The Journal of Immunology

Neonatal Exposure to Pneumococcal PhosphorylcholineModulates the Development of House Dust Mite Allergyduring Adult Life

Preeyam S. Patel and John F. Kearney

Currently, ∼20% of the global population suffers from an allergic disorder. Allergies and asthma occur at higher rates in

developed and industrialized countries. It is clear that many human atopic diseases are initiated neonatally and herald more

severe IgE-mediated disorders, including allergic asthma, which is driven by the priming of Th2 effector T cells. The hygiene

hypothesis attempts to link the increased excessively sanitary conditions early in life to a default Th2 response and increasing

allergic phenomena. Despite the substantial involvement of IgE Abs in such conditions, little attention has been paid to the effects

of early microbial exposure on the B cell repertoire prior to the initiation of these diseases. In this study, we use Ab-binding assays

to demonstrate that Streptococcus pneumoniae and house dust mite (HDM) bear similar phosphorylcholine (PC) epitopes. Neo-

natal C57BL/6 mice immunized with a PC-bearing pneumococcal vaccine expressed increased frequencies of PC-specific B cells in

the lungs following sensitizing exposure to HDM as adults. Anti-PC IgM Abs in the lung decreased the interaction of HDM with

pulmonary APCs and were affiliated with lowered allergy-associated cell infiltration into the lung, IgE production, development of

airway hyperresponsiveness, and Th2 T cell priming. Thus, exposure of neonatal mice to PC-bearing pneumococci significantly

reduced the development of HDM-induced allergic disease during adult life. Our findings demonstrate that B cells generated

against conserved epitopes expressed by bacteria, encountered early in life, are also protective against the development of allergic

disease during adult life. The Journal of Immunology, 2015, 194: 000–000.

In the past few decades, there has been a dramatic rise in theincidence of asthma and other atopic diseases among indi-viduals living in developed countries (1, 2). The hygiene

hypothesis (1) proposes that this increasing incidence may resultfrom a decreased frequency of childhood infection and perinatalexposure to microbes, leading to a long-lasting imbalance betweenthe Th1 and Th2 T cell subsets initiated at this early stage of life(3). However, empirical data supporting such a mechanism areconflicting (4, 5). We previously demonstrated that, in early life,the B cell repertoire diversity is more amenable to change bybacterial exposure than it is during adult life (6); however, little isknown about the long-term effects of such exposure on allergicdisease initiation. Increasing evidence suggests that primary sensi-tization to environmental Ags occurs early in life, but airway dis-ease may not develop until after elements of the respiratory immunesystem functionally mature (7). Because evidence is mounting that

the possibility of reversing the disease declines with time after onset(8), early therapeutic intervention is essential to achieve this goal.Approximately 40% of individuals with allergic rhinitis, the most

common allergic disease among adults (9), and 89% of asthmaticsdemonstrate sensitivity to indoor allergens derived from the housedust mite (HDM) species Dermatophagoides pteronyssinus (Der p)(10, 11). More than 75% of these individuals express IgE-mediatedsensitivity to the protease allergen Der p 1 (12). We and others haveobserved that HDM contains phosphorylcholine (PC) epitopes (13,14) similar to those integrated into the cell wall of Streptococcuspneumoniae (pneumococcus) bacteria (15). In mice, naturalTEPC15 (T15) idiotype-bearing natural anti-PC Abs generated bythe B1a B cell subset (16) are germline encoded and are protectiveagainst the development of pneumococcal disease and atheroscle-rosis (17, 18). These observations, and our previous studies on al-lergic airway responses to the fungus Aspergillus fumigatus (19),suggested that B cells and Abs with PC specificity might also beprotective against HDM-induced allergic disease development.In the current study, we investigated the effects of neonatal (day 3

of life) bacteria-associated PC exposure on the later induction ofHDM-induced allergic disease during adult life. Analysis of thesemice demonstrated that there was a broad decrease in cellular andhumoral mediators of allergic disease following challenge withHDM. The results we present argue strongly for a central role ofB cells, and their Ab products, in the protection against the de-velopment of HDM-induced allergic airway disease.

Materials and MethodsAnimals

C57BL/6 and mMT mice purchased from The Jackson Laboratory andTEPC15 (T15) IgH gene knock-in (KI) mice, initially generated in thelaboratory of K. Rajewsky (University of Cologne) (20), were maintainedunder specific pathogen-free conditions using approved animal protocolsfrom the Institutional Animal Care and Use Committee at the University ofAlabama at Birmingham.

Department of Microbiology, University of Alabama at Birmingham, Birmingham,AL 35294

Received for publication February 3, 2015. Accepted for publication April 15, 2015.

This work was supported by National Institutes of Health Grants AI14782-36 andAI100005-02 and initiated with support from a Senior Investigator Award from theAmerican Association for Asthma Research, previously the Sandler Program forAsthma Research.

Address correspondence and reprint requests to Dr. John F. Kearney, University ofAlabama at Birmingham, 1825 University Boulevard, Suite 410, Birmingham, AL35294. E-mail address: jfk@uab.edu

The online version of this article contains supplemental material.

Abbreviations used in this article: AHR, airway hyperresponsiveness; BALF, bron-choalveolar lavage fluid; DC, dendritic cell; HDM, house dust mite; i.t., intratracheal;KI, knock-in; MedLN, mediastinal lymph node; PC, phosphorylcholine; PFA, para-formaldehyde.

This article is distributed under The American Association of Immunologists, Inc.,Reuse Terms and Conditions for Author Choice articles.

Copyright� 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500251

Published May 8, 2015, doi:10.4049/jimmunol.1500251

HDM allergen particulate preparation

Milled natural Der p (RMB84M; Greer Laboratories) HDM powder wassonicated at 12 W at 4˚C in 30-min increments for 8 h before being passedthrough a 10-mm filter. HDM particulates were pelleted at 10,000 rpm for30 min prior to lyophilization. After weighing, the lyophilized particulateswere resuspended in sterile PBS at 10 mg/ml and stored at 280˚C or di-luted to 0.1 mg/ml at 4˚C prior to administration to mice. Particulate sizewas determined by flow cytometric comparison with 0.1–1 mm latex beadsas reference particles (Sigma-Aldrich LB1-LB30). HDM particulates werelabeled with Alexa Fluor 647 (Life Technologies), according to manu-facturer’s instructions.

In vivo intratracheal challenge and the HDM allergy model

For intratracheal (i.t.) challenge, 6- to 8-wk-old (adult) C57BL/6 mice wereanesthetized using 3–5% isoflurane before being immobilized on a verticalboard using a suture string looped around their upper incisors. The tonguewas extended from the oral cavity using blunt-end forceps so that liquidcould be pipetted into the oral cavity. The nares were manually pluggedto facilitate inhalation of the liquid suspension. Adult mice were challengedi.t. with 5 mg processed HDM particulate allergen resuspended in 50 mLPBS. Mice were rested for 7 d before being challenged i.t. daily for con-secutive 5 d with 5 mg HDM in 50 mL PBS. Following the last challengewith HDM allergen, mice were rested for 2 d before euthanization. Whenstated, the above procedure was also conducted using Alexa Fluor 647–la-beled HDM for all sensitizations and subsequent challenges. For in vivo Abexperiments, mice were given 5 mg Alexa Fluor 647–labeled HDM, AlexaFluor 647–labeled HDM, and 50 mg anti-PC IgM Ab or Alexa Fluor 647–labeled HDM and 50 mg irrelevant isotype control Ab i.t. and then eutha-nized 24 h later.

Pneumococcal vaccines and immunizations

S. pneumoniae strains R36A (PC bearing) and JY2190 (PC deficient) (21, 22)were grown to midlog phase at 37˚C in 5% CO2. R36A was grown in ToddHewitt Broth supplemented with 0.5% yeast extract (Difco). Pneumococcalstrain JY2190 was grown in chemically defined medium (Hazelton) supple-mented with 0.5% sodium bicarbonate (Fisher) and 0.15% cysteine hydro-chloride (Sigma-Aldrich). Bacteria were fixed with 1% paraformaldehyde(PFA) for 12 h and then resuspended in sterile PBS and stored at280˚C untiluse. For neonatal immunizations, 3- to 4-d-old C57BL/6 littermate pupswere immunized i.p. with 2 3 107 PFA-fixed pneumococcal strains R36Aor JY2190.

Bronchoalveolar lavage fluid, lung, and mediastinal lymphnode collection

Following sacrifice, trachea were cannulated to extract cellular infiltratesfrom the bronchoalveolar space via a 5-ml lavage with PBS. Mice wereperfused by cardiac puncture with PBS plus 1% heparin prior to lung re-moval. For cell isolation, lungs were minced and treated with 1 mg/mlcollagenase (Sigma-Aldrich) in 5 mL HBSS for 40 min at 37˚C, fol-lowed by 40-mm filtration and lymphocyte separation (Cellgro). To iden-tify CD138 plus IgM–secreting B cells and PC-specific B cells, lungs wereminced and treated with 5 mg collagenase plus 50 U DNase (Sigma-Aldrich) for 40 min at 37˚C. Mediastinal lymph node (MedLN) cellswere collected by mechanical isolation. All cells were manually enumer-ated using a hemocytometer.

