Whole-cell pertussis vaccine protects against Bordetella pertussis exacerbation of allergic asthma

UNCORRECTED PROOF IMLET 4082 1–10 Immunology Le e s xxx (2004) xxx–xxx Whole-cell pe ussis accine p o ec s agains Bo de ella pe ussis exace ba ion o alle gic as hma 3 4 Da en P. Ennisaa, Joseph P. Cassidyb, Be na d P. Mahona,∗ 5 aMucosal Immunology Labo a o y, Ins i u e o Immunology, NUI Maynoo h, I eland6bDepa men o Ve e ina y Pa hology, Uni e si y College Dublin, Dublin, I eland7 Recei ed 23 Augus 2004; ecei ed in e ised o m 1 Oc obe 2004; accep ed 4 Oc obe 2004 8 Abs ac 9 The p e alence o as hma and alle gic disease has inc eased in many coun ies and he e has been specula ion ha immuniza ion p omo es alle gic sensi iza ion. Bo de ella pe ussis in ec ion exace ba es alle gic as hma ic esponses. We in es iga ed whe he whole-cell pe ussis accine (Pw) enhanced o p e en ed B. pe ussis induced exace ba ion o alle gic as hma. G oups o mice we e immunized wi h Pw, in ec ed wi h B. pe ussis and/o sensi ized o o albumin. Immunological, pa hological and physiological changes we e measu ed o assess he impac o Pw immuniza ion on immune de ia ion and ai way unc ion. Pw immuniza ion modula ed o albumin-speci ic se um IgE p oduc ion, and educed local and sys emic IL-13 and o he cy okine esponses o sensi izing alle gen. His opa hological examina ion e ealed Pw immuniza ion educed he se e i y o ai way pa hology and dec eased b onchial hype eac i i y o me hacholine exposu e. Pw does no enhance ai way IL-13 and consequen ly does no enhance bu p o ec s agains he exace ba ion o alle gic esponses. We ind no e idence o Pw con ibu ing o alle gic as hma, bu a he p o ide e idence o a mechanism whe eby whole-cell pe ussis accina ion has a p o ec i e ole. 10 11 12 13 14 15 16 17 18 19 © 2004 Else ie B.V. All igh s ese ed. 20 Keywo ds: Bo de ella pe ussis; Vaccine; As hma; IL-13; Alle gen 21 22 1. In oduc ion23 As hma is a ch onic disease o he espi a o y ac o in-24 c easing p e alence in de eloped socie ies [1]. The cu en 25 unde s andingo alle gicas hmais ha i esul s omab eak-26 down in he no mal ole ance o inhaled an igens, associa ed 27 wi h Th2 cy okine p oduc ion [2,3]. The in lamma o y e-28 sponse in as hma is igh ly associa ed wi h ai way hype e-29 sponsi eness, inc eased mucus p oduc ion and an in il a ion30 o he b onchial mucosa wi h CD4+ T-cells [4]. The e is e -31 idence o an al e ed local T-cell esponse in a ou o Th232 cy okine elease(IL-4,IL-5andIL-13) esul inginB-celliso- 33 ype swi ching o IgE, ec ui men o eosinophils, basophils 34 andmas cellsandp oduc iono in lamma o ymedia o s[5]. 35 ∗Co esponding au ho . Tel.: +353 1 7083835; ax: +353 1 7086337. E-mail add ess: [email protected] (B.P. Mahon). The mu ine OVA model o ai way hype esponsi eness ex- 36 hibi s many o he ea u es o human as hma, including ai - 37 way hype eac i i y, in lamma ion and inc eased se um IgE 38 le els [6,7]. Th2 cells sec e ing IL-4, IL-5, and IL-13 play a 39 cen al oleinini ia ingandsus aining heas hma ic esponse 40 in his model [8]. While Th2 cells p omo e ai way in lamma- 41 ion in as hma, i has been p oposed ha Th1 cells p o ec 42 agains alle gic disease by an agonizing Th2 ac i i y. In ec- 43 ious diseases ha induce Th1 ype esponses, migh hampe 44 he de elopmen o alle gen-speci ic Th2 cells and p e en 45 alle gy [9].46 Epidemiologicalandclinicals udiesha esugges edalink 47 be ween he ela i e absence o in ec ious diseases and he 48 inc ease in alle gic diso de s [10,11]; his is e e ed o as he 49 ‘hygienehypo hesis’.I p edic s ha in ec ionsp e en hein- 50 duc ion o alle gen-speci ic Th2 cells h ough an agonism o 51 heinduc iono egula o yT-cells,pa icula lydu ingneona- 52 10165-2478/$ – see on ma e © 2004 Else ie B.V. All igh s ese ed. 2doi:10.1016/j.imle .2004.10.011 UNCORRECTED PROOF IMLET 4082 1–10 2D.P. Ennisa e al. / Immunology Le e s xxx (2004) xxx–xxx al and ea ly childhood de elopmen [10,12]. Howe e , he e 53 a e da a ha con ound his in e p e a ion, inc eased IFN-␥54 is seen in as hma ic pa ien s compa ed wi h no mal subjec s55 [3,13]. Alle gen-speci ic Th1 cells also ail o coun e ac ai - 56 way hype esponsi eness in mu ine models [14]. Fu he -57 mo e, se e al s udies ha e sugges ed ha i al/bac e ial in-58 ec ions do no p o ec bu exace ba e disease. Respi a o y 59 syncy ial i us, commonly associa ed wi