Prior Bordetella pertussis infection modulates allergen priming and the severity of airway pathology in a murine model of allergic asthma

UNCORRECTED PROOF P io Bo de ella pe ussis in ec ion modula es alle gen p iming and he se e i y o ai way pa hology in a mu ine model o alle gic as hma D. P. Ennis * , J. P. Cassidywand B. P. Mahon * * Mucosal Immunology Labo a o y, Ins i u e o Immunology, NUI Maynoo h, I eland and wDepa men o Ve e ina y Pa hology, Uni e si y College Dublin, I eland Summa y Backg ound I has been p oposed ha T helpe (Th)2-d i en immune de ia ion in ea ly li e can be coun e ed by Th1 inducing childhood in ec ions and ha such coun e - egula ion can p o ec agains alle gic as hma. Objec i e To es whe he Th1-inducing in ec ion wi h Bo de ella pe ussis p o ec s agains alle gic as hma using well-cha ac e ized mu ine models. Me hods G oups o mice we e sensi ized o o albumin (OVA) in he p esence o absence o B. pe ussis, a well-cha ac e ized Th1 inducing espi a o y in ec ion. Immunological, pa hological and physiological pa ame e s we e measu ed o assess he impac o in ec ion on immune de ia ion and ai way unc ion. Resul s We demons a e ha OVA sensi iza ion does no a ec he de elopmen o B. pe ussis- specific immune esponses domina ed by IgG2a and IFN-gand does no impai Th1-media ed clea ance o ai way in ec ion. In con as , B. pe ussis in ec ion a he ime o sensi iza ion modula ed he esponse o OVA and significan ly educed o al se um and OVA-specific IgE. The pa e n o cy okine esponses, in pa icula OVA-specific IL-5 esponses in he spleen was also modula ed. Howe e , B. pe ussis did no cause global supp ession as IL-10 and IL-13 le els we e enhanced in OVA-s imula ed spleen cell cul u es and in la age fluid om in ec ed co-sensi ized mice. His opa hological examina ion e ealed ha B. pe ussis in ec ion p io o OVA sensi iza ion esul ed in inc eased inflamma ion o b onchiola walls wi h accompanying hype plasia and mucous me aplasia o lining epi helia. These pa hological changes we e accompanied by inc eased b onchial hype - eac i i y o me hacholine exposu e. Conclusion Con a y o he abo e p emise, a Th1 esponse induced by a common childhood in ec ion does no p o ec agains b onchial hype - eac i i y, bu a he exace ba es he alle gic as hma ic esponse, despi e modula ion o immune media o s. Keywo ds alle gy, bac e ial, inflamma ion, lung, Th1/Th2 cells Submi ed &&2003; e ised &&2004; accep ed 26 May 2003 In oduc ion As hma is a ch onic disease o he espi a o y ac ha has inc eased d ama ically in p e alence in wes e n socie y [1]. The inflamma o y esponse in as hma is igh ly associa ed wi h ai way hype - esponsi eness (AHR), inc eased mucus p oduc ion and an infil a ion o he b onchial mucosa wi h CD4 1 T cells [2]. The e is e idence o an al e ed local T cell esponse in a ou o T helpe (Th)2 cy okine elease (IL-4, IL-5 and IL-13) esul ing in B cell iso ype swi ching o IgE, mas cell, eosinophil and basophil ec ui men and p oduc- ion o a wide ange o inflamma o y media o s [3]. The esul ing pulmona y inflamma ion leads o b onchocons ic- ion and ul ima ely o ai way emodelling [4]. The cu en unde s anding o he pa hophysiology o alle gic as hma is ha i esul s om a b eakdown in he no mal ole ance o inhaled an igens, associa ed wi h Th2 cy okine p oduc ion [5, 6]. The mu ine o albumin (OVA) model o AHR exhibi s many o he ea u es o human as hma, including ai way hype - eac i i y, ai way inflamma- ion and inc eased se um IgE le els [7, 8]. This model has been used ex ensi ely o p obe mechanisms o as hma [7, 9]. The inc eased incidence o as hma has been linked o imp o ed sani a ion in indus ialized socie ies, which in u n has educed he incidence o childhood in ec ions [10]. One cu en a emp o explain hese obse a ions, loosely e med he hygiene hypo hesis, s a es ha childhood as hma de elops as a esul o dec eased exposu e o in ec ious agen s du ing in ancy and ea ly childhood, which esul s in he pe sis ence o he neona al Th1 defici , he eby p edisposing he child o a opic disease [11]. While Th2 cells p omo e ai way inflam- ma ion in as hma, i has been p oposed ha because Th1 cells an agonize Th2 cell unc ion, immune de ia ion owa ds Th1 Jou nal: CEA HDisk used ED: Suma Copy ED: Guna Pgn by: su eshbabu A icle : 2042 Pages: 10 Despa ch Da e: 19/7/2004 Scan: Roopa Colou : Fig. 6 Q2 Co espondence: B. P. Mahon, Mucosal Immunology Labo a o y, Ins i u e o Immunology, Na ional Uni e si y o I eland Maynoo h, Maynoo h, Co. Kilda e, I eland. E-mail: bpm[email p o ec ed] Clin Exp Alle gy 2004 doi:10.1111/j.1365-2222.2004.02042.x 2004 Blackwell Publishing L d 1 BWUK CEA 2042.PDF 19-Jul-04 19:1 844323 By es 10 PAGES UNCORRECTED PROOF may be