ELISA

ELISAs were performed to measure anti-PC IgM and IgG3 Abs along withtotal IgE in the bronchoalveolar lavage fluid (BALF) and Der p 1–specificserum IgE. PC-specific ELISAs were conducted using high-binding enzymeimmunoassay/RIA plates (Costar) coated with 2 mg/ml PC-BSA (BiosearchTech) and detected with alkaline phosphatase–conjugated goat anti-mouseIgM, or IgG3 (Southern Biotechnology, Birmingham, AL), respectively.Standard curves were prepared using known quantities of PC-specific IgM(BH8) purified in our laboratory. PC-specific IgG3 concentration was cal-culated as predicted by the point of inflection for a curve generated by ODvalues at 405 nm. Der p 1–specific IgE was determined by coating plateswith 2 mg/ml natural Der p 1 (Indoor Biotechnologies), and Ab was detectedusing alkaline phosphatase–conjugated goat anti-mouse IgE (Southern Bio-technology Associates). Total IgE levels were determined by ELISA bycoating plates with 2 mg/ml rat anti-mouse IgE (Southern Biotechnology;clone 23G2), and standard curves were prepared with known concentrations ofIgE Abs (Southern Biotechnology; clone 0114-01). IgM bound to HDMparticles was calculated by incubating equal volumes of BALF with anti-Der p1–coated (2 mg/ml) plates (MA-5H8; Indoor Biotechnologies). Ab was

detected using alkaline phosphatase–conjugated goat anti-mouse IgM(Southern Biotechnology). Standard curves were prepared using knownquantities of PC-specific IgM (BH8)-coated HDM. For all ELISAs, p-nitro-phenyl phosphate substrate (Sigma-Aldrich) was added, and color develop-ment was detected with a SPECTROstarV Reader (BMG Labtech) at 405 nm.

ELISPOT assay

High-binding flat-bottom enzyme immunoassay/RIA plates (Costar) werecoated with either 5 mg/ml unlabeled goat anti-mouse IgM (Southern Bio-technology; catalogue 1021), goat anti-mouse IgA (Southern Biotechnology;catalogue 1040-01), or 5 mg/ml PC-BSA (Biosearch Technologies), re-spectively, to quantify IgM-secreting cells, IgA-secreting cells, or PC-specific Ab-secreting cells in the lung. Plates were coated overnight at 4˚Cand then blocked for 6 h at 37˚C with PBS plus 1% gelatin solution. Next,HDM-exposed mice were perfused by cardiac puncture, and their lungs weredigested with a mixture of 1 mg/ml collagenase (Sigma-Aldrich) with 50 UDNase in 5 mL HBSS (Life Technologies). Single-cell suspensions wereprepared in RPMI 1640 (Life Technologies) supplemented with 2% ultralowIgG FBS (Life Technologies), and then 53 106 cells were added to one welland diluted 2-fold in RPMI 1640 plus 2% low IgG FCS. Plates were thenincubated for 18 h at 37˚C, and cells were lysed with water plus 0.05%Tween-20. Plates were then washed three times with PBS plus 0.05%Tween-20, and then incubated with 500 mg/ml anti-IgM or anti-IgA alkalinephosphatase (Southern Biotechnology) in a solution of PBS plus 0.05%Tween-20 plus 1% gelatin for 2 h at 37˚C. Plates were then washed threetimes with PBS plus 0.05% Tween and developed for 18 h at 4˚C in substratebuffer (pH 10.25) containing 1 M 2-amino-2-methyl-1-propanol (Sigma-Aldrich), 0.1% Triton-X405 (Sigma-Aldrich), and 0.01 g/ml 5-bromo-4-chloro-3-indolyl-phosphate (Sigma-Aldrich). Plates were then washed indH2O, and resulting spots were enumerated visually.

Ab reagents, flow cytometry, and cell identification

Hybridomas and plasmacytomas were grown in serum-free RPMI 1640(hybridoma SFM; Life Technologies). IgM Abs were purified from super-natants on Sepharose-6B columns coupled with RS3.1 anti-IgH6a mAb, andIgA Abs were purified on Sepharose 4B affinity columns coupled with ratmonoclonal anti-IgA. HDM particulates and pneumococcal bacterial vaccinepreparations were stained with purified mAbs BH8 (anti-PC IgM) and S107(anti-PC IgA), followed by secondary goat anti-mouse FITC Abs againstrespective isotypes (Southern Biotechnology). BALF, MedLN, and lung cellswere stained for flow cytometry using fluorochrome-conjugated Abs specificfor the following molecules, transcription factors, and cytokines: CD3, CD4,CD5, CD11c, CD19, CD44, CD62L, CD86, CD117, CD127, CD138, B220,IgE, SiglecF, Ly6G, GATA3, IL-2, IL-12, IFN-g, IL-4, IL-6, and IL-13 fromBD Biosciences, and anti-IgM from eBioscience. Anti–CD11b-Alexa Fluor488, TC68-Alexa Fluor 647, BH8-Alexa Fluor 647, and PC-BSA (BiosearchTechnologies)-Alexa Fluor 647 were conjugated in our laboratory, accord-ing to manufacturer’s instructions. Leukocytes were identified using flowcytometry to detect specific markers based on the following gating strategy(see Supplemental Fig. 1): alveolar macrophages (Siglec-F+CD11c+), T cells(CD3+CD4+), B cells (B220+CD19+), eosinophils (Siglec-F+CD11c2),macrophages (CD11b+CD11c2), dendritic cells (DCs; CD11b2CD11c+),immature DCs (CD11b+CD11c+), neutrophils (CD11b+Ly6G+), IgE-boundB cells (B220+IgE+), basophils (B2202IgE+Ckit2), and mast cells (B2202

IgE+Ckit+). IgM-expressing lung B were identified by gating on B cells(B220+CD19+) that bound anti-mouse IgM; additionally, lung plasmablastswere detected by gating on viable B220lowIgM+ cells that also expressedCD138. PC-specific B cells were identified as B cells (B220+CD19+) thatbound both PC-BSA-Alexa Fluor 647 and the anti-idiotype reagent TC68Alexa Fluor 647. T cells (CD3+CD4+) from the MedLN were stained withisotype control Ab or anti-GATA3 Ab in conjunction with the eBioscienceFoxp3 staining buffer kit (00-5523-00). Following restimulation, per-meabilization, and fixation, MedLN T cells (CD3+CD4+) were stained withisotype control Ab as well as for production of Th1 and Th2 cytokines listedabove. Macrophages (CD11b+CD11c2), DCs (CD11b2CD11c+), immatureDCs (CD11b+CD11c+), and alveolar macrophages (Siglec-F+CD11c+) werealso analyzed for uptake of Alexa Fluor 647–labeled HDM. All flowcytometry analyses were performed on a FACSCalibur (BD Biosciences) orLSR II (BD Biosciences) and analyzed using FlowJo software (Tree Star).

Single-frequency forced oscillation technique for mechanicalventilation

Mice were anesthetized with ketamine xylazine (100 mg/kg) and pan-curonium bromide (0.8 mg/kg), and their tracheas were cannulated with an18-G tube connected to the inspiratory and expiratory ports of a Flexiventventilator (SCIREQ), in which mice were ventilated at a rate of 160 breaths

2 ANTI-PC Abs REDUCE DUST MITE–INDUCED ALLERGIC DISEASE

per minute. After initial resistance measurements (0 mg/ml methacholine), in-creasing concentrations (10–50 mg/ml) of methacholine (Sigma-Aldrich)were vaporized and total respiratory resistance was recorded every 12 s con-tinuously for up to 3 min. Averages from each methacholine dose were takenfrom four to six mice per group to determine airway hyperresponsiveness (AHR).

Histology and fluorescence staining

For paraffin-embedded sections, mouse lungs were fixed in 4% PFA,dehydrated by sequentially increasing concentrations of ethanol, and, afterxylene incubation, embedded in paraffin (Leica EG1150H). Six-micron lungsections were cut (Leica RM2235), rehydrated, and stained with either H&E(Sigma-Aldrich) or periodic acid–Schiff (Sigma-Aldrich) stain, accordingto manufacturer’s instructions, before being dehydrated and mounted in axylene-based mounting media (poly-mount xylene). For cryosections, mouselungs were inflated with a solution of 50% OCT compound (Tissue-Tek) and50% PBS before being tied off at the trachea and embedded in OCT com-pound. Lungs were cryosectioned (Leica CM-3050S) into 4-mm sections.These sections were fixed with acetone (Sigma-Aldrich), blocked with 5%BSA, and stained with goat anti-mouse: IgM (Life Technologies), laminin-biotin (Novus Biologicals), CD11c-488 (BioLegend), Siglec-F-PE (BD Bio-sciences), and CD138-PE (BD Biosciences), or PC-BSA-Alexa Fluor 647,and mounted in Fluoromount (Southern Biotechnology). Whole unmilledHDM species Der p (Greer) were cytocentrifuged onto slides and stained withBH8-Alexa Fluor 647 or isotype control Ab. For BALF stains, identicalvolumes of mouse BALF cell suspensions were cytocentrifuged (ShandonElliott) onto glass slides at 1000 rpm for 5 min before staining with modifiedWright’s stain (Sigma-Aldrich). Sections and mites were imaged with a Leica/Leitz DMRB fluorescence microscope equipped with appropriate filter cubes.

Ex vivo experiments

To isolate alveolar macrophages and pulmonary APCs, 10 ml sterile PBSbronchoalveolar lavages were pooled from five C57BL/6 mice. Mouse lungswere perfused and subjected to sterile collagenase digestion, followedby lymphocyte enrichment with lymphocyte separation medium (Cellgro;Sigma-Aldrich). BALF cells were seeded at 200 cells/well and lung digestsat 100,000 cells/well in 6-well plates and then rested for 12 h. BALF cellsfrom naive unmanipulated mice were primarily SiglecF+CD11c+ alveolarmacrophages, whereas leukocyte preparations from lung digests containedmultiple pulmonary APCs: CD11b+CD11c2 resident macrophages, CD11c+

CD11b+ DCs, and CD11c+Siglec-F+ alveolar macrophages. The RAW 264.7cell line (ATCC) was maintained in DMEM (Life Technologies) plus 10%FCS (Hyclone) and 100 mM 2-ME (Sigma-Aldrich) at 37˚C. For ex vivo Ab-mediated blocking experiments, 10 mg Alexa Fluor 647-HDMwas incubatedwith either 20 mg IgM isotype control Ab or 10 or 20 mg anti-PC IgM (BH8)for 30 min in serum-free DMEM before being added to cultures of seededBALF (alveolar macrophages) or lung digests (pulmonary APCs: residentmacrophages, DCs, and alveolar macrophages) and incubated for another3 h. The percentage of alveolar macrophages, pulmonary APCs, or RAW264.7 cells that had taken up Alexa Fluor 647–labeled HDM was determinedby flow cytometry in each case.