h lowe lung in-60 ec ions in in ancy, is known o exace ba e as hma [15,16].61 As does Bo de ella pe ussis [17]. Consequen ly, compe ing 62 in e p e a ions o he pa hogenesis o as hma ha e been p o-63 posed [18,19].64 B. pe ussis is a G am-nega i e bac e ium and he 65 causa i e agen o pe ussis o “whooping cough”, a espi- 66 a o y disease ha emains a signi ican cause o mo bidi y67 and mo ali y in in an s wo ldwide. I is a highly con agious 68 disease, and can occu a any age, hough se e e illness is69 mo e common in young un-immunized child en. B. pe ussis 70 in ec ion induces Th1 esponses [20,21] and can be mod-71 elled by espi a o y challenge o mice, which co ela es well72 o esponses in humans [22].73 The e has been specula ion abou he possible p omo ion74 o alle gy by common childhood accina ions [23,24].A75 subs an ial p opo ion o child en p edisposed o alle gy and 76 as hma may no be ully immunized because o public app e-77 hension su ounding immuniza ion [25]. A numbe o s ud- 78 ies ha e analysed he p e alence o alle gic sensi iza ion and 79 a opic disease in ela ion o immuniza ion [24,26]. G ube e 80 al. ound ha child en wi h highe immuniza ion co e age 81 seemed o acqui e ansien p o ec ion agains de elopmen 82 o a opy in he i s yea s o li e [26]. In con as , Hu wi z83 and Mo gens e n sugges ed ha diph he ia/pe ussis/ e anus84 (DTP) immuniza ion appea ed o be associa ed wi h an85 inc eased isk o subsequen as hma o o he alle gies86 [24]. 87 Two di e en ypes o pe ussis accine ha e been em- 88 ployed in in an immuniza ion p og ammes. The whole-cell 89 pe ussis accine (Pw) consis s o hea / o malin inac i a ed 90 i ulen whole bac e ia whe eas he pe ussis acellula ac- 91 cine (Pa) is composed o pu i ied componen s o he bac e ia92 (Pa), ypically including inac i a ed pe ussis oxin. Pw im-93 muniza ion has a high e icacy and is associa ed wi h he94 induc ion o an igen-speci ic Th1 cells [21,27,28], bu has95 been associa ed wi h eac ogenici y. In con as Pa immu- 96 niza ion induces a mixed Th1/Th2 esponse in child en and 97 in mu ine models, bu has educed eac ogenici y [29].I has 98 been sugges ed ha p omo ion o alle gy may occu di ec ly, 99 by adminis e ing po en ially p o-alle gic accines, o indi-100 ec ly, by hinde ing he Th1-p omo ing e ec o in ec ious101 agen s. Pe ussis accina ion ac s as an adju an o an igen-102 speci ic esponsesin labo a o yanimals[30];ac i epe ussis103 oxin, is known o enhance immunoglobulin E (IgE) o ma-104 ion in animal models [31] and has been linked wi h a shi 105 owa d Th2-like cy okines in humans [32,33]. 106 In ec ionwi hB.pe ussismodula esalle genp imingand107 he se e i y o ai way pa hology in a mu ine model o al- 108 le gic as hma [17] and we ha e p e iously shown ha Pw 109 immuniza ion induces a simila immune esponse o in ec- 110 ion [34] and ha al hough a iables such as ou e, dose 111 and iming in luence T-cell esponses in animal models, Pw 112 is a consis en induce o Th1 esponses [20]. In o de o 113 es whe he immuniza ion wi h Pw exace ba ed as hma, we 114 employed a well-cha ac e ized mu ine model o whole-cell 115 pe ussis accina ion and B. pe ussis in ec ion in combina- 116 ion wi h he mu ine OVA model o ai way hype esponsi e- 117 ness. We show ha al hough Pw induces a Th1 ype im- 118 mune esponse o B. pe ussis in ec ion, i does no exac- 119 e ba e pa hology in a model o alle gic as hma. Ou ind- 120 ings demons a e ha Pw immuniza ion p e en s B. pe us- 121 sis enhancemen o OVA-induced IL-10 and IL-13, which 122 esul s in a subsequen dec ease in ai way hype esponsi e- 123 ness and pa hology. This s udy inds no e idence o a mech- 124 anism o suppo specula ion linking Pw immuniza ion and 125 as hma. 126 2. Ma e ials and me hods 127 2.1. Animals and expe imen al app oach 128 Six- o 8-week-old emale BALB/c (Ha lan, UK) mice 129 we e used unde he guidelines o he I ish Depa men 130 o Heal h and he esea ch e hics commi ee o he Na- 131 ional Uni e si y o I eland Maynoo h. The expe imen al 132 app oach is ou lined in Table 1, b ie ly g oups o mice 133 we e immunized wi h whole-cell pe ussis accine (Pw), 134 in ec ed wi h B. pe ussis, and hen sensi ised o o albu- 135 min (OVA) a he peak o in ec ion as de ailed below. Con- 136 ol mice ecei ed simila ea men in which 0.9% (w/ ) 137 (aq) NaCl (he ea e e med Saline) eplaced expe imen al 138 ea men . 