p o ec i e in as hma [12, 13]. One p edic ion a ising om he abo e hypo hesis is ha a powe ul Th1-inducing in ec ion du ing o p io o ai way sensi iza ion should diminish o p o ec agains Th2-media ed alle gic as hma. Bo de ella pe ussis is a G am-nega i e bac e ium ha causes he se e e in an disease whooping cough. B. pe ussis espi a o y challenge o mice is a well-cha ac e ized model o ai way Th1-induced immuni y, which co ela es well o immuni y in humans [14]. Reco e y om in ec ion is associa ed wi h he de elopmen o B. pe ussis-specific Th1 cells in bo h humans and mice [15]. Th1 cells p oducing IFN- gplay an essen ial, non- edundan ole in he clea ance o he bac e ia om he espi a o y ac [16]. Mu ine espi a o y challenge by ae osol adminis a ion o he bac e ia has been used ex ensi ely o s udies o B. pe ussis immuni y and pa hogenesis and al hough mice lack he cha ac e is ic cough, in o he espec s he cou se o in ec ion and many o he sys emic e ec s a e simila o hose obse ed in in an s [17]. In o de o es he abo e hypo hesis, we used he Th1 d i ing B. pe ussis model, in combina ion wi h he mu ine OVA model o alle gic as hma. Based on he p edic ion abo e, p io in ec ion wi h B. pe ussis migh be expec ed o educe OVA-induced Th2-media ed AHR and immunopa hology. Ou findings demons a e ha al hough dampening o he Th2 esponse was seen a he local and sys emic le el, OVA sensi iza ion du ing B. pe ussis in ec ion led o enhanced p oduc ion o ai way IL-10 and IL-13, coupled wi h a subsequen inc ease in AHR and pa hology. This sugges s ha al hough IL-10 may be ega ded as a egula o y cy okine, i has b oade unc ions ha may no always p o ec agains inflamma o y disease. These da a ha e implica ions wi h ega d o he alidi y o he hygiene hypo hesis and aise conce ns ega ding he apies based on he con e sion o Th2-domina ed alle gic inflamma o y esponses in o Th1-domina ed esponses based on p o ec i e e ec s o Th1 cells in alle gy and as hma. Ma e ials and me hods Animals Six- o eigh -week old emale BALB/c (Ha lan, UK) mice we e used unde he guidelines o he I ish Depa men o Heal h and he esea ch e hics commi ee o he Na ional Uni e si y o I eland Maynoo h. Ae osol in ec ion Respi a o y in ec ion was ini ia ed by ae osol challenge wi h B. pe ussis s ain W28, ollowing g ow h unde agi a ion condi ions a 37 1C in S aine –Schol e liquid medium. Bac e ia om a log-phase cul u e we e esuspended a a concen a ion o 2 10 10 CFU/mL in 1% (w/ ) casein in 0.9% (w/ ) saline. The challenge inoculum was adminis e ed o wo g oups o mice on 0 day (Bp and BpOVA g oups). Adminis a ion was by ae osol o e a pe iod o 15 min using a nebulize . G oups o ou o mo e mice we e killed a a ious ime poin s a e ae osol challenge o assess he numbe o iable B. pe ussis in he lungs. Remaining mice ecei ed a simila ae osol o s e ile saline alone. Immuniza ion and ai way deli e y o OVA Two g oups o 6–8-week-old emale BALB/c mice (OVA and BpOVA) we e sensi ized by i.p. injec ion o 100 mg OVA (G ade V; Sigma, Do se , UK) emulsified in 2% Alhyd ogel s adju an (Supe os Biosec o , Sweden) a 10 and 24 days a e bac e ial o saline challenge. Con ol g oups (C l and Bp) ecei ed saline alone (i.p.). On 35, 36 and 37 days, OVA and BpOVA-sensi ized mice ecei ed 50 mg OVA in a–nasally, whe eas C l and Bp g oups ecei ed saline only. Enume a ion o iable bac e ia in he lungs Lungs we e emo ed asep ically in o 1 mL o s e ile physiological saline wi h 1% casein. One hund ed mic o- lio es o se ially dilu ed homogena e om indi idual lungs we e placed on o iplica e Bo de –Gengou aga pla es and he numbe o CFU de e mined a e incuba ion a 37 1C o 4 days. Resul s a e epo ed as he mean numbe o B. pe ussis CFU o indi idual lungs om ou o mo e mice. B onchoal eola la age B onchoal eola la age (BAL) fluids we e ob ained by epea adminis a ion and aspi a ion o 0.5 mL olumes ( o al 5 mL) o phospa e-bu e ed saline (PBS) ia cannula ion o he achea o mice om h ee expe imen s (n55). Cells om he la age fluid we e eco e ed by cen i uga ion a 300 g o 6 min and esuspended in PBS; o al leucocy es we e coun ed and cy ospin p epa a ions we e s ained wi h a combined Alcian blue/Discombe’s s ain o de e mine he di e en ial cell coun . Supe na an s we e collec ed o cy okine analysis and s o ed a 80 1C. Measu emen o o albumin- and Bo de ella pe ussis- speci ic an ibody OVA- and B. pe ussis-specific-IgG1, IgG2a, IgG2b and IgG3 p esen in collec ed se a we e measu ed by ELISA as p e iously desc ibed [16, 18]. B iefly, pla es we e coa ed wi h OVA p o ein (5 mg/mL) o sonica ed B. pe ussis an igen (1 mg/ mL) o e nigh a 4 1C. A e blocking and he addi ion o se um samples, alkaline phospha ase-labelled a an i-mouse IgG1, IgG2a, IgG2b