BALF cytokine analysis

The first milliliter of fluid extracted from mouse BALF was used to measurethe levels of 25 cytokines and chemokines using the Milliplex Map mousecytokine/chemokine magnetic bead panel kit (Millipore MCYTOMAG-70K-PMX), according to the manufacturer’s instructions. The data wereanalyzed using the Milliplex Analyst 5.1 software.

T cell restimulations

MedLN cells were separated by mechanical disruption and incubatedwith 0.081 mM PMA, 1.34 mM ionomycin (eBioscience Cell StimulationCocktail 00-4970), and 3.0 mg/ml brefeldin A (eBioscience 00-4506-51)for 6 h. Following restimulation, cells from the MedLN were treatedwith the BD Cellular Fixation/Permeabilization Kit (554714), accordingto manufacturer’s instructions, before staining with Abs directed againstthe Th1 and Th2 cytokines mentioned above.

Sample size and statistics

Values represent the mean 6 SEM from three to five independent experi-ments with 5–10 mice per group. Statistical calculations described belowwere performed with Prism 4.0 software (GraphPad). Comparison of threeor more groups was performed by a one-way ANOVA test, followed byTukey’s post hoc analysis. Data from only two groups were analyzed bya two-tailed unpaired t test to determine statistical significance. In the figures,statistically significant results are represented as values of *p , 0.05, **p ,0.01, and ***p , 0.001.

ResultsNeonatal exposure to PC-bearing R36A reduces infiltration ofcells into the BALF and lungs following adult exposure toHDM

To better understand the relationship between early microbial ex-posure and the development of allergic disease, we began by testing

HDM for reactivity with mAbs raised against conserved epitopes ona panel of bacteria. In this work, we found that PC-specific IgM andIgAAbs bound small milled and sonicatedHDMparticles in additionto intact HDM (Fig. 1C–E). Because the bacterially induced anti-PCAbs reacted with a significant human allergen, we asked whether

early-life microbial PC exposure could reduce the development ofHDM-induced allergic disease (Fig. 1F). Three-day-old individualC57BL/6 littermate mouse pups were immunized with R36A, a PC-bearing unencapsulated pneumococcus strain (Fig. 1A); JY2190, anunencapsulated PC-deficient pneumococcal mutant (Fig. 1B); ortreated with PBS alone. In this study, we used littermate mice to

avoid the variability in immune responses resulting from differingcommensal microbiota among separately caged mice. In addition tothese mice, we included groups of age-matched T15 IgH gene KI(T15 KI) mice. T15 KI mice contained a high frequency of PC-specific B cells in the absence of deliberate microbial exposure. At

6–8 wk of age, these groups of mice were sensitized i.t. with HDMon day 0 and then challenged i.t. with HDM particulates daily ondays 7–11 to induce allergic disease (Fig. 1F). Mice were eutha-nized 14 d following initial sensitization (day 0), and all analyseswere performed at this time point, unless otherwise stated.We observed that bronchoalveolar lavage of naive unmanipulated

mice contained mostly alveolar macrophages (data not shown);however, HDM sensitization and challenge resulted in the accumu-lation of T cells, eosinophils, neutrophils, basophils, andmast cells intoboth the bronchoalveolar space and the pulmonary parenchyma

(Figs. 1G–L, 2A, 2B). BALF from mice immunized with PC-bearingR36A as neonates and from the T15 KI mice had half as manyT cells, eosinophils, neutrophils, APCs, mast cells, and basophilsinfiltrating the bronchoalveolar space following HDM exposure asadults compared with mice immunized with JY2190 as neonates ortreated with PBS alone (Fig. 1G, 1H). The numbers of alveolar

macrophages collected from all groups of mice were statisticallysimilar, as they are normally resident cells of the lung, and are notknown to be mobilized in large numbers after HDM exposure(Fig. 1H). Wright stains of BALF cytospins confirmed that all groupsof mice had similar numbers of alveolar macrophages per field

(Fig. 1I–L). However, fewer neutrophils, eosinophils, and lymphocyteswere detected in the BALF of C57BL/6 mice immunized with R36Aas neonates and the T15 KI mice compared with mice exposed toJY2190 or PBS as neonates (Fig. 1I–L). Thus, mice immunized withPC-bearing R36A as neonates and the T15 KI mice had a significantlydecreased infiltration of allergy-associated cells into their bron-

choalveolar space following HDM exposure compared with C57BL/6mice exposed to PC-deficient JY2190 or PBS early in life.Prior to HDM exposure, the lungs of naive unmanipulated mice

contained 10 times fewer T cells, neutrophils, macrophages, and

DCs compared with those exposed to HDM. Following HDMsensitization and challenge, we enumerated inflammatory and al-lergy-associated cells in the pulmonary parenchyma. Both C57BL/6mice immunized with R36A as neonates and the T15 KI micehad significantly decreased numbers of infiltrating T cells, eosino-

phils, neutrophils, APCs, mast cells, and basophils in their pulmo-nary parenchyma compared with mice receiving JY2190 or PBSearly in life (Fig. 2A, 2B). The infiltration of allergy-associated cellswas validated by fluorescence microscopy of cryosections of lungsfrom mice exposed to HDM (Fig. 2C–F). C57BL/6 mice vaccinated

The Journal of Immunology 3

with R36A as neonates and the T15 KI mice had decreased eo-sinophilic infiltration surrounding their bronchioles compared withmice immunized with JY2190 or treated with PBS early in life(Fig. 2C–F). Because initiation of HDM-induced allergic diseasedepends on the activation of DCs in the lung (23), we assessed APCactivation by quantifying alveolar and tissue-resident DC andmacrophage expression of CD86, a MHC-II costimulatory mole-cule. Tissue-resident APCs, but not alveolar macrophages, in thepulmonary parenchyma of C57BL/6 mice immunized with R36Aand the T15 KI mice expressed significantly less CD86 comparedwith C57BL/6 mice immunized with JY2190 or treated with PBS asneonates (Fig. 2G, 2H). Therefore, exposure to PC-bearing R36A asneonates and the presence of anti-PC Abs in T15 KI mice eachsignificantly inhibited lung-resident APC activation and diminishedthe infiltration of allergy-associated cells into the pulmonary pa-renchyma following adult exposure to HDM compared withC57BL/6 mice exposed to PC-deficient JY2190 or PBS early in life.

Neonatal exposure to PC-bearing R36A diminishes mediastinallymph node T cell activation, IgE secretion, AHR, and allergicdisease development

Upon natural inhalation of allergen, scavenging APCsmigrate fromthe pulmonary parenchyma and present allergen to naive T cells indraining lymph nodes to prime a Th2-polarized response (24). Toassess HDM priming of this Th2 response, we collected the me-diastinal lymph node (MedLN) following HDM sensitization andchallenge. The MedLN of HDM-exposed C57BL/6 mice were∼30 times larger (∼2.8 mm) in diameter than those of naive un-manipulated mice (∼0.1 mm) (Fig. 3A and data not shown).

C57BL/6 mice immunized as neonates with R36A and the T15 KImice each possessed MedLNs that were half the size and weightof those collected from C57BL/6 mice immunized with JY2190or treated with PBS as neonates (Fig. 3A, 3B). As expected, thesmaller size of MedLNs corresponded with lower lymph nodecellularity, including half as many T cells, B cells, neutrophils,eosinophils, and basophils (Fig. 3C, 3D). In addition to the de-creased T cell population in the MedLN of C57BL/6 mice immu-nized with R36A as neonates and of the T15 KI mice, these T cellsexpressed significantly decreased levels of CD44, a marker of Agexperience (Fig. 3E–G). Thus, in C57BL/6 mice exposed to PC-bearing R36A as neonates, and in T15 KI mice, T cell activation inthe MedLNs is inhibited following HDM exposure compared withC57BL/6 mice exposed to PC-deficient JY2190 or PBS early in life.T cells primed in the MedLN can induce B cell class switching

to IgE, a hallmark of allergic disease (25). We thus quantified theamount of total IgE from the BALF and Der p 1–specific IgE inthe serum. Mice immunized with PC-bearing R36A as neonatesand the T15 KI mice each had significantly decreased total IgElevels in their lung (Fig. 4A) and Der p 1–specific IgE in the serum(Fig. 4B) compared with C57BL/6 mice exposed to PC-deficientJY2190 or PBS early in life.Th2-associated cytokines that induce IgE production are also

significant contributors to the AHR development, production ofmucus in bronchioles, and recruitment of allergy-mediating cellsaround bronchioles (26). To assess AHR development, followingHDM exposure, mice were mechanically ventilated and a forcedoscillation technique was used to measure total resistance of therespiratory system after vaporized doses of methacholine (Fig. 4C).

A B CD E

F

G H

I J K L

FIGURE 1. Anti-PC Abs bind both HDM and

R36A, and neonatal exposure to PC-bearing R36A

reduced cellular infiltration into the bronchoalveolar

space following adult exposure to HDM. Anti-PC

IgM (BH8), anti-PC IgA (S107) Abs, or their re-

spective isotype control Abs were incubated with

(A) pneumococcal bacterial strains PC-bearing R36A

or (B) PC-deficient JY2190 or (C) with processed

HDM allergen. (A–C) Ab binding was detected by

flow cytometry. Whole mounts of HDM were stained

with (D) isotype control Ab or (E) fluorescently

labeled anti-PC IgM Ab (BH8) and viewed with

a Leica/Leitz DMRB microscope. (F) Littermate

C57BL/6 mice that were either treated i.p. with PBS,

or immunized with JY2190 or R36A at day 3 of life,

as well as the T15 KI mice were sensitized with

HDM on day 0 and challenged with HDM daily on

days 7–11 at 6–8 wk of age. Following challenge

with HDM, mice were euthanized on day 14, tra-

cheas were cannulated, and a 5-ml wash of the

bronchoalveolar space was removed. (G and H) Cells

in the BALF were enumerated and identified by flow

cytometry, as stated in Materials and Methods, as

well as demonstrated in Supplemental Fig. 1. (I–L)

Equal volumes of BALF were cytocentrifuged onto

glass slides and stained with modified Wright stain

to identify alveolar macrophages (A.M.), eosinophils

(eo), and neutrophils (neut). Values represent the

mean 6 SEM from five independent experiments

with 5–10 mice per group. Data were analyzed by

ANOVA, in which statistically significant results are

represented as **p , 0.01 and ***p , 0.001.