139 2.2. Immuniza ion, sensi iza ion and ai way deli e y o 140 OVA 141 Fou g oups o a leas hi y- i e 6–8-week-old emale 142 BALB/c mice (Pw, PwBp, PwOVA and PwBpOVA) we e 143 immunized i.p. wi h 0.16I.U. o whole-cell pe ussis accine 144 (Pw) (Thi d In e na ional S anda d, 1998, pe ussis whole- 145 cell accine, NIBSC, UK), equi alen o 1/25 h o he hu- 146 man dose acco ding o he schedule ou lined in Table 1.A 147 0 day mice we e in ec ed wi h B. pe ussis, selec ed g oups 148 we e hen sensi ized wi h o albumin (OVA). Sensi iza ion 149 in ol ed 100␮g OVA (G ade V; Sigma, Do se , UK) emul- 150 si ied in Alhyd ogel®adju an (Supe os Biosec o , Swe- 151 den) (1mg/mouse aluminium hyd oxide) adminis e ed as 152 0.2ml i.p. a 10 and 24 days. Con ol g oup (C l) ecei ed 153 saline alone (i.p.). On 35, 36, and 37 days, PwOVA and 154 PwBpOVA sensi ized mice ecei ed 10␮l con aining 50␮g155 OVA in a-nasally (i.n.) whe eas emaining g oups ecei ed 156 saline only (Table 1). All expe imen s we e epea ed a leas 157 wice. 158 UNCORRECTED PROOF IMLET 4082 1–10 D.P. Ennisa e al. / Immunology Le e s xxx (2004) xxx–xxx 3 Table 1 Expe imen al design Time (days) −42 −14 0 10 24 35/36/37 G oupa Pw Pw Pw Saline ae osol Saline (i.p.) Saline (i.p. and i.n.) Saline (i.n.) Pw and B. pe ussis in ec ion (PwBp) Pw Pw B. pe ussis ae osol in ec ion Saline (i.p.) Saline (i.p. and i.n.) Saline (i.n.) Pw and OVA sensi iza ion (PwOVA) Pw Pw Saline ae osol OVA (i.p.) OVA (i.p and i.n.) OVA (i.n.) Pw, B. pe ussis in ec ion and OVA sensi iza ion (PwBpOVA) Pw Pw B. pe ussis ae osol in ec ion OVA (i.p.) OVA (i.p and i.n.) OVA G oupb Con ol (C l) Saline (i.p.) Saline (i.p.) Saline ae osol Saline (i.p.) Saline (i.p. and i.n.) Saline (i.n.) B. pe ussis in ec ion (Bp) Saline (i.p.) Saline (i.p.) B. pe ussis ae osol in ec ion Saline(i.p.) Saline (i.p. and i.n.) Saline (i.n.) OVA sensi iza ion (OVA) Saline (i.p.) Saline (i.p.) Saline ae osol OVA (i.p.) OVA (i.p and i.n.) OVA (i.n.) B.pe ussis in ec ionandOVAsen- si iza ion (BpOVA) Saline (i.p.) Saline (i.p.) B. pe ussis ae osol in ec ion OVA (i.p.) OVA (i.p and i.n.) OVA (i.n.) aG oups o 6–8-week-old emale BALB/c mice we e immunized (i.p.) and boos ed wi h whole-cell pe ussis accine (Pw) a −42 and −14 days. On 0 day, mice we e ei he sham in ec ed o in ec ed wi h B. pe ussis (Bp) by ae osol. A 10 and 24 days selec ed g oups we e sensi ised o OVA by i.p alone o i.p and i.n. ou es, espec i ely. On 35, 36 and 37 days mice we e exposed o ei he saline o OVA by he i.n. ou e (i.e. 25, 26, and 27 days pos -OVA p iming and a e bac e ial clea ance). Bac e ial bu dens in he ai ways we e measu ed be ween 0 and 37 days. All o he eadou s, including ple hysmog aphy we e pe o med a 37 days. bFo compa ison, u he g oups o unimmunized (sham immunized) mice we e ea ed as ollows: con ol (C l) mice we e sham in ec ed on 0 day and sham sensi ised wi h saline. A second g oup (Bp) we e in ec ed wi h B. pe ussis a 0 day and sham sensi ized. The hi d g oup (OVA) we e sham in ec ed bu sensi ized wi h OVA (100␮g, i.p.) a 10 and 24 days and again (50␮g i.n.) a 24, 35, 36, and 37 days. A sepa a e g oup (BpOVA) we e in ec ed wi h B. pe ussis on 0 day, and sensi ized as abo e. Each expe imen was epea ed a leas wice, on each occasion n>35 mice pe g oup, bac e ial bu dens we e measu ed be ween 0 and 37 days, all o he eadou s we e a 37 days. 2.3. B. pe ussis ae osol in ec ion 159 Respi a o y in ec ion was ini ia ed by ae osol challenge160 wi h B. pe ussis s ain W28, ollowing g ow h unde agi a-161 ion condi ions a 37◦C in S aine -Schol e liquid medium.162 Bac e ia om a log-phase cul u e we e esuspended a a con-163 cen a ion o 2×1010 CFU/ml in 1% (w/ ) casein in 0.9%164 (w/ ) saline. The challenge inoculum was adminis e ed o165 g oups o mice on 0 day (Bp, PwBp and PwBpOVA g oups).166 Adminis a ion was by ae osol o e a pe iod o 15min using 167 a nebulize . G oups o ou o mo e mice we e killed a a i- 168 ous ime poin s a e ae osol challenge o assess he numbe 169 o iable B. pe ussis in he lungs. Remaining mice ecei ed 170 a simila ae osol o s e ile saline alone. 