and IgG3 (Pha mingen) we e used o de ec OVA- and B. pe ussis-specific an ibody as p e iously desc ibed [19]. To al and OVA-specific IgE was measu ed using a a an i-mouse IgE monoclonal an ibody (Pha min- gen). The IgE concen a ion was exp essed as mic og ams pe millili e a e compa ison wi h mu ine IgE s anda ds. T cell p oli e a ion assays Spleen cells (2 10 6 /mL) om in ec ed, sensi ized and con ol mice (n54 o mo e pe g oup) we e es ed o in i o p oli e a ion agains hea -inac i a ed B.pe ussis (1 10 4 CFU/mL), OVA (20 mg/mL), Concana alin A (Con A) (5 mg/mL, posi i e con ol), o medium alone (nega i e con ol). A e 72 h, cell p oli e a ion was assessed by liquid scin illa ion coun ing o [ 3 H]-Thymidine inco po a ion and esul s we e exp essed as mean CPM o iplica e wells SE. A he 72-h ime-poin , cul u e supe na an s we e sampled o cy okine analysis, al hough he kine ics o cy okine p oduc- Q3 Q4 Q5 2D. P. Ennis e al. CEA : 2042 2004 Blackwell Publishing L d, Clinical and Expe imen al Alle gy BWUK CEA 2042.PDF 19-Jul-04 19:1 844323 By es 10 PAGES UNCORRECTED PROOF ion a ies his ime-poin has p e iously p o ed accep able o de ec ion o mos cy okines [14]. Cy okine measu emen Concen a ions o IL-4, IL-5, IL-10, IL-13 and IFN-g om spleen and b oncho-al eola la age fluid (BALF) we e assessed by ELISA (Pha mingen). Cy okine concen a ions we e calcula ed by compa ison wi h known cy okine s an- da ds as p e iously desc ibed [16]. Whole-body ple hysmog aphy Ai way esponsi eness was assessed by me hacholine (MCh)- induced ai flow obs uc ion om conscious mice using whole- body ple hysmog aphy (Buxco Elec onics, USA) as p e- iously desc ibed [20]. Pulmona y ai flow obs uc ion was measu ed by enhanced pause (PenH), a alue de e mined om he a io o expi a o y ime and elaxa ion ime o peak expi a o y flow and peak inspi a o y flow and hough o co ela e wi h ai way esponsi eness. Measu emen s we e ob ained a e exposu e o mice o 3 min o PBS (baseline) ollowed by inc emen al doses (3.3–50 mg/mL) o MCh deli e ed by ae osol [21]. Respi a o y ac his ology Animals (n55 pe g oup pe expe imen ) we e killed a 37 days. Lungs we e emo ed, fixed in a pa a o maldehyde/ lysine/pe ioda e fixa i e, pa a fin embedded, sec ioned and s ained using he haema oxylin and eosin (H&E), Discombe’s (iden ifica ion o eosinophils)/Alcian blue (iden ifica ion o mucus), Pe iodic Acid-Schi (assessmen o basemen mem- b ane hickness), azu e-A (iden ifica ion o mas cells) and Van Gieson (iden ifica ion o fib osis) me hods. His opa ho- logical changes e iden we e g aded acco ding o a semi- quan i a i e sco ing sys em as mild, mode a e o se e e by wo esea che s wi hou p io knowledge o he ea men g oup (Table 2). All expe imen s we e pe o med a leas wice (n55) on each occasion. Resul s O albumin sensi iza ion does no impai T helpe 1- media ed mechanisms o bac e ial clea ance In o de o examine he e ec s o immune c oss- egula ion we es ed he e ec o OVA sensi iza ion upon he de elopmen o a p o ec i e Th1 esponse o in ec ion. G oups o mice we e sensi ized o OVA in he p esence o absence o a p io B. pe ussis in ec ion (Table 1). The kine ics o bac e ial clea ance om he lungs o expe imen al animals we e moni o ed by pe o ming colony coun s on whole-lung homogena es a di e en imes pos -bac e ial challenge. Mice ecei ed ei he saline (C l and OVA) o iable B. pe ussis (Bp and BpOVA) by ae osol. This was ollowed by OVA (OVA and BpOVA) o sham sensi iza ion (Table 1). G oups in ec ed wi h B. pe ussis (Bp and combined BpOVA g oups) showed simila kine ics o bac e ial clea ance. No bac e ia we e eco e ed om he OVA o C l g oup (Fig. 1), which we e unin ec ed bu ecei ed saline by ae osol. Bac e ial bu den in he Bp and BpOVA g oups peaked a 10 days and declined he ea e . By 35 days-pos -challenge, bo h he Bp and he BpOVA g oups showed comple e bac e ial clea ance (Fig. 1). The e o e, sensi iza ion wi h OVA does no impai he e ec o unc ion associa ed wi h he Th1-media ed clea ance o a bac e ial disease. Table 1.Expe imen al design Desc ip ion * Time (days) 0 10 24 35/36/37 Con ol Saline ae osol Saline (i.p.) Saline (i.p. and i.n.) Saline (i.n.) Bp in ec ion Bp ae osol in ec ion Saline (i.p.) Saline (i.p. and i.n.) Saline (i.n.) OVA sensi iza ion Saline ae osol OVA (i.p.) OVA (i.p and i.n.) OVA (i.n.) Bp in ec ion and OVA sensi iza ion (BpOVA) Bp ae osol in ec ion OVA (i.p.) OVA (i.p and i.n.) OVA (i.n.) * G oups o 6–8-week-old emale BALB/c mice (n54 o mo e pe eplica e pe ime-poin ) we e ea ed as ollows: Con ol (C l) mice we e sham in ec ed on 0 day and sham sensi ized a 10 days and 24 days. A second g oup (Bp) we e in ec ed wi h Bo de ella pe ussis a 0 day and sham sensi ized a 10 day and 