4 ANTI-PC Abs REDUCE DUST MITE–INDUCED ALLERGIC DISEASE

Following exposure to HDM, C57BL/6 mice immunized with PC-bearing R36A as neonates and the T15 KI mice had airway resis-tance measurements similar to naive mice that had not receivedHDM (Fig. 4C). These C57BL/6 mice had significantly lower air-way resistance prior to methacholine exposure and following thehighest dose (50 mg/ml) of methacholine compared with micetreated with PC-deficient JY2190 or PBS as neonates (Fig. 4C).Periodic acid–Schiff–stained lung paraffin sections demon-

strated that HDM-treated, but not naive unmanipulated mice, de-veloped increased mucin-producing goblet cell frequencies inbronchioles throughout the upper and lower airways (Fig. 4D, 4F,4H, 4J). However, mucin-producing goblet cells in the bronchioles(magenta) of C57BL/6 mice immunized with R36A as neonates(Fig. 4H) and T15 KI mice (Fig. 4J) were dramatically lower innumber compared with C57BL/6 mice exposed to JY2190 or PBSas neonates (Fig. 4D, 4F). H&E-stained lung sections of naivemice not treated with HDM were free of inflammatory infiltrates(results not shown). The extensive leukocytic infiltrates associatedwith the bronchioles of PBS- or JY2190-immunized HDM-exposed C57BL/6 mice (Fig. 4E, 4G) were not observed aroundthe bronchioles of mice first treated with R36A as neonates or ofthe T15 KI mice (Fig. 4I, 4K). These results demonstrate thatneonatal exposure to PC-bearing R36A and endogenous produc-tion of anti-PC Ab in the T15 KI mice reduces IgE production,AHR development, overproduction of mucin in the bronchioles,and infiltration of leukocytes around the airways compared withmice treated with PC-deficient JY2190 or PBS as neonates.

IgM-secreting PC-specific B cells dominate the local immuneresponse to HDM among mice immunized with PC-bearingR36A as neonates

Although mice immunized with R36A as neonates and the T15 KImice have reduced numbers of allergy-associated cells infiltrating

their lungs, they had approximately twice as many B cells in theirpulmonary parenchyma following HDM exposure compared withmice first treated with JY2190 or PBS as neonates (Fig. 5A). Morethan 90% of these B cells expressed the IgM isotype (Fig. 2B, 2G).The frequencies of lung IgM+B220+CD19+ B cells and IgM+

CD138+B220low plasmablasts in both C57BL/6 mice immunizedwith R36A as neonates and in T15 KI mice were higher comparedwith those in C57BL/6 mice immunized with JY2190 as neonates(Fig. 5C, 5D). ELISPOT analysis demonstrated that C57BL/6 miceimmunized with R36A as neonates and T15 KI mice have morethan twice as many IgM-secreting cells in their pulmonary paren-chyma compared with mice immunized with JY2190 (Fig. 5E, 5F).To determine the location of these B cells, lung cryosections werestained for IgM (green) and laminin (gray) (Fig. 5I–L). C57BL/6mice immunized with R36A early in life and the T15 KI mice bothhad higher numbers of IgM+ cells proximal to their bronchiolescompared with mice immunized with JY2190 (Fig. 5H–L). Thus,mice immunized with PC-bearing R36A as neonates and the T15 KImice had an increased frequency of IgM-expressing B cells in theirlungs located proximal to their bronchioles compared with micetreated with PC-deficient JY2190 or PBS as neonates.PC is the immunodominant epitope on unencapsulated pneu-

mococcus such as R36A (27–29). Therefore, we next probed for PC-specific B cells in the lung. ELISPOT assays following repeatedHDM exposure revealed that mice immunized with R36A early inlife had ∼10-fold more PC-specific IgM-secreting cells in theirlungs compared with mice immunized with JY2190 as neonates(Fig. 6A, 6B). By contrast, none of the groups of mice analyzed haddetectable numbers of PC-specific IgA-secreting cells in their lungs(data not shown). Using flow cytometry, PC-specific B cells wereidentified as B cells that bound both PC-BSA and TC68, an Abreagent that detects the PC-specific T15 IgH (VHT15) (Fig. 6D, 6E).Similar to our ELISPOT analysis, mice immunized with R36A as

A B

C D E F

G H

FIGURE 2. Neonatal exposure to PC-bearing R36A reduced the activation of APCs in the lung and cellular infiltration of allergic mediators into the

pulmonary parenchyma. Following exposure to HDM, mouse lungs were perfused and enzymatically digested. (A and B) Cells in the lung were enumerated

and identified by flow cytometry, as stated in Materials and Methods, as well as demonstrated in Supplemental Fig. 1. Following exposure to HDM, (C–F)

cryosections of lungs were stained with Siglec-F (red) and CD11c (green) to visualize eosinophils (Siglec-F+CD11c2), and bronchioles (Br) and vessels (v)

are labeled as such. CD86 expression on tissue-resident DCs, immature DCs, macrophages, and alveolar macrophages from the lungs of these mice together

with expression prior to HDM exposure is displayed as (G) histograms and (H) quantitatively as CD86 mean fluorescence intensity. These APCs were

identified by flow cytometry, as stated in Materials and Methods. Values represent the mean6 SEM from five independent experiments with 5–10 mice per

group. Data were analyzed by ANOVA, in which statistically significant results are represented as *p , 0.05, **p , 0.01, and ***p , 0.001.

The Journal of Immunology 5

neonates and the T15 KI mice had an increased percentage of PC-specific B cells in their lung compared with mice immunized withJY2190 or PBS as neonates (Fig. 6D). These PC-specific B cellswere stained for CD5 and CD138 expression to determine their B1aor plasmablast phenotypes, respectively. This analysis demonstrated

that PC-specific B cells did not express CD5 (data not shown);however, these cells did express CD138 (Fig. 6F). To furtherdetermine the phenotype and distribution of these PC-specificB cells following HDM exposure, we stained lung cryosectionswith anti-mouse IgM (green), anti-CD138 (red), PC-BSA (yellow),

A B

C D F

E G

FIGURE 3. The number and proportion of Ag-

experienced CD44highCD4+ T cells were diminished

in the MedLNs of mice exposed to PC-bearing

R36A as neonates. Following exposure to HDM, (A)

MedLNs were removed and (B) weighed. Cells re-

moved from the MedLN were (C and D) enumerated

and identified by flow cytometry, as stated in

Materials and Methods, as well as demonstrated in

Supplemental Fig. 1. CD44 expression on CD4+

T cells from MedLNs are presented as (E) flow plots

and (F) histograms and (G) quantified as CD44 mean

fluorescence intensity. Values represent the mean 6SEM from five independent experiments with 5–10

mice per group. Data were analyzed by ANOVA, in

which statistically significant results are represented

as *p , 0.05, **p , 0.01, and ***p , 0.001.

PBSJY2190R36AT15 KI0

100

200

300

*

***

PBSJY2190R36AT15 KI0.0

0.5

1.0

1.5

ng/m

L To

tal I

gE

*

**

0 10 20 30 40 50

1

2

3

4

Vaporized Methacholine (mM)

Rrs

(cm

H2O

.s/m

L)

**

D

E

F

G

H

I

J

K

C

100 m

50 m

50 m

PBS R36A T15 KIJY2190Vaporized Methacholine (mg/mL)

Rrs

(cm

H2O

.s/m

L) PBSJY2190R36AT15 KINo HDM

Tota

l IgE

(ng

/mL)

PBS JY2190 R36A T15 KI PBS JY2190 R36A T15 KI

Der

p1-I

gE (

ng/m

L)

A B

FIGURE 4. Mice exposed to PC-bearing

R36A as neonates produced decreased IgE,

did not develop severe AHR, and exhibited

diminished mucin production in their bron-

chioles and cellular infiltration around these

airways. Following exposure to HDM, (A)

total IgE Ab in the BALF was quantified

from the first milliliter of the BALF and (B)

Der p 1–specific IgE from the serum by

ELISA. (C) Groups of HDM-exposed mice

were mechanically ventilated, and total airway

resistance was measured following challenges

with vaporized methacholine. Following

exposure to HDM, 6-mm paraffin-embedded

lung sections were stained with (D, F, H,

and J) periodic acid–Schiff stain or (E, G, I,

and K) H&E. (D, F, H, and J) Upper images

are magnifications of marked areas in lower

periodic acid–Schiff–stained bronchioles.

Values represent the mean 6 SEM from

three to five independent experiments with

5–10 mice per group. Data were analyzed by

ANOVA, in which statistically significant

results are represented as *p , 0.05, **p ,0.01, and ***p , 0.001.