171 2.4. Enume a ion o iable bac e ia in he lungs 172 Lungs we e emo ed asep ically in o 1ml o s e ile phys-173 iological saline wi h 1% casein. Hund ed mic oli es o se i-174 ally dilu ed homogena e om indi idual lungs we e placed175 on o iplica e Bo de -Gengou aga pla es and he numbe o 176 CFU de e mined a e incuba ion a 37◦C o 4 days. Re-177 sul s a e epo ed as he mean numbe o B. pe ussis CFU178 (±S.E.M.) o indi iduallungs,eachde e minedin iplica e,179 om ou o mo e mice pe ime poin . All expe imen s we e180 epea ed wice. 181 2.5. B onchoal eola la age 182 B onchoal eola la age luids (BALF) we e ob ained by183 cannula ion o he achea ollowed by epea adminis a ion184 and aspi a ion o 0.5ml PBS pe mouse. This was pooled 185 om i e mice ( o al 2.5ml) pe expe imen al g oup. All ex- 186 pe imen s we e pe o med a leas wice. Dilu ed BALF was 187 assessed o he p esence o cy okines. 188 2.6. Measu emen o OVA and B. pe ussis-specific 189 an ibody 190 OVA and B. pe ussis-speci ic IgG1, 2a, 2b, and 3 p esen 191 in collec ed se a we e measu ed on day 37 by ELISA as p e- 192 iously desc ibed [35,36]. To al and OVA-speci ic IgE was 193 measu ed using a a an i-mouse IgE monoclonal an ibody 194 (BD, Pha mingen, San Diego, CA, USA). The IgE concen- 195 a ion was exp essed as ␮g/ml a e compa ison o mu ine 196 IgE s anda ds. 197 2.7. T-cell p oli e a ion assays 198 Spleen cells (2×106/ml) om in ec ed, sensi ized and 199 con ol mice (n=4 o mo e pe g oup) we e es ed o 200 in i o p oli e a ion agains hea -inac i a ed B.pe ussis 201 (1×104CFU/ml), OVA (20␮g/ml), Concana alin A (Con 202 A) (5␮g/ml, posi i e con ol), o medium alone (nega i e 203 con ol). A e 72h, cell p oli e a ion was assessed by liquid 204 scin illa ion coun ing o [3H]- hymidine inco po a ion and 205 esul swe eexp essedasmeanCPMo iplica ewells±S.E. 206 A he 72h ime poin , cul u e supe na an s we e sampled o 207 cy okine analysis, al hough he kine ics o cy okine p oduc- 208 ion a ies his ime poin has p e iously p o ed accep able 209 o de ec ion o mos cy okines [22].210 UNCORRECTED PROOF IMLET 4082 1–10 4D.P. Ennisa e al. / Immunology Le e s xxx (2004) xxx–xxx 2.8. Cy okine measu emen 211 Concen a ions o IL-5, IL-10, IL-13 and IFN-␥ om 212 spleen, and BALF we e assessed by ELISA (BD, Pha min-213 gen, San Diego, CA, USA). Cy okine concen a ions we e 214 calcula ed by compa ison wi h known cy okine s anda ds as215 p e iously desc ibed [35], all de e mina ions we e made in216 iplica e, esul s a e p esen ed as mean cy okine concen a-217 ion (±S.E.M.). 218 2.9. Whole body ple hysmog aphy 219 Ai way esponsi enesson37dayswasassessedbyme ha- 220 choline (MCh) induced ai low obs uc ion om conscious221 mice using whole-body ple hysmog aphy (Buxco Elec on-222 ics, Sha on, CT, USA) as p e iously desc ibed [37]. Pul- 223 mona yai lowobs uc ionwasmeasu edbyenhancedpause224 (PenH), a alue de e mined om he a io o expi a o y ime225 and elaxa ion ime o peak expi a o y low and peak inspi-226 a o y low and hough o co ela e wi h ai way esponsi e-227 ness. Measu emen s we e ob ained a e exposu e o mice228 o 3min o PBS (baseline) ollowed by inc emen al doses 229 (3.3mg–50mg/ml) o MCh deli e ed by ae osol [38]. 230 2.10. Respi a o y ac his ology231 Animals (n=5 pe g oup pe expe imen ) we e sac i iced232 a 37 days. Lungs we e emo ed, ixed in a pa a o malde-233 hyde/lysine/pe ioda e ixa i e, pa a in embedded, sec ioned234 and s ained using he haema oxylin and eosin (H&E), Dis-235 combes (iden i ica ion o eosinophils), alcian blue (iden- 236 i ica ion o mucus), PAS (assessmen o basemen mem- 237 b ane hickness), azu e-A (iden i ica ion o mas cells) and 238 Van Gieson (iden i ica ion o ib osis) me hods. His opa ho- 239 logical changes e iden we e g aded acco ding o a semi-240 quan i a i e sco ing sys em as mild, mode a e o se e e by241 wo esea che s wi hou p io knowledge o he ea men 242 g oup using a p e iously es ablished sco ing sys em [17].243 All expe imen s we e pe o med a leas wice.244 2.11. S a is ical me hods 245 Resul s a e exp essed as he mean±S.E.M. o he indi-246 ca ednumbe o animals.AS uden ’s - es wasused ode e - 247 mine signi icance among he g oups. A alue o P<0.05 was248 conside ed signi ican . Analyses we e pe o med using he249 G aph-Pad P ismTM so wa e (G aphPad, San Diego, CA).250 3. Resul s 251 3.1. O albumin sensi iza ion does no impai 252 accine-media ed clea ance o B. pe ussis253 The mu ine OVA model o ai way hype esponsi eness 254 induces a powe ul Th2 esponse [7] whe eas bo h B. pe -255 Fig. 1. Cou se o B. pe ussis in ec ion in expe imen al and con ol mice. G oups o mice we e sac i iced 