24 days. The hi d g oup (OVA) we e sham in ec ed bu sensi ized wi h OVA (100 mg, i.p.) a 10 and 24.days and hen again (50 mg i.n.) a 24, 35, 36, and 37 days. The inal g oup (BpOVA) we e in ec ed wi h B. pe ussis on 0 day, and sensi ized as abo e. Table 2.His ological assessmen o ai way pa hology T ea men g oup Epi helial mucous me aplasia Epi helial hype plasia Smoo h muscle hype ophy Pe i-ai way in lamma ion * O e all E N L M F Con ol      Bp 11 1111 OVA 11 11 11 11 1 11 11  BpOVA 111 111 11 111 11 111 111  A semi-quan i a i e sco e ( , absen ; 1, mild; 11, mode a e; 111, se e e) was assigned o ea u es o ai way pa hology obse ed. * Pe i-ai way in lamma ion assessed in e ms o o e all deg ee and o numbe s o in il a ing eosinophils (E), neu ophils (N), lymphocy es, plasma cells and mac ophages (L), mas cells (M) and in e ms o ci cumsc ibing ib osis (F). Obse a ions a e ep esen a i e o a leas wo expe imen s whe e n55 o mo e in each case. Q1 Q6 Q7 Bo de ella pe ussis in ec ion 3 CEA : 2042 2004 Blackwell Publishing L d, Clinical and Expe imen al Alle gy BWUK CEA 2042.PDF 19-Jul-04 19:1 844323 By es 10 PAGES UNCORRECTED PROOF Bo de ella pe ussis supp esses o albumin-speci ic humo al immune esponses Al hough OVA-induced sensi iza ion does no impai Th1 clea ance o B. pe ussis, we wan ed o examine he influence o B. pe ussis in ec ion on esponses associa ed wi h OVA Th2 sensi iza ion. OVA-specific IgG was no de ec ed om mice in ec ed wi h B. pe ussis only; simila ly B. pe ussis- specific IgG could no be de ec ed in OVA-sensi ized animals, sugges ing no significan c oss- eac ion be ween he wo immunogens occu ed (Figs 2a and b). An analysis o an ibody subclasses e ealed ha in ec ion induced g ea e se um i es o B. pe ussis-specific IgG2a han IgG1 (Figs 2a and b), consis en wi h ou p e ious findings [19]. Sensi iza- ion o in ec ed mice wi h OVA (BpOVA) did no signifi- can ly al e his p ofile. OVA sensi iza ion in he absence o in ec ion esul ed in s ong an ibody esponses almos exclusi ely o he IgG1 subclass, consis en wi h a Th2 model o p iming. In con as o he minimal influence o OVA on he esponse o in ec ion (Fig. 2a), bac e ial B. pe ussis in ec ion supp essed o modula ed immuni y o OVA. Fo example, OVA-specific IgG1 i es we e significan ly lowe (Po0.001) in he combined g oup (Fig. 2b). Supp ession was no confined o IgG subclasses. To al se um and OVA- specific IgE was also significan ly educed (Po0.001) be ween he OVA and he Bp/OVA g oups (Figs 2c and d), suppo ing a ole o B. pe ussis in supp essing o egula ing he immune esponse o an igen exposu e du ing in ec ion. O albumin sensi iza ion du ing Bo de ella pe ussis in ec ion enhances ai way In e leukin-10 and In e leukin-13 In o de o dissec he na u e o B. pe ussis supp ession, cell- media ed immune esponses we e examined om spleen cul u es. As p e iously epo ed [22], B. pe ussis in ec ion induced s ong splenic p oli e a i e and IFN-g esponses bu e y li le IL-5 (Figs 3a–c). This was consis en wi h he obse ed p o ec ion (Fig. 1) and he an ibody subclass da a (Fig. 2). Again, IFN-gwas no significan ly educed by OVA sensi iza ion (Fig. 3c). As expec ed, OVA sensi iza ion alone induced significan IL-5 bu no IFN-g(Figs 3a and c). Howe e B. pe ussis did influence cy okine esponses o OVA. Reduced le els o IL-5 we e de ec ed in he combina- ion g oup sugges ing ha B. pe ussis supp essed Th2 esponses o OVA, mi o ing he educ ion in i e o specific an ibody de ec ed. In e es ingly B. pe ussis in ec ion did induce specific IL-10 as well as IL-13 esponses (Figs 3b and d). While OVA sensi iza ion had li le o he e ec on he immune esponse induced by in ec ion, i significan ly enhanced he le els o IL-10 and IL-13 (Po0.01 and o0.01, espec i ely) p oduced in esponse o he bac e ium (Figs 3b and d). To ex end hese findings, we examined he le els o cy okines p esen in BALF. OVA sensi iza ion, bu no in ec ion, induced IL-4 (Fig. 4a); howe e , p io in ec ion wi h B. pe ussis supp essed his. B. pe ussis in ec ion induced local IFN-g, which was no educed by OVA sensi iza ion (Fig. 4d). In e es ingly, while OVA sensi iza ion induced IL-10 and IL-13 de ec able in BALF, his was significan ly (Po0.01 and o0.01, espec i ely) enhanced i sensi iza ion ollowed in ec ion (Figs 4b and c). A T-helpe 1 in ec ion in he espi a o y ac does no p o ec , bu exace ba es he alle gic as hma ic esponse I has been p oposed ha p io Th1 esponses o bac e ial in ec ions p o ec agains alle gic disease by dampening he ac i i y o Th2 e ec o cells. I migh also be p edic ed ha he supp essi e o modula o y e ec s o B. pe ussis ou lined abo e would p o ec agains Th2-d i en pa hology. We used whole-body ple hysmog aphy in o de o