6 ANTI-PC Abs REDUCE DUST MITE–INDUCED ALLERGIC DISEASE

and anti-laminin (gray) (Fig. 6G–J). IgM+ CD138+ cells were de-tected in lung sections from mice treated with JY2190 or PBSas neonates, but these cells did not react with PC (Fig. 6G, 6H).However, in the lungs of mice treated with R36A as neonates(Fig. 6I) and the T15 KI (Fig. 6J) mice, large CD138+ IgM-expressing PC-specific cells were detected at a higher frequencythan those identified in PBS-treated mice (Fig. 6G) or those im-munized with JY2190 as neonates (Fig. 6H). Mice immunizedwith R36A as neonates also had some clusters of non-PC–bindingIgM-expressing cells in their lungs (data not shown). Thus, miceimmunized with PC-bearing R36A as neonates and the T15 KImice had an increased frequency of CD138+ IgM-expressing PC-specific cells in their lungs following exposure to HDM comparedwith mice immunized with PC-deficient JY2190 or treated withPBS as neonates.We next quantified the amount of PC-specific IgM in the BALF

of these mice after HDM exposure (Fig. 6C). Prior to HDM ex-posure, IgM, IgA, and IgG3 anti-PC Abs in the BALF and PC-specific B cells in the pulmonary parenchyma of these micewere undetectable (data not shown). However, following repeatedHDM exposure, mice immunized with R36A as neonates secretedhigher amounts of PC-specific IgM and IgG3 Ab, but not IgA, intotheir bronchoalveolar spaces compared with those immunizedwith JY2190 (Fig. 6C and data not shown). Thus, in mice exposedto PC-bearing R36A early in life and in T15 KI mice, there was anincreased secretion of PC-specific Abs into the bronchoalveolarspace compared with mice treated with PC-deficient JY2190 orPBS as neonates.

Anti-PC IgM Abs decrease ex vivo uptake of HDM by residentAPCs and the in vivo trafficking of HDM to the MedLN andpriming of a Th2 response

In the presence of complement, anti-PC IgM Abs are efficient atpromoting the opsonization of PC-bearing microbes and apoptotic

cells (30). However, the lung contains 500 times less the concen-

tration of complement components C3, C5, and factor B compared

with those present in serum (31). To better understand the function

of anti-PC IgM Abs in the lung, we designed an ex vivo system to

determine mechanisms involved in the modulation of APC activa-

tion by Ab interactions with HDM. Alveolar macrophages and

pulmonary APCs isolated from mouse lungs were cultured ex vivo.

Next, 10 mg Alexa Fluor 647–labeled HDM was combined with

either 20 or 10 mg purified monoclonal anti-PC IgM (BH8), or

20 mg isotype control Ab was added to serum-free cultures contain-

ing alveolar macrophages and pulmonary APCs (resident DCs and

macrophages), and to the mouse macrophage cell line (RAW 264.7

cells) for 3 h. HDM uptake by APCs under these conditions was

determined by flow cytometry. Anti-PC IgM Abs decreased the

uptake of Alexa Fluor 647–labeled HDM by alveolar macrophages,

pulmonary APCs, and RAW 264.7 cells in a dose-dependent

manner (Fig. 7A). In addition to decreased uptake of HDM par-

ticulates, anti-PC IgM Abs also decreased the activation of alveolar

macrophages and pulmonary APCs as demonstrated by decreased

expression of CD86 (Fig. 7B).To determine whether these IgM Abs functioned similarly in vivo,

purified monoclonal anti-PC IgM Ab (BH8) or isotype-control Ab

A B C D E

FG H

I J K L

FIGURE 5. IgM-expressing B cells dominated the local immune response to HDM among mice immunized with PC-bearing R36A as neonates. Fol-

lowing exposure to HDM, lungs were perfused and enzymatically digested to enumerate B cells in the lung. (A) B220+CD19+ cells that (B and C) expressed

IgM as well as (D) CD138+B220low cells that expressed IgM were quantified and also identified from the lung by (G) flow cytometry, as stated in Materials

and Methods, as well as demonstrated in Supplemental Fig. 1. Cells isolated from perfused lungs were incubated on anti-IgM–coated plates for ELISPOT

analysis and were (E and F) visually enumerated. (I–L) Cryosections of lungs from these mice were stained for IgM (green) and laminin (gray), and

bronchioles (Br) and vessels (v) are labeled as such. These (H) IgM+ cells were quantified from 50 fields per section. Values represent the mean 6 SEM

from three to five independent experiments with 5–10 mice per group. Data were analyzed by ANOVA, in which statistically significant results are

represented as *p , 0.05, **p , 0.01, and ***p , 0.001.

The Journal of Immunology 7

(ISO) were administered i.t. to mice along with Alexa Fluor 647–labeled HDM. Uptake of HDM by APCs in the lung was measuredby flow cytometry at 24 h. Purified anti-PC IgM mAb (BH8) de-creased the interaction of HDMwith alveolar macrophages, residentDCs, and macrophages in the lung by about half compared withmice treated with HDM and isotype control (ISO) or HDM alone(Fig. 7C). Alveolar macrophages collected from the BALF of micetreated with HDM plus anti-PC IgM Ab also expressed decreasedamounts of CD86 compared with mice treated with HDM plusisotype control Ab (Fig. 7C). Collectively, these results demonstratethat anti-PC IgM interferes with the uptake of HDM by APCsin vitro, ex vivo, and in vivo.We next determined the amount of Ab coated on HDM particles

in the lung following HDM sensitization and challenge. We used

a sandwich ELISA technique to capture HDM particles from theBALF and quantified the amount of IgM bound to these particles.Significantly more IgM was bound to HDM from the BALF ofC57BL/6 mice immunized with R36A and from T15 KI mice thanwas bound to HDM from mice immunized with JY2190 (Fig. 7D).To trace the uptake and trafficking of HDM by APCs in the lung,we sensitized and challenged mice with Alexa Fluor 647–labeledHDM. Labeling of HDM with Alexa Fluor 647 did not affect theoutcome of HDM-induced allergic disease (data not shown). Weobserved a decreased percentage of alveolar macrophages, andlower numbers of resident DCs, macrophages, and neutrophils inthe lungs of C57BL/6 mice immunized with R36A and the T15 KImice that had taken up Alexa Fluor 647–labeled HDM (Fig. 7E,7F). Additionally, these mice also had 10 times fewer in number and

TC68+ PC BSA+0.0

0.5

1.0

1.5

2.0

2.5***

Laminin

CD138

PC-BSA

50 m

B6 JY2190R36AT15 KI0

3

650

100150200

*

**

PBSJY2190R36AT15 KI0

10

20

30

ng

/mL

anti-

PC

IgM **

*NS

CD138

F B220+PBS

JY2190R36A

T15 KI

PBS JY2190 R36A T15 KI

ng/m

L an

ti-P

C Ig

M

% o

f Max

C

Spo

ts p

er 5

x106

Cel

ls

PBS JY2190 R36A T15 KI

A PBS JY2190

R36A T15 KI

B

T15 KIJG H IPBS JY2190 R36A IgM

V

V

Br

Br

V

D

E

PC-BSA

TC

68

PBS JY2190 R36A T15 KI

TC68+PC-BSA+

Per

cent

of B

Cel

ls

PBSJY2190

R36AT15 KI

IgM

FIGURE 6. Neonatal exposure to PC-bearing R36A resulted in the accumulation of CD138+ IgM-expressing PC-specific cells in the lung following exposure to

HDM. Following exposure to HDM, lungs were perfused and enzymatically digested. Cells isolated from perfused lungs were incubated on PC-coated plates for

ELISPOTanalysis and were (A and B) visually enumerated. (C) The first milliliter of the BALF was collected to quantify (A) anti-PC IgM by ELISA. (D and E) The

percentage of B cells that were PC specific was identified by flow cytometry, as stated in Materials and Methods, as well as demonstrated in Supplemental Fig. 1.

These PC-specific B cells were also stained for (F) CD138. (G–J) Cryosections of lungs from these groups of mice were also stained for IgM (green), CD138 (red),

PC-BSA binding (yellow), and laminin (gray) and viewed with a Leica/Leitz DMRB microscope. Values represent the mean6 SEM from three to five independent

experiments with 5–10 mice per group. Data were analyzed by ANOVA, in which statistically significant results are represented as *p , 0.05 and **p , 0.01.

8 ANTI-PC Abs REDUCE DUST MITE–INDUCED ALLERGIC DISEASE

4 times lower percentage of HDM-bearing DCs in the MedLNcompared with DCs isolated from the MedLN of C57BL/6 miceimmunized with JY2190 as neonates (Fig. 7G, 7I). To determinewhether early exposure to R36A influences the degree of Th2

priming in adults, we quantified the number of MedLN T cellsexpressing GATA3, a Th2-commitment factor. C57BL/6 mice im-munized with R36A and the T15 KI mice had 4 times less GATA3-expressing T cells in their MedLN compared with mice immunized

HDM only

HDM+ISO

HDM+BH8Med

ia0.0

0.5

1.0

1.5

CD

86 M

FI (x

103 ) ***

Alv Mac Pulm APCsMac cell line 0

5

1020

40

60

80HDM-ISOHDM-10 BH8HDM-20 BH8

***

***

***HDM only

0

20

40

60

80

IgM

(ng

/mL)

Coa

ted

HD

M

******C D

Alv Mac DC Neuts0.0

0.2

0.4

12345

HD

M P

os C

ells

(x10

4 )

*****

*

0.00.10.20.3

1234

***

***

0

1

2

3*****

GA

TA3+

T C

ells

(x1

04)

PBS R36A T15 KIJY2190

IgM

(ng

/mL)

Coa

ted

HD

M E

F G H

I

MedLN MedLNHD

M P

ositi

ve D

Cs

(x10

4 )

BALF

PBSJY2190R36AT15 KI

HDMBH8ISO

HDM

CD

11c

CD86

BAH

DM

Pos

itive

Cel

ls (

%)

% o

f Max

CD

86 M

FI (

x103

)

Alv Mac Pulm APCs Mac cell line

4321

1 HDM2 HDM+ISO3 HDM+BH84 Media

Alv Mac DCs Macs0

2

45

10

15

20

HD

M P

ositi

ve C

ells

(%

)

*

****

0

1

2

3

4

CD

86 M

FI (

x103 )

**

Alv Mac Alv Mac0

20

40

60

80

100

HD

M P

ositi

ve (%

)

***

FIGURE 7. Anti-PC IgM Abs decreased ex vivo uptake of HDM by resident APCs and the in vivo trafficking of HDM to the MedLN, and priming of a Th2

response. Alveolar macrophages and pulmonary APCs, in an ex vivo system, along with a mouse macrophage cell line (RAW 264.7 cells), were incubated with