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 imen s and a e p e- sen ed as mean (±S.E.M.) CFU in he lungs, de e mined indi idually om ou mice a each ime poin , and o each expe imen al g oup. Da a o C l and OVA g oups ha e been o se om ze o o cla i y. ussis in ec ion and Pw immuniza ion induce a powe ul Th1 256 esponse [29]. In o de o examine immune c oss- egula ion 257 and po en ial in e e ence wi h immuniza ion, we es ed he 258 e ec o OVA sensi iza ion upon he de elopmen o a p o- 259 ec i e esponse o in ec ion in Pw immunized and non- 260 immunized mice. Mice ecei ed combina ions o OVA sen- 261 si iza ion, Pw immuniza ion and ae osol challenge wi h i - 262 ulen B. pe ussis (Table 1). G oups o mice in ec ed wi h 263 B. pe ussis (Bp and BpOVA) showed simila kine ics o 264 bac e ial clea ance (Fig. 1), indica ing ha OVA sensi iza- 265 ion does no in luence bac e ial clea ance a es. Likewise, 266 OVA sensi ized and non-sensi ized mice ha had been im- 267 munized p io o bac e ial challenge (PwBpOVA and PwBp, 268 espec i ely) showed iden ical kine ics o clea ance. No bac- 269 e ia we e eco e ed om he OVA sensi ised o con ol 270 (C l) g oups, which we e unin ec ed bu ecei ed saline by 271 ae osol (Fig. 1). The bac e ial bu den in he Bp and BpOVA 272 g oups peaked a 10 days and declined he ea e . Pw im- 273 munized mice clea ed subsequen in ec ion by B. pe us- 274 sis by 7 days. In con as unimmunized mice (Bp and he 275 BpOVA g oups) only showed comple e bac e ial clea ance 276 by 35 days (Fig. 1). The e o e sensi iza ion wi h OVA did 277 no impai accine-media ed clea ance o B. pe ussis in his 278 model. 279 3.2. OVA-specific IgE p oduc ion is modula ed by Pw 280 immuniza ion 281 The goal o his s udy was o examine he in luence o 282 Pw immuniza ion on esponses associa ed wi h alle gic sen- 283 si iza ion. Al hough OVA-induced sensi iza ion does no im- 284 pai accine-media ed clea ance o B. pe ussis, i was pos- 285 sible ha Pw in luenced alle gic sensi iza ion. OVA-speci ic 286 IgG was no de ec ed om mice in ec ed wi h B. pe ussis 287 UNCORRECTED PROOF IMLET 4082 1–10 D.P. Ennisa e al. / Immunology Le e s xxx (2004) xxx–xxx 5 Fig. 2. Se um IgE and IgG subclasses elici ed by Pw accina ion, bac e ial in ec ion and alle gic sensi iza ion. (A) B. pe ussis o (B) OVA-speci ic se um an ibody esponses by IgG subclasses elici ed in whole-cell B. pe ussis (Pw), accina ed plus Bp in ec ion (PwBp), accina ed plus sensi ized (PwOVA), o in mice accina ed hen in ec ed wi h B. pe ussis p io o sensi iza ion (PwBpOVA), esponse om mice ha ecei ed OVA sensi iza ion alone (OVA) o combined wi h in ec ion (BpOVA) a e shown o compa ison. (C) B. pe ussis-speci ic se um IgE and (D) OVA-speci ic se um IgE p esen om each expe imen al g oup. Resul s a e ep esen a i e o wo expe imen s exp essed as geome ic mean i e o ng/ml o an ibody (±S.E.M.) om ou animals each de e mined independen ly in iplica e. *P<0.05 compa ed o he PwOVA ea ed g oup. only; simila ly B. pe ussis-speci ic IgG could no be de-288 ec ed in OVA sensi ized animals, sugges ing no signi ican 289 c oss- eac ion be ween he wo immunogens (Fig. 2A and 290 B). An analysis o he se um an ibody subclasses e oked291 e ealed ha Pw p edominan ly induced pe ussis-speci ic292 IgG2a (Fig. 2A) as p e iously epo ed [28,34]. IgG2a e-293 mained he dominan subclass o pe ussis-speci ic an i- 294 body when Pw immuniza ion was combined wi h OVA sen-295 si iza ion (PwOVA) o B. pe ussis in ec ion (PwBp) al- 296 hough IgG1 and IgG3 was de ec ed in he la e g oups297 (Fig. 2A). Pw immuniza ion p io o OVA sensi iza ion298 (PwOVA) did no signi ican ly al e he dominan OVA-299 speci ic IgG1 esponse. In ec ion o hese mice wi h B. pe -300 ussis (PwBpOVA) did no b oaden he subclasses o OVA-301 speci ic an ibody de ec ed al hough he i e was inc eased302 (Fig. 2B). Taken oge he , hese da a indica e ha Pw im-303 muniza ion does no modula e se um IgG subclasses in-304 duced by alle gic sensi iza ion. In con as o he conse -305 a i e e ec s on se um IgG subclasses, signi ican di e -306 ences we e obse ed in he induc ion o IgE (Fig. 2C and 307 D). Pw induces li le B. pe ussis-speci ic IgE, and his is 308 no al e ed by OVA sensi iza ion o in ec ion (Fig. 2C). 