measu e ai way eac i i y in mice in ec ed wi h B. pe ussis p io o OVA sensi iza ion in compa ison wi h con ols (Fig. 5). We ound ha con a y o he abo e p emise, a Th1 esponse induced by in ec ion in he espi a o y ac did no p o ec agains b onchial hype - eac i i y bu a he exace ba ed he alle gic as hma ic esponse. S a is ical analysis using wo-way ANOVA showed ha mice sensi ized o OVA ollowing B. pe ussis in ec ion displayed significan ly g ea e b onchial hype - eac i i y compa ed wi h OVA sensi ized alone (Po0.001) (Fig. 5). This demons a es ha in ec ion wi h B. pe ussis o he espi a o y ac p io o sensi iza ion esul s in inc eased ai way eac i i y and exace ba es he alle gic esponse. B. pe ussis in ec ion also modula ed he quali y o he inflamma o y influx o he espi a o y ac . The e was a ma ked educ ion in eosinophil numbe s obse ed in BpOVA compa ed wi h OVA-sensi ized ai ways (Table 3). His ologi- cal examina ion o lung issue showed ha pa hological changes we e la gely ocussed on b onchioles and adjacen pe ib onchiola blood essels wi h a ying deg ees o ai way wall inflamma ion and smoo h muscle hype ophy accom- panied by a ying deg ees o epi helial hype plasia and mucous me aplasia (Fig. 6). A semi-quan i a i e his opa ho- logical sco ing sys em was used o acili a e compa isons be ween g oups (Table 2). Minimal changes we e obse ed in 0 7 14 21 28 35 0 1 2 3 4 5 6 Bp BpOVA C l OVA Days a e challange Bac e ial bu den (Log10 CFU/lung) Fig. 1.Cou se o Bo de ella pe ussis in ec ion in expe imen al and con ol (C l) mice. G oups o mice we e killed a in e als a e challenge and he numbe o iable bac e ia es ima ed by pe o ming colony coun s on indi idual lung homogena es. Resul s a e ep esen a i e om wo expe i- men s and a e p esen ed as mean CFU in he lungs de e mined indi idually pe g oup om ou mice a each ime poin . Da a o C l and o albumin (OVA) g oups ha e been o se om ze o o cla i y. 4D. P. Ennis e al. CEA : 2042 2004 Blackwell Publishing L d, Clinical and Expe imen al Alle gy BWUK CEA 2042.PDF 19-Jul-04 19:1 844323 By es 10 PAGES UNCORRECTED PROOF mice challenged wi h B. pe ussis only, as in ec ion had esol ed by he 37 days ime-poin . OVA sensi iza ion esul ed in ypical inflamma ion o ai way walls wi h infil a ion o eosinophils, neu ophils and lymphocy es (Fig. 6c). Howe e , BpOVA mice displayed mo e se e e ai way wall inflamma ion wi h a g ea e deg ee o bo h epi helial hype plasia and mucous me aplasia han Bp, OVA o con ol mice (Fig. 6d). Gi en ha ai way esis ance (R)is in e sely p opo ional o he ou h powe o he ai way luminal adius ( 4 ) e en minimal na owing caused by p ocesses such as ansmu al inflamma ion, epi helial hype - plasia o mucus exuda ion subsequen o epi helial mucous me aplasia can p o oundly inc ease pulmona y esis ance as indica ed by ple hysmog aphy (Fig. 5). Discussion The p esen s udy demons a es ha p io in ec ion wi h iable B. pe ussis modula es he immune esponse induced by alle gen sensi iza ion. In ec ion supp esses an ibody and cell- C l Bp OVA BpOVA 0 1000 2000 3000 B. pe ussis-speci ic IgG C l Bp OVA BpOVA 0 1000 2000 3000 IgG1 IgG2a IgG2b IgG3 OVA-speci ic IgG C l Bp OVA BpOVA 0 10 20 30 * To al se um IgE (µg/mL) C l Bp OVA BpOVA 0 250 500 750 1000 * OVA-speci ic IgE (ng/mL) (a) (b) (c) (d) Fig. 2.Se um and o albumin (OVA)-speci ic IgE and IgG sub- classes elici ed by bac e ial in ec- ion and alle gic sensi iza ion. (a) Bo de ella pe ussis and (b) OVA- speci ic se um an ibody esponses by IgG subclass elici ed in con ol (C l), in ec ed (Bp), sensi ized (OVA) mice, o in mice in ec ed wi h B. pe ussis p io o sensi iza ion (BpOVA); exp essed as geome ic mean i e o an ibody (SE). (c) To al IgE and (d) OVA-speci ic IgE p esen in se a om each expe i- men al g oup exp essed as mg/mL o ng/mL, espec i ely. OVA-speci- ic IgE (ng/mL) is a ela i e mea- su e de e mined by adap a ion o a s anda d IgE ELISA. Resul s a e ep esen a i e o h ee expe imen s om ou animals pe o med inde- penden ly in iplica e. * S a is ical signi icance, Po0.001 compa ed wi h BpOVA- ea ed g oup. IL-5 C l Bp OVA BpOVA 0 250 500 750 1000 1250 1500 1750 2000 (a) Conc. IL-5 (pg/mL) Conc. IL-13 (pg/mL) IL-13 C l Bp OVA BpOVA 0 250 500 750 1000 C Bp OVA + (b) IFN - γ C l B p OVA B p OVA 0 200 400 600 800 1000 (c) Conc. IFN - γ γ (pg/mL) IL-10 C l Bp OVA BpOVA 0 200 400 600 800 1000 (d) Conc. IL-10 (pg/mL) Fig. 3.Cell-media ed immune esponses om spleen, elici ed by bac e ial in ec ion and alle gic sensi- iza ion. IL-5 (a), IL-13 (b), IFN-g(c), and IL-10 (d) esponses om spleen cell cul u es s imula ed wi h medium alone (nega i e con ol, e ical shading), hea inac i a ed Bo de ella pe ussis sonica e a 1 10 4 CFU/ mL (ha ched ba ), o albumin (OVA) (20 mg/mL) (open ba ) o Con A (posi i e con ol, black ba ). Re- sponses a e ep esen a i e o ipli- ca e expe imen s each assay was pe o med in iplica e on indi idual samples om ou mice pe g oup and esul s a e exp essed as mean (SE). Bo de ella pe ussis in ec ion 5 CEA : 2042 2004 Blackwell Publishing L d, Clinical and Expe imen al Alle gy BWUK CEA 2042.PDF 19-Jul-04 19:1 844323 By es 10 PAGES UNCORRECTED PROOF media ed esponses agains OVA locally and sys emically, while enhancing he le els o he egula o y cy okine IL-10 and IL-13. Despi e his modula ion, B. pe ussis exace ba es OVA-induced ai way pa hology, leading o he de elopmen o mo e p onounced alle gen-induced ai way inflamma ion as well as he induc ion o enhanced AHR. As hma is a ch onic inflamma o y disease o he ai ways, he p e alence o which has inc eased subs an ially in ecen decades [24, 25]. The explana ion ha has a ac ed mos a en ion is he hygiene hypo hesis, which sugges s ha he inc ease in alle gic disease is caused by a cleane en i onmen and ewe childhood in ec ions [26]. The goal o his s udy was o es he hypo hesis ha a powe ul Th1-media ed in ec ion such as B. pe ussis would diminish o p o ec in a mu ine model o alle gic as hma. Zuany-Amo im e al. [27] used a simila model, bu employed hea -killed Mycobac e - ium accae ha was e ec i e in blocking alle gic inflamma- ion bu by a mechanism independen o IFN-g. The same au ho s wen on o find ha mycobac e ia induce IL-10- p oducing egula o y T cells. In iguing ecen da a om McGui k e al. [15] ha e shown ha he filamen ous haemaglu inin componen o B. pe ussis beha es in a simila way. Ou obse a ion o IL-10 in la age fluid and ollowing in i o s imula ion o spleen cells suppo s he la e finding. In he case o he p o ec ion gene a ed by mycobac e ial exposu e, i is p oposed ha IL-10 has an essen ial ole in modula ing he immune sys em by inducing a shi om an alle gen-specific Th2 esponse [28]. In he p esen s udy, we obse e a simila modula ion bu his does no esul in p o ec ion agains ai way hype - eac i i y, implying ha al hough he es o a ion o a pu a i e balance be ween Th1 and Th2 is an a ac i e heo y, i is unlikely o p o ide a uni e sal explana ion o he pa hogenesis o as hma. S udies wi h Th1 and Th2 cells in diabe es melli us and au oimmune encephalomyeli is indica e ha c oss- egula ion does no always ope a e and in some ins ances can be unexpec edly ha m ul [29]. Fo example, Genain e al. [30] showed in a model o mul iple scle osis ha a shi in cy okine p oduc ion om a Th1 o a Th2 pa e n inc eased concen a ions o pa hogenic au oan ibodies and in some ins ances exace ba ed au oimmune disease. Pakala e al. [31] also showed ha immune de ia ion owa ds a Th2 esponse did no educe, bu a he exace ba ed pa hology and disease. In he p esen s udy, he Th2-associa ed e ec o OVA sensi iza ion had li le influence on he Th1-media ed clea ance o a bac e ial in ec ion o he ai ways. In con as , B. pe ussis in ec ion supp esses IgG and IgE esponses associa ed wi h OVA sensi iza ion. This p o ides compelling IL-4 C l Bp OVA BpOVA 0 10 20 30 40 50 60 (a) Conc. IL- 4 (pg/mL) IL-10 C l Bp OVA BpOVA 0 100 200 300 400 500 * (b) Conc. IL-10 (pg/mL) IL-13 C l Bp OVA BpOVA 0 100 200 300 400 500 600 700 * (c) Conc. IL-13 (pg/mL) IFN-γ C l Bp OVA BpOVA 0 250 500 750 (d) Conc. IFN-γ γ (pg/mL) Fig. 4.Bo de ella pe ussis in ec ion modula es he local cy okine esponse o alle gen. G oups o mice we e ea ed as desc ibed in he legend o Fig. 1. A 37 days, dilu ed b onchoal eola la age was pooled om i e mice pe g oup and concen a ions o IL-4 (a), IL-10 (b), IL-13 (c) and IFN-g(d) esponses de e mined by enzyme immunoassay. Resul s a e ep esen a i e o iplica e expe imen s, assays we e pe o med in iplica e, alues a e exp essed as mean SE be ween expe imen al and con ol g oups. * Po0.01 1 10 10 0 0 1 2 3 4 5 C l Bp OVA BpOVA MCh Conc. (m g /mL) Ai way eac i i y (PenH) Fig. 5.Bo de ella pe ussis exace ba es b onchial hype - esponsi eness o sensi izing an igen. G oups o mice we e ea ed as desc ibed in he legend o Fig. 1. A 37 days, ai way hype - eac i i y in esponse o inc easing concen a ions o inhaled me hacholine (MCh) was measu ed by whole-body ple hysmog aphy. Resul s a e ep esen a i e o h ee expe i- men s (n54 pe g oup) and alues a e exp essed as mean enhanced pause (PenH) SE. 6D. P. Ennis e al. CEA : 2042 2004 Blackwell Publishing L d, Clinical and Expe imen al Alle gy BWUK CEA 2042.PDF 19-Jul-04 19:1 844323 By es 10 PAGES UNCORRECTED PROOF e idence ha B. pe ussis powe ully modula es he esponse o he hi d pa y an igens. These da a a e again consis en wi h s udies in which M. accae injec ion o OVA-immunized mice significan ly supp