(A) Alexa Fluor 647–labeled HDM (10 mg) and increasing concentrations (10 or 20 mg) of anti-PC IgM Ab (BH8) or isotype control Ab (20 mg). Percentage of

APCs that had taken up Alexa Fluor 647–labeled HDM was determined by flow cytometry, as stated in Materials and Methods, as well as demonstrated in

Supplemental Fig. 1. (B) CD86 expression from pulmonary APC was also determined. (C) Mice were treated i.t. with Alexa Fluor–labeled HDM, HDM with

anti-PC IgM Ab (BH8), or HDM with isotype control Ab, and then rested for 24 h. Cells were collected from the BALF, and lungs were perfused and en-

zymatically digested. The percentage of alveolar macrophages and resident DCs and macrophages containing HDM were enumerated and identified by flow

cytometry, as stated in Materials and Methods. (C) Activation of the alveolar macrophages was determined by expression of CD86. (D–I) Mice sensitized and

challenged with Alexa Fluor–labeled HDM to induce allergic disease were euthanized 14 d following initial sensitization, as previously described. (D) IgM

bound to HDM from the BALF was quantified by ELISA. (E) The percentage of alveolar macrophages containing HDM in the BALF (F) and the number of

pulmonary APCs containing HDM in the lung were quantified. In addition, the (G) number and (I) percentage of DCs containing HDM in MedLN, as well as

(H) number of GATA3-expressing T cells in the MedLN, were enumerated and identified by flow cytometry, as stated in Materials and Methods, as well as

demonstrated in Supplemental Fig. 1. Values represent the mean 6 SEM from three to five independent experiments with 5–10 mice per group. Data were

analyzed by ANOVA, in which statistically significant results are represented as *p , 0.05, **p , 0.01, and ***p , 0.001.

The Journal of Immunology 9

with JY2190 or treated with PBS as neonates (Fig. 7H). Theseresults demonstrate that mice immunized with PC-bearing R36A aswell as the T15 KI mice have decreased HDM trafficking by DCsand a significantly inhibited generation of a Th2 response in theMedLN compared with mice treated with PC-deficient JY2190 orPBS as neonates.

Early microbial exposure does not influence the Th1 or Th2T cell subset balance

It has been hypothesized that early microbial exposures generatea robust Th1 T cell response capable of suppressing the overtdevelopment of Th2 responses (3, 4). BALF collected from naiveunmanipulated mice prior to HDM exposure did not containdetectable levels of cytokines or chemokines (data not shown).Following HDM exposure, the C57BL/6 mice immunized witheither pneumococcal strain as neonates had statistically similarlevels of Th1-associated cytokines, such as IL-2, IL-12, and IFN-g,in the airways (Fig. 8A). There was also no statistical difference inthe frequency of IL-2–, IL-12–, or IFN-g–producing MedLNT cells (Fig. 8D). However, the lungs of C57BL/6 mice treatedwith R36A as neonates and the T15 KI mice contained signifi-cantly lower concentrations of Th2-associated cytokines, such asIL-4, IL-5, IL-6, IL-13, and IL-9 compared with mice exposed toJY2190 or PBS as neonates (Fig. 8B). These protected mice alsohad a significantly decreased frequency of IL-4–, IL-6–, and IL-13–producing T cells in their MedLN (Fig. 8E). Therefore, miceimmunized with PC-bearing R36A as neonates secrete signifi-cantly decreased Th2-associated cytokines in their airways andhad a significantly reduced frequency of Th2 cytokine-producingcells in their MedLN compared with mice immunized with PC-deficient JY2190. A comparison of Th1- and Th2-associated cyto-kine production in C57BL/6 mice treated with PBS, and thoseimmunized with JY2190 or with R36A, as well as T15 KI micedemonstrated no statistical differences in the amounts of Th1-as-sociated cytokines secreted into the lungs or produced by T cells

in the MedLN (Fig. 8B, 8E). Additionally, nonspecific early mi-crobial exposure, in the case of mice that received JY2190 asneonates, is not sufficient to significantly decrease the productionof Th2-associated cytokines or Th2 T cell generation.Because the decreased secretion of Th2-associated cytokines

into the lung (Fig. 8B) could result from either a lower number ofcells secreting Th2-associated cytokines or impaired productionof chemokines that recruit these cells, we quantified the level ofchemokines produced in the lung that are potentially capable ofrecruiting Th2-associated cells into the lung. C57BL/6 mice im-munized with R36A as neonates and the T15 KI mice secreteddecreased concentrations of CXCL1, CXCL2, RANTES, andCCL4 compared with C57BL/6 mice exposed to JY2190 or PBSas neonates (Fig. 8C). Therefore, neonatal immunization with PC-bearing R36A leads to decreased chemokine secretion into mouselungs compared with immunization with PC-deficient JY2190.These observations suggest that immunization with PC-containingformulations can decrease the influx of allergy-associated cellsinto the lung.

DiscussionThere is a clear association between the rise in incidences of atopicconditions and decreased rates of infection among newborns (1, 32);however, a mechanism to explain this trend has remained elusive.Although a permanent skewing of the Th1 and Th2 T cell subsetshas long been proposed (3, 4, 33), empirical evidence to support thisclaim has been confounding. Less effort has been made to under-stand how the developing neonatal immune system is affected byearly exposure to microbes. We, and others, have demonstrated thatneonatal bacterial exposure can select rare B cell clones for ex-pansion, permanently skewing the overall diversity of the devel-oping B cell repertoire (6, 34, 35). As IgE Ab producers, B cellsinitiate multiple manifestations of allergic pathologies, and are thusclearly considered important instigating components of atopic dis-eases (25). In this study, we now demonstrate that B cells, and their

IL-2 IL-12 IFN-g0.0000.0050.0100.015

0.20.40.60.8

5

IL-4 IL-6 IL-130.0

0.5

1.0

1.5

2.0

2.5

Cel

ls in

the

MLN

(x10

5 )

JY2190R36A

B6

T15

N.D. N.D. N.D.

****

**

IL-2 IL-12p70 IFNg0

1

2

3

4

5

Cyt

okin

es in

the

BA

LF (

pg/m

L)

IL-4 IL-5 IL-6 IL-13 IL-90123

20

40

60

Cyt

okin

es in

the

BA

LF (

pg/m

L)

* **** * *

**

*

CXCL1CXCL2RANTESCCL40

2

4

10

20

30

Cyt

okin

es in

the

BA

LF (

pg/m

L)

*

**

****

***

*

A B C

IL-2 IL-12p70 IFN- IL-4 IL-5 IL-6 IL-13 IL-9 CXCL1 CXCL2 RANTES CCL4

BA

LF C

ytok

ines

(pg

/mL)

BA

LF C

ytok

ines

(pg

/mL)

BA

LF C

ytok

ines

(pg

/mL) PBS

JY2190R36AT15 KI

Cel

ls in

the

Med

LN (

x105

)

Cel

ls in

the

Med

LN (

x105

)

IL-4 IL-6 IL-13

D E F

TH2

B IgE

Microbial Phosphorylcholine

Phosphorylcholine-specific B cells TH2 response

IL-2 IL-12 IFN-

FIGURE 8. Early microbial exposure did not influence the Th1 or Th2 T cell subset balance. Following HDM exposure, the first milliliter of the BALF

was collected for quantification of (A) Th1-associated and (B) Th2-associated cytokines and (C) chemokines using Milliplex magnetic beads and MIL-

LIPLEX Analyst V5.1 software. (D and E) T cells isolated from the MedLN were restimulated with PMA + ionomycin for 6 h before being fixed and

permeabilized to stain for the production of (D) Th1 and (E) Th2 cytokines, as stated in Materials and Methods, as well as demonstrated in Supplemental

Fig. 1. Data were analyzed by ANOVA, in which statistically significant results are represented as *p , 0.05, **p , 0.01, and ***p , 0.001. (F) Exposure

to PC-bearing microbes during early life generates PC-specific B cells that secrete Abs locally in the lung following incidence with PC-bearing HDM.

These B cells and their non-IgE Ab products diminished the development of Th2 responses and allergic pathology.

10 ANTI-PC Abs REDUCE DUST MITE–INDUCED ALLERGIC DISEASE

Ab products, can exert an opposing effect to this accepted patho-logical function by impeding the development of allergic disease inresponse to the PC-bearing HDM allergen.Mice exposed to PC-bearing pneumococcus as neonates had

10-fold increased CD138+ IgM-expressing PC-specific B cells inthe pulmonary parenchyma, and higher concentrations of secretedAbs in their lung following exposure to HDM compared with miceexposed to PC-deficient pneumococcus. When passively admin-istered or endogenously produced, these anti-PC IgM Abs reducedthe interaction of HDM with APCs in the lung. Increased anti-PCIgM Ab titers were also associated with decreased trafficking ofthe allergen to the draining lymph node and significantly reduceda Th2 T cell response. Because Th2-associated cytokines induce thepathology and inflammation associated with allergic disease (36),we hypothesize that the diminished priming of a Th2 response inthe MedLN was directly responsible for the observed decrease inIgE production and the recruitment of allergy-associated inflam-matory mediators into the lung. This effect was accompanied bya moderate development of airway resistance, inhibited productionof mucin-producing goblet cells, and reduced numbers of allergy-associated effector cells in the bronchoalveolar space and pulmo-nary parenchyma. In addition to an overall decreased cellular in-filtration, APCs and effector T cells in lung tissues expressedlower levels of cell surface markers indicative of inflammation andactivation. Thus, a single immunization of neonatal mice with PC-bearing pneumococcus 3 d after birth had a potent and long-lastingimpact on susceptibility to HDM-induced allergic inflammation inadult mice (Fig. 8F).A tenet of the hygiene hypothesis is that microbial exposure dur-

ing development tempers the initiation of allergic diseases later inadult life via a global redirection of Th2 to Th1 responses (1, 4, 37).However, C57BL/6 mice immunized with PC-deficient JY2190pneumococcus as neonates developed manifestations of allergicdisease, upon HDM challenge, similar to those seen in mice treatedwith PBS. Additionally, the limited development of allergic diseaseobserved in the T15 KI mice that had received no deliberatebacterial exposure, which could alter the Th1/Th2 balance,also strongly supports a protective contribution of bacterial-inducedspecific Ab in this model of HDM-induced allergic disease. Basedon these findings, we infer that neonatal exposure to PC-bearingpneumococcus modulates the development of HDM-induced aller-gic disease by B cell–dependent mechanisms, and not the stimula-tion of Th1 cells. To further strengthen our hypothesis that B cellsand Abs play a pivotal role in the protection against allergic airwaydisease development, we immunized 3-d-old B cell–deficientmMT mice with PC-bearing pneumococcus R36A, or PC-deficientJY2190, or PBS alone (Supplemental Fig. 2). At 8 wk of age,these mice were challenged with HDM. mMT mice challengedwith HDM developed similar allergic manifestations whether theyhad been treated with PBS, or immunized with JY2190 or R36Aas neonates. Compared with our observations in similarly treatedC57BL/6 mice, these results demonstrate that R36A-triggered pro-tection against HDM-induced allergic disease does not occur in theabsence of B cells (Supplemental Fig. 2).Our studies also demonstrate that the decreased development of