309 OVA sensi iza ion induces high le els o OVA-speci ic IgE 310 bu his is signi ican ly educed by p io Pw immuniza ion 311 (Fig. 2D). Howe e , a combina ion o immuniza ion and in- 312 ec ion p io o OVA sensi iza ion esul ed in a signi ican e-313 duc ion o IgE (P<0.05) (Fig. 2D) compa ed o he PwOVA 314 g oup.315 3.3. Pw immuniza ion p e en s B. pe ussis 316 enhancemen o OVA-induced IL-10 and IL-13 317 B. pe ussis in ec ion enhances OVA-induced IL-10 and 318 IL-13 [17]. Pw immuniza ion has hi he o been ega ded as 319 inducing essen ially simila immune esponses o hose in- 320 duced by in ec ion [22]. In o de o dissec he in luence o 321 immuniza ion on ai way hype esponsi eness, we examined 322 cell-media edimmune esponses in he a iouss udy g oups. 323 Pw immuniza ion alone o in combina ion wi h B. pe ussis 324 in ec ion (Pw o PwBp) induced e y li le IL-5 bu s ong 325 IFN-␥ esponses (Fig. 3A and B). This was consis en wi h 326 he p o ec ion obse ed ea lie (Fig. 1) and p e ious da a 327 [34]. Pw immuniza ion educed le els o IL-5, IL-13 and 328 IFN-␥(Fig. 3A–C) in all immunized g oups sugges ing ha 329 Pw immuniza ion p e en s li e B. pe ussis enhancemen o 330 hese indices; mi o ing he educ ion in OVA-speci ic IgE 331 (Fig. 2D). In e es ingly, p e ious esul s ha e shown ha B. 332 pe ussis in ec ion induced speci ic IL-10 as well as IL-13 333 esponses [17]. He e we demons a e ha in con as o in- 334 ec ion, Pw-immuniza ion esul ed in signi ican ly educed 335 le els o IL-10, and IL-13 (Fig. 3A–D). 336 To ex end hese indings, we examined he le els o cy- 337 okines p esen in b onchoal eola la age luid (BALF) om 338 each g oup o mice. Pw immuniza ion alone induced li le o 339 no de ec able cy okines in BALF. As expec ed, OVA sensi i- 340 za ion induced high le els o IL-5, -10 and -13 bu he le els 341 o IL-10 and -13 in pa icula , known o ise in in ec ed mice, 342 UNCORRECTED PROOF IMLET 4082 1–10 6D.P. Ennisa e al. / Immunology Le e s xxx (2004) xxx–xxx Fig.3. Cellmedia edimmune esponses omspleen,elici edbyPw accina ion,bac e ialin ec ionandalle gicsensi iza ion.IL-5(A),IFN-␥(B),IL-13(C)and IL-10 (D) esponses om spleen cell cul u es s imula ed wi h medium alone (− e con ol, ho izon al shading), hea inac i a ed B. pe ussis a 1×104CFU/ml (ha ched ba ), OVA (open ba ) o Con A (+ e con ol, black ba ). Responses a e ep esen a i e o duplica e expe imen s each pe o med in iplica e on indi idual samples om ou mice pe g oup and a e exp essed as mean (±S.E.M.). *P<0.05 compa ed o he PwOVA ea ed g oup. we e educed in immunized mice ha had been in ec ed wi h343 B. pe ussis (PwBpOVA) (Fig. 4A–D).344 3.4. Pw immuniza ion p io o B. pe ussis in ec ion345 dec eases b onchial hype esponsi eness o sensi izing346 an igen 347 I has been p oposed ha p io Th1 esponses o bac e- 348 ial in ec ions p o ec agains alle gic disease howe e , Th1-349 inducing B. pe ussis in ec ion exace ba es ai way hype e-350 sponsi eness in OVA sensi ized mice. I migh be p edic ed351 ha Pw, which induces a e y simila immune esponse o B.352 pe ussiswouldha easimila exace ba ingin luence.In ac , 353 his is no he case. We used whole body ple hysmog aphy as 354 a su oga e measu e o ai way eac i i y in mice immunized 355 wi h Pw and in ec ed wi h B. pe ussis p io o OVA sensi i- 356 za ion(Fig.5).P io immuniza ionwi hPwdoesno enhance 357 bu p o ec s agains B. pe ussis exace ba ed ai way hype e-358 sponsi eness in compa ison o con ols. S a is ical analysis 359 using wo-way analysis o a iance (ANOVA) showed ha 360 mice accina ed wi h Pw, and sensi ized o OVA ollowing 361 B.pe ussisin ec ion(PwBpOVA)displayedsigni ican ly e-362 duced b onchial hype eac i i y compa ed o BpOVA sensi- 363 ized animals (P<0.05) (Fig. 5D). Thus demons a ing ha 364 accina ion wi h Pw p o ec s agains B. pe ussis exace ba- 365 ion o alle gic as hma. 366 B. pe ussis in ec ion is known o modula e he quali y 367 o he in lamma o y in lux o he espi a o y ac , wi h a 368 ma ked educ ion in eosinophil numbe s accompanied by 369 a ying deg ees o epi helial hype plasia, mucus me aplasia, 370 and ai way pa hology [17]. Lung issue was assessed his o- 371 logically (Table 2). Minimal pa hology was obse ed in mice 372 immunized wi h Pw o hose immunized and in ec ed wi h 373 B. pe ussis (PwBp) (Fig. 6A and B). Pw and OVA sensi ized 374 (PwOVA) mice illus a ed mode a e mu al and pe i-ai way 375 in lamma ionwi haccompanyingmildmucusme aplasiaand 376 mode a e hype plasia o he epi helium (Fig. 6C). The com- 377 