essed se um IgE [32] bu a e in di ec con as o Zuany-Amo im e al. [27] in which hey ailed o see any e ec on IgG2a le els o se um IgE. Al hough M. accae and B. pe ussis p o oke simila immune esponses, adically di e en pa hologies a e induced. The esolu ion o his pa adox may lie in he di e en ae iologies o bo h M. accae and B. pe ussis. Respi a o y challenge by M. accae causes minimal epi helial damage and limi ed ai way pa hology. In s a k con as B. pe ussis causes significan damage o he epi helial lining o he ai ways [33]. This is media ed h ough a bac e ial i ulence ac o called acheal cy o oxin, which induces IL-1band eac i e ni ogen in e media es ha b ing abou cilios asis, ollowed by ai way emodelling [34, 35]. Thus, while M. accae and B. pe ussis induce e y simila immune esponses, i may be ha du ing B. pe ussis in ec ion he e is a combina ion o epi helial damage, IFN-g, IL-10 and IL-13 p oduc ion ha has p o ound influences on he epi helium and i s local en i onmen , which se es o exace ba e a he han p o ec agains as hma. In as hma, he b onchial epi helium is highly abno mal wi h s uc u al changes in ol ing sepa a ion o columna cells om hei basal a achmen s and unc ional changes esul ing in inc eased exp ession and elease o p o-inflamma o y cy okines and g ow h ac o s [36]. Benea h he damaged and dys unc ional epi helium lie inc eased numbe s o subepi he- lial myofib oblas s ha deposi in e s i ial collagens causing hickening o he basemen memb ane [4]. These e ec s a e seen in he p esen s udy in he OVA g oup (Fig. 6c) bu a e mo e p ominen in he combina ion g oup ha ecei ed B. pe ussis p io o OVA sensi iza ion (Fig. 6d). E idence sugges s ha he epi helium should no be iewed in isola ion, as ai way smoo h muscle cells con ibu e o he pe pe ua ion o ai way inflamma ion and ai way emodelling [37]. G un- s ein e al. [38] has sugges ed ha IL-10 may play an impo an ole in alle gic as hma by ac ing di ec ly on he sensi ized ai way smoo h muscle i sel . The p esen s udy sugges s ha al hough IL-10 may be a egula o y cy okine, i has b oade unc ions ha may no always p o ec agains inflamma o y disease, pa icula ly i he e has been damage o he supe ficial epi helium. Lee e al. [39] ha e also demons a ed ha IL-10 induces IL-13 p oduc ion in i o and ha his induc ion was esponsible o he mucus, bu no he inflamma o y and fib o ic e ec s o IL-10. This would appea o be consis en wi h ou own da a (Figs 3b and d, 4b and c) whe e we see an inc ease in bo h IL-10 and IL-13 a he sys emic and local le el. The dec ease in eosinophilia (Table Table 3.Leucocy es p esen in BAL luid G oup To al leucocy es (10 4 ) Eosinophils (10 4 ) Mac ophages (10 4 ) Lymphocy es (10 4 ) Neu ophils (10 4 ) Con ol 5.0 0.5 o0.005 3.9 2.0 0.2 0.1 o0.005 Bp 5.0 0.4 o0.005 4.6 0.9 1.1 0.7 0.2 0.1 OVA 8.0 1.7 4.1 0.9 2.9 1.2 0.6 0.1 0.5 0.2 BpOVA 5.0 0.8 2.4 0.8 * 2.1 0.8 0.4 0.1 0.5 0.3 G oups o mice we e killed (37 days), b onchoal eola la age (BAL) cells we e collec ed, coun ed, and cy ospin p epa a ions s ained o ob ain he di e en ial leucocy e coun . Da a ep esen mean (SE) alues; n55–8 mice pe expe imen . * Po0.05 s. OVA g oup. Fig. 6.Bo de ella pe ussis inc eases he se e i y o ai way pa hology o sensi izing an igen. Pho omic o- g aphs a–d illus a e ep esen a i e mo phological changes in ans e se sec ions o b onchioles a 37 days (n55 pe g oup). (a) Con ol g oup; (b) B. pe ussis in ec ed g oup (mild mu al and pe i-ai way in lamma ion e iden ); (c) o albumin (OVA)-sensi ized g oup illus a ing mode a e mu al and pe i-ai way in lamma ion wi h ac- companying mode a e mucus me aplasia (blue s aining goble cells) and hype plasia o epi helium; (d) Combined B. pe ussis/OVA- ea ed g oup illus a ing se e e mu al and pe i-ai way in lamma ion, mode a e epi helial hype - plasia and se e e mucous me aplasia wi h accompanying mucus plugging o he lumen (P). All sec ions s ained wi h a combined Discombes/Alcian blue s ain. O iginal mag- ni ica ion 400. Bo de ella pe ussis in ec ion 7 CEA : 2042 2004 Blackwell Publishing L d, Clinical and Expe imen al Alle gy BWUK CEA 2042.PDF 19-Jul-04 19:1 844323 By es 10 PAGES UNCORRECTED PROOF 2) obse ed in he BpOVA g oup is mos likely because o he modula ion o IL-5 esponses in hese mice (Fig. 3a). IL-5 is a cy okine necessa y o he egula ion o eosinophil g ow h, di e en ia ion, ac i a ion and su i al and plays a c i ical ole in he ec ui men o eosinophils o he lung [40]. Simila esponses we e obse ed by Wu e al. [41] using mu ine cy omegalo i us in ec ion in conjunc ion wi h he mu ine model o OVA-induced alle gic ai way disease. P e ious s udies in humans ha e demons a ed ha IL-13 mRNA and p o ein le els a e ele a ed in he lungs o a opic