht llergen sensitiztion through skin with nd, ut not with Poly(I:C), dereses Th2-type llergi responses nd irwy retivity. TLR4 lignd ws the only lignd to shift the lne towrd Th1-type immunity y induing IFN-g prodution from CD8 þ T ells nd y reduing Th2-type ytokines. The suppressive effet ws stritly dependent on IFN-g. These findings shed light on the intertion of innte immunity-tivting miroil omponents nd the derml route of sensitiztion during llergi inflmmtion. Our results emphsize the importne of restoring the skin rrier funtion nd lso highlight the role of ross-tlk etween the derml miroiome nd the host in the development of llergies. MATERIALS AND METHODS Mie Femle Bl/ mie (N-SCB AB, Sollentun, Sweden) ged 6 8 weeks were mintined on -free diets nd wter d liitum. All niml experiments were pproved y The Soil nd Helth Cre Deprtment of the Stte Provinil Offie of Southern Finlnd. 97 Journl of Investigtive Dermtology (213), Volume 133

8 R Hpkoski et l. TLR lignds Ultr Pure E. oli (strin 111:B4, #6B24-MT) nd High Moleulr Weight Poly(I:C) (endotoxin level o1.25 EU/ml, #9L2- MT) were purhsed from InvivoGen (Sn Diego, CA). (PmCys-SKKKK, L2) ws purhsed from EMC miroolletions GmH (Tüingen, Germny). Tretment protools Mie were nesthetized nd their ks were shved nd tpestripped three times efore the injetions to introdue the skin injury. The mie were sensitized weekly intrdermlly with 5 mg of in 1 ml, or with only for 4 weeks. Groups of mie were treted with 1 mg or1mg of onurrent with 5 mg of ( nd groups), with 1 mg or 1mg of ( nd groups), or with 1 mg, 1 mg, or 1 mg of Poly(I:C) ( nd Poly(I:C) groups). On dys 28, 29, nd 3 ll mie were hllenged with intrnsl (5 mg in 5 ml of). For kineti studies, the ks of the mie were shved nd tpe stripped three times, nd they were treted either with 5 mg of- Alex647 (Invivogen, Sn Diego, CA) lone or in omintion with 1 mg of eh TLR lignd intrdermlly. After different time points (,2,4,6,9,24,nd48hours),themiewerekilledndthedrining LNs were olleted for FACS nlysis. CD8 T ells were depleted y 2 mg of nti-mouse CD8 ma intrperitonelly 4, 2, nd 1 dy efore the 3-dy eliittion period. Control mie were treted with the sme mount of rt IgG2. IFN-g ws neutrlized y 2 mg ofnti-mouseifn-g ma or ontrol ma intrperitonelly on dy 27 (1 dy efore the irwy hllenge) nd gin three times intrnslly (5 mg of nti-ifn-g A or together with 5 mg of ) on dys 28, 29, nd 3. Determintion of irwy retivity to methholine Airwy resistne nd ompline were mesured from sensitized nd hllenged mie s previously desried (Hpkoski et l., 211). BAL nd lung histology BAL inflmmtory ells nd lung histology were hndled nd ounted s previously desried (Hpkoski et l., 211). RT-PCR nlysis Totl RNA extrtions with Trisure Regent (Bioline, London, UK), omplementry DNA synthesis, nd rel-time PCR (RT-PCR) were performed s previously desried (Lehto et l., 23). Mesurement of ytokines y ELISA nd flow ytometry The ytokine levels of IL-13 were mesured y ELISA from BAL fluids nd from superntnts of ultured LN nd from splenoytes ording to the mnufturer s instrutions (R&D Systems, Minnepolis, MN for IL-13). The detetion limit ws 8 pg ml 1. Flow ytometri nlyses BAL ells were wshed one with /2% FBS; F reeptors were loked with nti-mouse CD16/32 (ebiosiene, Sn Diego, CA); nd ells were stined with nti-tcr, nti-cd3, nti-cd4, nd nti-cd8 ntiodies (BD Phrmingen, Sn Diego, CA). For intrellulr ytokines, BAL ells were stimulted t 37 1C for 4 hours with phorol myristte ette (2 ng ml 1 )ndionomyin(1mgml 1 ) (Sigm, St. Louis, MO), inluding refeldin A (Sigm). After wshing, F loking, nd surfe stining, ells were permeilized using the Fix nd Perm kit (Cltg, Burlingme, CA) nd stined with nti- IFN-g (BD Phrmingen) nd nti-il-13 (ebiosiene). For kineti studies, rhil LNs were removed, tesed with glss slides, nd filtered through 7-mm ell striner; therefter, the totl numer of ells ws ounted. Single-ell suspensions were F loked, stined with mas for CD11, CD11, MHCII, CD4, CD8, nd CD86 (ebiosiene), nd run on FACSCntoII instrument(betondikinson,frnklinlkes,nj).dtwereproessed using FlowJo Softwre (Tree Str, Ashlnd, OR). Mesurement of serum ntiodies -speifi IgE nd IgG2 ntiodies were mesured from serum using ELISA s previously desried (Lehto et l., 23). Sttistis Single-group omprisons were mde using the nonprmetri Mnn Whitney U-test. Results re expressed s men±sem nd P-vlues of o.5 were onsidered sttistilly signifint. Sttistil nlyses were performed using GrpPdPrism (version 4 GrphPd Softwre, L Joll, CA). CONFLICT OF INTEREST The uthors stte no onflit of interest. ACKNOWLEDGMENTS We thnk Suli Svukoski for his expertise in immunohistologil work. We lso thnk Päivi Alnder nd Snttu Hirvikorpi for their exellent tehnil ssistne. This reserh ws funded y the Ademy of Finlnd, y the Europen Union s FP7, grnt greement numer HEALTH-F , nd y the Nummel Foundtion. SUPPLEMENTARY MATERIAL Supplementry mteril is linked to the online version of the pper t REFERENCES Akri O, Stok P, Meyer E et l. (23) Essentil role of NKT ells produing IL-4 nd IL-13 in the development of llergen-indued irwy hyperretivity. Nt Med 9:582 8 Akir S, Uemtsu S, Tkeuhi O (26) Pthogen reognition nd innte immunity. Cell 124: Bh JF (22) The effet of infetions on suseptiility to utoimmune nd llergi diseses. N Engl J Med 347:911 2 Bthelor JM, Grindly DJ, Willims HC (21) Wht s new in topi ezem? An nlysis of systemti reviews pulished in 28 nd 29. Clin Exp Dermtol 35:823 7 Bisws SK, Lopez-Collzo E (29) Endotoxin tolerne: new mehnisms, moleules nd linil signifine. Trends Immunol 3: Brun-Fhrländer C, Riedler J, Herz U et l. (22) Environmentl exposure to endotoxin nd its reltion to sthm in shool-ge hildren. 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