bina iono Pwimmuniza ion,B.pe ussisin ec ionandOVA 378 sensi iza iondid no showenhancedpa hology bu onlymod- 379 e a e mucus me aplasia and mode a e hype plasia o he ep- 380 i helium (Fig. 6D). Gi en ha p e ious wo k has shown ha 381 B. pe ussis in ec ion in combina ion wi h OVA sensi iza ion 382 (BpOVA) displayed mo e se e e ai way in lamma ion wi h 383 a g ea e deg ee o bo h epi helial hype plasia and mucous 384 me aplasia, i can be clea ly seen he e ha Pw immuniza- 385 ion educes he se e i y o ai way pa hology (Fig. 6D) and 386 UNCORRECTED PROOF IMLET 4082 1–10 D.P. Ennisa e al. / Immunology Le e s xxx (2004) xxx–xxx 7 Fig. 4. Pw immuniza ion modula es he local cy okine esponse o B. pe ussis in ec ion and OVA sensi iza ion. Dilu ed BALF (0.5ml pe mouse) was pooled om i e mice pe g oup and concen a ions o IL-5 (A), IFN-␥(B), IL-13 (C) and IL-10 (D) we e de e mined by EIA. Resul s a e ep esen a i e o duplica e expe imen s. Cy okine concen a ions we e assayed in iplica e wi h alues exp essed as mean cy okine concen a ion in dilu ed BALF (±S.E.M.). *P<0.05 compa ed o he PwOVA ea ed g oup. educes pulmona y esis ance as indica ed by ple hysmog a- 387 phy (Fig. 5D). 388 4. Discussion389 The p esen s udy demons a es ha Pw immuniza ion 390 p o ec s agains B. pe ussis exace ba ion o OVA-induced391 Table 2 His ological assessmen o ai way pa hology T ea men g oup Mucous me aplasia o ai way epi helium Hype -plasia o ai way epi helium Smoo h muscle hype ophy o ai way wall Pe i-ai way/ ascula in lamma iona O e all deg ee E N L M F C l −−−−−−−−− Bp −+++−+++−− O a++++++++ b+++++ c−− BpOVA +++ +++ ++ +++b++ +++ +++c−− Pw −−−−−−−−− PwBp −++−−−−−− PwOVA + ++ + ++b++ ++ ++c−− PwBpOVA ++ ++ ++ ++b++ ++ ++c−− Asemi-quan i a i e sco e(−absen , + mild, ++ mode a e,+++ se e e)wasassigned o ea u es o ai way pa hologyobse ed acco ding o p e iously desc ibed c i e ia [17]. aPe i-ai way/ ascula in lamma ion was 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). bIn lamma ion ex ending in o su ounding pulmona y in e s i ium and al eola spaces. cMac ophage gian cells o m pa o in lamma o y exuda es wi hin su ounding al eola spaces. ai way hype esponsi eness in a mu ine model. Pw immu- 392 niza ion supp esses an ibody and cell media ed esponses 393 agains OVA sensi iza ion in combina ion wi h B. pe ussis 394 in ec ion a bo h he local and sys emic le el, coupled wi h 395 a subsequen educ ion in ai way eac i i y and pa hology. 396 I has been p e iously shown ha B. pe ussis exace ba es 397 OVA-induced ai way pa hology leading o he de elopmen 398 o mo e p onounced alle gen-induced ai wayin lamma ion UNCORRECTED PROOF IMLET 4082 1–10 8D.P. Ennisa e al. / Immunology Le e s xxx (2004) xxx–xxx Fig. 5. Pw immuniza ion p io o B. pe ussis in ec ion dec eases b onchial hype esponsi eness o sensi izing an igen. 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. (A) Con ol and Bp in ec ed mice (B), Pw and PwBp (C), OVA and PwOVA (D), BpOVA and PwBpOVA g oups, espec i ely. Resul s a e ep esen a i e o wo expe imen s (n=4) and alues a e exp essed as mean enhanced pause (PenH)±S.E.M., in g oups whe e no e o s a e isible, e o ba s a e sho e han he size o he da a poin symbol. [17]. We demons a e ha Pw immuniza ion p o ec s agains 399 his as well as educing ai way hype esponsi eness.400 The p e alence o as hma and alle gic disease has in- 401 c eased in many coun ies [39,40] and he e has been spec- 402 ula ion as o possible causes [41,42], including he possible 403 oleo immuniza ioninp omo ingalle gicsensi iza ion[43]. 404 Fo example, pe ussis accina ion ac s as an adju an o 405 an igen-speci ic esponses in labo a o y animals [30,33];a406 speci ic IgE esponse o pe ussis oxin i sel has been iden-407 i ied in child en ecei ing pe ussis immuniza ion [44]; and408 accina ion wi h some o he o ganisms such as Haemophilus409 influenzae enhances his amine elease in labo a o y animals 410 [45]. Ac i e pe ussis oxin has a simila e ec [46].Inad- 411 di ion, wo s udies ha e sugges ed ha pe ussis in ec ion 412 inc eased he isk o a opy [47,48]. I is he e o e heo e i- 413 cally possible ha Pw immuniza ion migh con ibu e o he414 de elopmen o alle gic disease.415 The goal o his s udy was o es whe he immuniza ion416 wi h whole-cell pe ussis (Pw) accine would p o ec agains 417 B. pe ussis exace ba ion o alle gic as hma. B. pe ussis