HDM-induced allergy by early bacterial exposure required the PCepitope to be expressed by both the challenging bacteria and theallergen. The T15 KI mice and C57BL/6 mice treated with pneu-mococcal strains JY2190 or R36A were not protected from allergicdisease resulting from repeated exposure to the PC-deficient timothygrass pollen allergen (data not shown). We also investigated the sig-nificance of timing of microbial exposure. Adult mice immunizedwith either strain of pneumococcus exhibited similar development ofHDM-induced allergic disease (Supplemental Fig. 3). These findings

are consistent with our previous results demonstrating that neonatal,but not adult, exposure to group A streptococci significantly dam-pens development of Aspergillus-induced airway disease (19). Wefurther determined whether anti-PC IgM Abs were sufficient todampen the development of HDM-induced allergic disease. Miceadministered anti-PC IgM Abs 1 h prior to sensitization and everychallenge with HDM displayed significantly decreased developmentof HDM-induced allergic disease compared with mice administeredisotype control Ab (Supplemental Fig. 4). Thus, our combined ob-servations of 1) the protective effects elicited by neonatal exposure toPC-bearing pneumococcus but not PC-negative bacteria in B cell–sufficient mice; 2) the optimal protection in T15 KI mice that havenot been deliberately immunized with PC-containing bacteria; 3) thelack of protection observed in mMT mice; and 4) the protectionafforded by passive administration of highly purified IgM anti-PCmAbs argue strongly for the central role for B cells and IgM Abs inprotection against the development of allergic airway disease in thisHDM model of allergic airway disease.PC is a conserved molecule detectable on HDM particles con-

taining allergenic cargo such as HDM-associated Der p 1 (38).Mammalian leukocytes have multiple innate receptors for PC,including CD36 and platelet-activating factor receptor on leuko-cytes, epithelial cells, and APCs (39). Because PC associated withHDM particles can potentially ligate these PC-recognizing innatereceptors, CD36 and platelet-activating factor receptor may pro-vide entry points for phagocytosis on cells that express theseproteins. Subsequent processing could then prepare the Der p 1allergenic cargo for presentation, resulting in the activation of Th2T cells. Although numerous pulmonary cells bearing PC-ligatingreceptors are potentially involved in the initiation and developmentof allergic disease, DCs are specifically essential in the developmentof HDM-induced allergic disease (40). Thus, interference of HDMallergen uptake by the DCs may be one way that anti-PC Absdisrupt a critical step in the induction of allergic disease. Thus, wehypothesize that Abs, by impeding the interaction of PC-decoratedHDM particles with APCs in the lung, play a major role in inhib-iting the development of HDM-induced allergic disease.Pneumococcal infections among infants are a major cause of

morbidity and mortality in both underdeveloped and developedcountries (41). Despite the recent introduction of neonatal vacci-nation with Prevnar 13 and decreased infection rates of theserotype-specific pneumococcal strains covered by this vaccine(42), serotype replacement is prevalent (43), with the consequencethat pneumococcal infections still occur, even in developedcountries. Neonatal responses to purified polysaccharides are poor,but, in children colonized or infected with pneumococcus, somemount high Ab responses to PC, even at 2–3 y of age, whereasothers do not (44). Whether childhood pneumococcal infectionscorrelate with decreased HDM sensitivity is unknown; however,there is evidence that high IgE titers to pneumococcus are associ-ated with decreased risk for asthma (45). Additionally, non-asthmatics have Abs to a larger number of pneumococcal serotypescompared with asthmatics, and when PBMCs from these non-asthmatics were cultured with HDM, they released less IL-5 thanthose from asthmatics (46). Many other organisms such as Neisseriasp, Haemophilus influenzae, Pseudomonas aeruginosa, and hel-minths also contain PC epitopes (39). Helminth infections are in-versely correlated with allergic disease incidence (47). This inversecorrelation has mostly been attributed to the potent induction ofregulatory T cells; however, alternative mechanisms such as an Abresponse to PC-associated glycoprotein, ES-62, could also bemodulating allergic disease development (48).Anti-PC Abs secreted by B1a cells have been shown to modulate

the outcome of both pneumococcal disease and atherosclerosis (49).

The Journal of Immunology 11

Although our PC-specific B cells do not express CD5, B1a cells losetheir CD5 expression as they become plasma cells (50); therefore,we cannot exclude the possibility that B1a cells induced or expandedby our PC-based vaccine modulate the development of HDM-induced allergic disease. Currently, there is no Food and DrugAdministration–approved PC-based vaccine or cure for allergies orasthma, and management of most atopic diseases involves treat-ment of symptoms. Immunotherapy has successfully induced allergentolerance; however, this anti-inflammatory response is restricted tothe highly variable peptides and extracts administered during im-munotherapy. Immunotherapy generates blocking Abs that can po-tentially inhibit allergen-induced mediator release from mast cellsand basophils or Ag presentation to T cells (51). However, themechanism by which these Abs reduce allergic disease developmentis limited to the Ag specificity of IgE Abs, and would most likelyonly function after IgE generation and progression of allergic dis-ease. Because we have shown that the PC-specific B cells generatedby neonatal exposure to PC-containing bacteria have the potential toinhibit HDM-induced allergic disease development, we suggest thata regimen of suitable vaccine exposure, informed microbiota ma-nipulation, or probiotic use in at-risk children could be used toprevent the development or further progression of respiratory aller-gies and atopic diseases later in life.

AcknowledgmentsWe thank Drs. Rodney King, Peter Burrows, and Denise Kaminski for crit-

ical reading of this manuscript; current and former members of the Kearney

Laboratory; Janet Yother at University of Alabama at Birmingham for the

generous donation of PC-mutant strain JY2190; Davide Botta for assistance

with the Milliplex assay; and the University of Alabama at Birmingham Pro-

gram in Protease and Matrix Biology, specifically Kim Estell and Drs. Amit

Gagger and Chad Steele, for assistance with the AHR measurements.

DisclosuresThe authors have no financial conflicts of interest.

References1. Strachan, D. P. 1989. Hay fever, hygiene, and household size. BMJ 299: 1259–

1260.2. Cookson, J. B. 1987. Prevalence rates of asthma in developing countries and

their comparison with those in Europe and North America. Chest 91: 97S–103S.3. Romagnani, S. 1992. Human TH1 and TH2 subsets: regulation of differentiation and

role in protection and immunopathology. Int. Arch. Allergy Immunol. 98: 279–285.4. Okada, H., C. Kuhn, H. Feillet, and J. F. Bach. 2010. The ‘hygiene hypothesis’

for autoimmune and allergic diseases: an update. Clin. Exp. Immunol. 160: 1–9.5. Hansen, G., G. Berry, R. H. DeKruyff, and D. T. Umetsu. 1999. Allergen-specific

Th1 cells fail to counterbalance Th2 cell-induced airway hyperreactivity butcause severe airway inflammation. J. Clin. Invest. 103: 175–183.

6. Kearney, J. F., P. Patel, E. K. Stefanov, and R. G. King. 2015. Natural antibodyrepertoires: development and functional role in inhibiting allergic airway dis-ease. Annu. Rev. Immunol. 33: 475–504.

7. Holt, P. G., J. W. Upham, and P. D. Sly. 2005. Contemporaneous maturation ofimmunologic and respiratory functions during early childhood: implications fordevelopment of asthma prevention strategies. J. Allergy Clin. Immunol. 116: 16–24, quiz 25.

8. Holt, P. G., and P. D. Sly. 2007. Prevention of allergic respiratory disease ininfants: current aspects and future perspectives. Curr. Opin. Allergy Clin.Immunol. 7: 547–555.

9. Pawankar, R., G. W. Canonica, S. T. Holgate, and R. F. Lockey. 2011. WAOWhite Book on Allergy 2011-2012: Executive Summary. World Allergy Orga-nization. Available at: http://www.worldallergy.org/publications/wao_white_book.pdf.

10. Chivato, T., E. Valovirta, R. Dahl, J. de Monchy, A. Bloch Thomsen,S. Palkonen, and L. Jacobsen. 2012. Allergy, living and learning: diagnosis andtreatment of allergic respiratory diseases in Europe. J. Investig. Allergol. Clin.Immunol. 22: 168–179.

11. Nelson, R. P., Jr., R. DiNicolo, E. Fernandez-Caldas, M. J. Seleznick,R. F. Lockey, and R. A. Good. 1996. Allergen-specific IgE levels and mite al-lergen exposure in children with acute asthma first seen in an emergency de-partment and in nonasthmatic control subjects. J. Allergy Clin. Immunol. 98:258–263.