and non- a opic as hma ics [42, 43], sugges ing ha o e p oduc ion o IL-13 may p edispose owa d he de elopmen o bo h ypes o as hma [43]. Wal e e al. [44] showed ha an OVA-specific Th2 line gene a ed om IL-13 / mice, which p oduced high le els o IL-4 and IL-5, bu no IL-13, ailed o induce AHR, demons a ing he essen ial ole o IL-13 in he de elopmen o AHR. IL-13 may also inc ease AHR di ec ly. IL-13 induces smoo h muscle p oli e a ion in i o [45] and can aid con ac ions o acheal smoo h muscle [46]. Ai way smoo h muscle cells ha e also been shown o exp ess IL-13 ecep o s, including bo h componen s o he IL-13R complex [47]. Am ani e al. [48] ha e sugges ed ha inc eased le els o IFN-gin as hma ic indi iduals may p omo e AHR and exace ba e as hma by di ec ly modula ing con ac ile e- sponses. OVA sensi iza ion o in ec ed mice in he combina- ion g oup induced significan le els o IFN-gde ec able in he BALF (Fig. 4d), sugges ing ha IFN-gmay con ibu e o he obse ed exace ba ion o pa hology (Fig. 6d). This co ela es well wi h s udies in humans whe e inc eased IFN-g was seen in as hma ic pa ien s compa ed wi h no mal subjec s [49, 50]. I has also been shown ha AHR can mani es independen ly o pulmona y inflamma ion [51] al hough his was no seen in his s udy. Respi a o y syncy ial i us (RSV), commonly associa ed wi h lowe lung in ec ions in in ancy is also known o exace ba e as hma [52, 53]. Ma suse e al. [54] ound ha he e ec o RSV in ec ion a ies depending upon he inflamma o y con ex o he lung. Bo h p ima y and ecu en RSV in ec ions augmen ongoing alle gic inflam- ma ion, howe e , in he absence o alle gic sensi iza ion, he e ec s o RSV we e ansien . Compa able wi h ou own s udy wi h B. pe ussis, Lukacs e al. [55] ound ha an ini ial RSV in ec ion can ini ia e a p o-as hma ic en i onmen ha p omo es a mo e se e e as hma ic esponse, e en when he alle gic esponse is ini ia ed a a ime a e clea ance o he RSV-induced eac ions. Likewise, OVA sensi iza ion o mice in ec ed in a enously wi h Lis e ia monocy ogenes, con e s a non-le hal in ec ion o a le hal disease. In ha model IL-10 plays a c i ical ole in he supp ession o an i-lis e ial esis ance in OVA-immunized mice [56]. O he g oups ha e shown ha hea -killed Mycobac e ium bo is-BCG supp essed he de elopmen o OVA-induced ai way eosinophila [57, 58]. In many o hese s udies he iming o sensi iza ion and in ec ion influence he ou come, howe e , he exace ba ion media ed by B. pe ussis appea s o be pe sis en and long li ed (da a no shown). Taken oge he , hese s udies sugges ha pa hogens may induce an al e ed cy okine en i onmen in he con ex o ai way emodelling ha ul ima ely p o ides o an exace ba ed as hma ic- ype esponse [55]. Immune coun e - egula ion based on Th1/Th2 mechanisms o e en egula o y T cells sec e ing IL-10 ha e been sugges ed as mechanisms ha could p o ec agains as hma. We show he e ha his esponse mus be iewed in he b oade con ex o he hos –pa hogen in e ac ion. B. pe ussis ulfils many o he c i e ia o a po en immunomodula o ha should p o ec agains as hma. Howe e , he po en influence on ai way emodelling du ing in ec ion means ha his bac e - ium has he opposi e e ec . This s udy clea ly shows ha while he hygiene hypo hesis is an a ac i e heo y, i is o limi ed alidi y as cu en ly s a ed. Fu he mo e, ou esul s aise conce ns ega ding immunomodula o y he apies aimed a he con e sion o Th2-domina ed alle gic inflamma o y esponses in o Th1-domina ed esponses based on coun e - egula ion ha may be o limi ed e ficacy, o e en ha m ul. Acknowledgemen s We hank M s Sheila Wo ell, M Joseph B ady and M s Be nade e Ruane o hei expe echnical assis ance. This wo k was suppo ed by g an s om he I ish HEA PRTL p og amme (Da en Ennis). Be na d Mahon is a Wellcome T us /HRB new blood ellow (GR 054236). Re e ences 1 Ume su DT, McIn i e JJ, Akba i O, Macaubas C, DeK uy RH. As hma: an epidemic o dys egula ed immuni y. Na Immunol 2002; 3:715–20. 2 Wilde JA, Collie DD, Wilson BS, Bice DE, Lyons CR, Lipscomb MF. Dissocia ion o ai way hype esponsi eness om immuno- globulin E and ai way eosinophilia in a mu ine model o alle gic as hma. Am J Respi Cell Mol Biol 1999; 20:1326–34. 3 Ying S, Humbe M, Ba kans J e al. 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Ele a ed elease o umo nec osis ac o -alpha and in e e on-gamma by b onchoal eola leukocy es om pa ien s wi h b onchial as hma. Am Re Respi Dis 1993; 147:291–5. 51 Makela MJ, Kanehi o A, Bo ish L e al. IL-10 is necessa y o he exp ession o ai way hype esponsi eness bu no pulmona y Q8 Q9 Bo de ella pe ussis in ec ion 9 CEA : 2042 2004 Blackwell Publishing L d, Clinical and Expe imen al Alle gy BWUK CEA 2042.PDF 19-Jul-04 19:1 844323 By es 10 PAGES