in-418 ec ionmodula esalle genp imingand hese e i yo ai way419 pa hology in a mu ine model[17]. I has been p oposed ha 420 IL-10 plays an essen ial ole in modula ing he immune e-421 sponses by inducing owa ds egula o y T-cell esponses[49] 422 howe e Leee alha edemons a ed ha IL-10inducesIL-13 423 p oduc ion in i o and ha his is esponsible o he mucus, 424 bu no he in lamma o y/ ib o ic e ec s o IL-10 [50].In 425 he p esen s udy, we obse e ha Pw immuniza ion p e en s 426 induc ion o IL-10 and IL-13 and p o ec s agains ai way hy- 427 pe eac i i y. Al hough IL-10 is known o ac in an immune 428 egula o y manne , we and o he s ha e sugges ed ha i has 429 b oade unc ions ha mayno alwaysp o ec agains in lam- 430 ma o y disease [17,51]. Fo example, G uns ein e al. ha e 431 sugges ed ha IL-10 may play an impo an ole in alle gic 432 as hma by ac ing di ec ly on he ai way smoo h muscle i - 433 sel [51]. P e ious s udies in humans ha e demons a ed ha 434 IL-13 mRNA and p o ein le els a e ele a ed in he lungs o 435 a opic and non-a opic as hma ics [52] sugges ing ha o e 436 exp ession o IL-13 may p edispose owa d he de elopmen 437 o bo h ypes o as hma [53]. The educ ion in bo h IL-10 438 and IL-13 a he sys emic and local le els sugges s ha by 439 emo ing hedamaginge ec so pe ussisin ec ionandcon- 440 sequen ly educing IL-10 and IL-13, Pw exe s a p o ec i e 441 e ec .In e es ingly, e y ecen wo kbyKim e al has shown 442 ha componen s o B. pe ussis can inhibi ai way hyepe e- 443 sponsi eness [54]. Tha s udy demons a ed ha unme hy- 444 la ed CpG sequences om B. pe ussis DNA inhibi ed Th2 445 cy okines in he ai ways ia a TLR9 in e ac ion [54]. Ou 446 UNCORRECTED PROOF IMLET 4082 1–10 D.P. Ennisa e al. / Immunology Le e s xxx (2004) xxx–xxx 9 Fig. 6. Whole-cell B. pe ussis accine educes he se e i y o ai way pa hology o sensi izing an igen bo h in he p esence and absence o B. pe ussis in ec ion. Rep esen a i e mo phological changes a 37 days in ans e se sec ions o b onchioles om (A) Pw immunized mice showing no changes e iden ; (B) Pw/Bp mice, minimal changes e iden ; (C) Pw/OVA ea ed mice, illus a ing mode a e mu al and pe i-ai way in lamma ion wi h accompanying mild mucous me aplasia (blue s aining goble cells) and mode a e hype plasia o epi helium; (D) Combined Pw/Bp/OVA ea ed g oup, illus a ing a mode a e mu al andpe i-ai wayin lamma ion, mode a emucousme aplasiaandmode a eepi helial hype plasia; (E)OVAsensi izedg oup illus a ing mode a emu al andpe i- ai way in lamma ion wi h accompanying mode a e mucus me aplasia (blue s aining goble cells) and hype plasia o epi helium; (F) 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. All sec ions a e ep esen a i e o g oups o i e mice pe expe imen , pe o med a leas wice. Sec ions s ained wi h a combined Discombe’s/Alcian blue s ain, o iginal magni ica ion 400×. obse a ion o educed in lamma ion and OVA-speci ic IgE 447 (Figs. 2 and 6) in PwBpOVA mice is consis en wi h ha 448 inding. TLR9 media ed educ ion in Th2 cy okines can be449 in oked in his si ua ion, because al hough hese mice will be450 exposed obac e ialDNAinacon ex whe ep io immuniza-451 ion will limi issue damage o he o he immunomodula o y452 e ec s o iable B. pe ussis [17,54].453 The mos common o mula ion o he hygiene hypo hesis454 is based upon a lack o immunological s imuli in ea ly in-455 ancy ha esul s in abe an Th2 esponses [10,12]. Clea ly456 in an pe ussis immuniza ion o in ec ion could po en ially457 in luence his p ocess. S udies in neona al mice suppo ou 458 indings ha Pwisas ongTh1induce ,bu i is lesse ec i e 459 ininducingan ibody esponses du ing his pe iod [28,55,56].460 A s udy by G ube e al. e ealed no e idence o an alle gy 461 p omo inge ec o commonchildhood accinesinap ospec- 462 i ely ollowed a opy isk-enhanced bi h coho [26]. Mo e- 463 o e , hey ound ha child en wi h a be e accina ion co - 464 e age seemed o be be e p o ec ed agains he de elopmen 465 o a opy in hei second and hi d yea s o li e. In pa icu- 466 la , measles/mumps, pe ussis, and diph he ia/ e anus immu- 467 niza ion we e associa ed wi h a ansien educ ion o a opy, 468 whe eas immuniza ion agains polio and H. influenzae had 469 no e ec . Fu he mo e, immuniza ion o child en wi h Pw 470 down egula ed he IgE esponse o co-adminis e ed diph he- 471 ia and e anus oxoids [57]. In con as , a s udy in ol ing he 472