12. Trombone, A. P., K. R. Tobias, V. P. Ferriani, J. Schuurman, R. C. Aalberse,A. M. Smith, M. D. Chapman, and L. K. Arruda. 2002. Use of a chimeric ELISA to

investigate immunoglobulin E antibody responses to Der p 1 and Der p 2 in mite-allergic patients with asthma, wheezing and/or rhinitis. Clin. Exp. Allergy 32:1323-1328.

13. Baldo, B. A., T. C. Fletcher, and G. Uhlenbeuck. 1977. Reaction of house dustmite extracts with mouse anti-phosphorylcholine and anti-galactan myelomaproteins. Naturwissenschaften 64: 594–595.

14. Baldo, B. A., S. Krilis, and T. C. Fletcher. 1979. Phosphorylcholine-containingallergens. Naturwissenschaften 66: 623–625.

15. Tomasz, A. 1967. Choline in the cell wall of a bacterium: novel type of polymer-linked choline in Pneumococcus. Science 157: 694–697.

16. Masmoudi, H., T. Mota-Santos, F. Huetz, A. Coutinho, and P. A. Cazenave.1990. All T15 Id-positive antibodies (but not the majority of VHT15+ anti-bodies) are produced by peritoneal CD5+ B lymphocytes. Int. Immunol. 2: 515–520.

17. Briles, D. E., C. Forman, S. Hudak, and J. L. Claflin. 1982. Anti-phosphorylcholine antibodies of the T15 idiotype are optimally protectiveagainst Streptococcus pneumoniae. J. Exp. Med. 156: 1177–1185.

18. Gronwall, C., J. Vas, and G. J. Silverman. 2012. Protective roles of natural IgMantibodies. Front. Immunol. 3: 66.

19. Kin, N. W., E. K. Stefanov, B. L. Dizon, and J. F. Kearney. 2012. Antibodiesgenerated against conserved antigens expressed by bacteria and allergen-bearingfungi suppress airway disease. J. Immunol. 189: 2246–2256.

20. Taki, S., M. Meiering, and K. Rajewsky. 1993. Targeted insertion of a variableregion gene into the immunoglobulin heavy chain locus. Science 262: 1268–1271.

21. Yother, J., and J. M. White. 1994. Novel surface attachment mechanism of theStreptococcus pneumoniae protein PspA. J. Bacteriol. 176: 2976–2985.

22. Yother, J., K. Leopold, J. White, and W. Fischer. 1998. Generation and propertiesof a Streptococcus pneumoniae mutant which does not require choline or analogsfor growth. J. Bacteriol. 180: 2093–2101.

23. Hammad, H., B. N. Lambrecht, P. Pochard, P. Gosset, P. Marquillies,A. B. Tonnel, and J. Pestel. 2002. Monocyte-derived dendritic cells inducea house dust mite-specific Th2 allergic inflammation in the lung of humanizedSCID mice: involvement of CCR7. J. Immunol. 169: 1524–1534.

24. Nakano, H., M. E. Free, G. S. Whitehead, S. Maruoka, R. H. Wilson, K. Nakano,and D. N. Cook. 2012. Pulmonary CD103(+) dendritic cells prime Th2 responsesto inhaled allergens. Mucosal Immunol. 5: 53–65.

25. Gould, H. J., and B. J. Sutton. 2008. IgE in allergy and asthma today. Nat. Rev.Immunol. 8: 205–217.

26. Kinyanjui, M. W., J. Shan, E. M. Nakada, S. T. Qureshi, and E. D. Fixman. 2013.Dose-dependent effects of IL-17 on IL-13-induced airway inflammatoryresponses and airway hyperresponsiveness. J. Immunol. 190: 3859–3868.

27. Nicoletti, C., C. Borghesi-Nicoletti, X. H. Yang, D. H. Schulze, and J. Cerny.1991. Repertoire diversity of antibody response to bacterial antigens in agedmice. II. Phosphorylcholine-antibody in young and aged mice differ in both VH/VL gene repertoire and in specificity. J. Immunol. 147: 2750–2755.

28. Mosser, J. L., and A. Tomasz. 1970. Choline-containing teichoic acid asa structural component of pneumococcal cell wall and its role in sensitivity tolysis by an autolytic enzyme. J. Biol. Chem. 245: 287–298.

29. Fischer, W., T. Behr, R. Hartmann, J. Peter-Katalinic, and H. Egge. 1993. Tei-choic acid and lipoteichoic acid of Streptococcus pneumoniae possess identicalchain structures: a reinvestigation of teichoid acid (C polysaccharide). Eur. J.Biochem. 215: 851-857.

30. Ehrenstein, M. R., and C. A. Notley. 2010. The importance of natural IgM:scavenger, protector and regulator. Nat. Rev. Immunol. 10: 778–786.

31. Bolger, M. S., D. S. Ross, H. Jiang, M. M. Frank, A. J. Ghio, D. A. Schwartz, andJ. R. Wright. 2007. Complement levels and activity in the normal and LPS-injured lung. Am. J. Physiol. Lung Cell. Mol. Physiol. 292: L748–L759.

32. Vargas, M. H. 2006. Ecological association between scarlet fever and asthma.Respir. Med. 100: 363–366.

33. Strachan, D. P. 2000. Family size, infection and atopy: the first decade of the“hygiene hypothesis.” Thorax 55(Suppl. 1): S2–S10.

34. Vakil, M., D. E. Briles, and J. F. Kearney. 1991. Antigen-independent selectionof T15 idiotype during B-cell ontogeny in mice. Dev. Immunol. 1: 203–212.

35. Stohrer, R., and J. Kearney. 1984. Ontogeny of B cell precursors responding toalpha 1- greater than 3 dextran in BALB/c mice. J. Immunol. 133: 2323–2326.

36. Paul, W. E., and J. Zhu. 2010. How are T(H)2-type immune responses initiatedand amplified? Nat. Rev. Immunol. 10: 225–235.

37. Kuo, C. H., H. F. Kuo, C. H. Huang, S. N. Yang, M. S. Lee, and C. H. Hung.2013. Early life exposure to antibiotics and the risk of childhood allergic dis-eases: an update from the perspective of the hygiene hypothesis. J. Microbiol.Immunol. Infect. 46: 320-329.

38. Wills-Karp, M., A. Nathan, K. Page, and C. L. Karp. 2010. New insights into innateimmune mechanisms underlying allergenicity. Mucosal Immunol. 3: 104–110.

39. Clark, S. E., and J. N. Weiser. 2013. Microbial modulation of host immunity withthe small molecule phosphorylcholine. Infect. Immun. 81: 392–401.

40. Plantinga, M., M. Guilliams, M. Vanheerswynghels, K. Deswarte, F. Branco-Madeira, W. Toussaint, L. Vanhoutte, K. Neyt, N. Killeen, B. Malissen, et al.2013. Conventional and monocyte-derived CD11b(+) dendritic cells initiate andmaintain T helper 2 cell-mediated immunity to house dust mite allergen. Im-munity 38: 322–335.

41. Lozano, R., M. Naghavi, K. Foreman, S. Lim, K. Shibuya, V. Aboyans,J. Abraham, T. Adair, R. Aggarwal, S. Y. Ahn, et al. 2012. Global and regionalmortality from 235 causes of death for 20 age groups in 1990 and 2010: a sys-tematic analysis for the Global Burden of Disease Study 2010. Lancet 380:2095–2128.

42. Andrews, N. J., P. A. Waight, P. Burbidge, E. Pearce, L. Roalfe, M. Zancolli,M. Slack, S. N. Ladhani, E. Miller, and D. Goldblatt. 2014. Serotype-specific ef-

12 ANTI-PC Abs REDUCE DUST MITE–INDUCED ALLERGIC DISEASE

fectiveness and correlates of protection for the 13-valent pneumococcal conjugate

vaccine: a postlicensure indirect cohort study. Lancet Infect. Dis. 14: 839–846.43. O’Brien, K. L., L. J. Wolfson, J. P. Watt, E. Henkle, M. Deloria-Knoll,

N. McCall, E. Lee, K. Mulholland, O. S. Levine, and T. Cherian, Hib and

Pneumococcal Global Burden of Disease Study Team. 2009. Burden of disease

caused by Streptococcus pneumoniae in children younger than 5 years: global

estimates. Lancet 374: 893–902.44. Gray, B. M., H. C. Dillon, Jr., and D. E. Briles. 1983. Epidemiological studies of

Streptococcus pneumoniae in infants: development of antibody to phosphocho-

line. J. Clin. Microbiol. 18: 1102–1107.45. Hollams, E. M., B. J. Hales, C. Bachert, W. Huvenne, F. Parsons, N. H. de Klerk,

M. Serralha, B. J. Holt, S. Ahlstedt, W. R. Thomas, et al. 2010. Th2-associated

immunity to bacteria in teenagers and susceptibility to asthma. Eur. Respir. J. 36:

509–516.

46. Jung, J. A., H. Kita, R. Dhillon, R. M. Jacobson, M. H. Nahm, M. Park,C. Tsigrelis, and Y. J. Juhn. 2010. Influence of asthma status on serotype-specificpneumococcal antibody levels. Postgrad. Med. 122: 116–124.

47. Amoah, A. S., D. A. Boakye, R. van Ree, and M. Yazdanbakhsh. 2014. Parasiticworms and allergies in childhood: insights from population studies 2008-2013.Pediatr. Allergy Immunol. 25: 208-217.

48. Harnett, W., and M. M. Harnett. 2008. Parasitic nematode modulation of allergicdisease. Curr. Allergy Asthma Rep. 8: 392–397.

49. Baumgarth, N. 2011. The double life of a B-1 cell: self-reactivity selects forprotective effector functions. Nat. Rev. Immunol. 11: 34–46.

50. Reynolds, A. E., M. Kuraoka, and G. Kelsoe. 2015. Natural IgM is produced byCD5- plasma cells that occupy a distinct survival niche in bone marrow. J.Immunol. 194: 231–242.

51. Flicker, S., and R. Valenta. 2003. Renaissance of the blocking antibody conceptin type I allergy. Int. Arch. Allergy Immunol. 132: 13–24.

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