1. The Journal of Immunology
Neonatal Exposure to Pneumococcal Phosphorylcholine
Modulates the Development of House Dust Mite Allergy
during 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.
I
n the past few decades, there has been a dramatic rise in the
incidence 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 result
from a decreased frequency of childhood infection and perinatal
exposure to microbes, leading to a long-lasting imbalance between
the Th1 and Th2 T cell subsets initiated at this early stage of life
(3). However, empirical data supporting such a mechanism are
conflicting (4, 5). We previously demonstrated that, in early life,
the B cell repertoire diversity is more amenable to change by
bacterial exposure than it is during adult life (6); however, little is
known about the long-term effects of such exposure on allergic
disease 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 immune
system 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 asthmatics
demonstrate sensitivity to indoor allergens derived from the house
dust mite (HDM) species Dermatophagoides pteronyssinus (Der p)
(10, 11). More than 75% of these individuals express IgE-mediated
sensitivity to the protease allergen Der p 1 (12). We and others have
observed that HDM contains phosphorylcholine (PC) epitopes (13,
14) similar to those integrated into the cell wall of Streptococcus
pneumoniae (pneumococcus) bacteria (15). In mice, natural
TEPC15 (T15) idiotype-bearing natural anti-PC Abs generated by
the B1a B cell subset (16) are germline encoded and are protective
against 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 be
protective 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 of
HDM-induced allergic disease during adult life. Analysis of these
mice demonstrated that there was a broad decrease in cellular and
humoral mediators of allergic disease following challenge with
HDM. The results we present argue strongly for a central role of
B cells, and their Ab products, in the protection against the de-
velopment of HDM-induced allergic airway disease.
Materials and Methods
Animals
C57BL/6 and mMT mice purchased from The Jackson Laboratory and
TEPC15 (T15) IgH gene knock-in (KI) mice, initially generated in the
laboratory of K. Rajewsky (University of Cologne) (20), were maintained
under specific pathogen-free conditions using approved animal protocols
from the Institutional Animal Care and Use Committee at the University of
Alabama 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 and
AI100005-02 and initiated with support from a Senior Investigator Award from the
American Association for Asthma Research, previously the Sandler Program for
Asthma Research.
Address correspondence and reprint requests to Dr. John F. Kearney, University of
Alabama at Birmingham, 1825 University Boulevard, Suite 410, Birmingham, AL
35294. 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 Ó 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

2. HDM allergen particulate preparation
Milled natural Der p (RMB84M; Greer Laboratories) HDM powder was
sonicated at 12 W at 4˚C in 30-min increments for 8 h before being passed
through a 10-mm filter. HDM particulates were pelleted at 10,000 rpm for
30 min prior to lyophilization. After weighing, the lyophilized particulates
were 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 size
was determined by flow cytometric comparison with 0.1–1 mm latex beads
as reference particles (Sigma-Aldrich LB1-LB30). HDM particulates were
labeled 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 were
anesthetized using 3–5% isoflurane before being immobilized on a vertical
board using a suture string looped around their upper incisors. The tongue
was extended from the oral cavity using blunt-end forceps so that liquid
could be pipetted into the oral cavity. The nares were manually plugged
to facilitate inhalation of the liquid suspension. Adult mice were challenged
i.t. with 5 mg processed HDM particulate allergen resuspended in 50 mL
PBS. 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 challenge
with HDM allergen, mice were rested for 2 d before euthanization. When
stated, the above procedure was also conducted using Alexa Fluor 647–la-
beled HDM for all sensitizations and subsequent challenges. For in vivo Ab
experiments, mice were given 5 mg Alexa Fluor 647–labeled HDM, Alexa
Fluor 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 Todd
Hewitt Broth supplemented with 0.5% yeast extract (Difco). Pneumococcal
strain 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 at 280˚C until
use. For neonatal immunizations, 3- to 4-d-old C57BL/6 littermate pups
were immunized i.p. with 2 3 107
PFA-fixed pneumococcal strains R36A
or JY2190.
Bronchoalveolar lavage fluid, lung, and mediastinal lymph
node collection
Following sacrifice, trachea were cannulated to extract cellular infiltrates
from the bronchoalveolar space via a 5-ml lavage with PBS. Mice were
perfused by cardiac puncture with PBS plus 1% heparin prior to lung re-
moval. For cell isolation, lungs were minced and treated with 1 mg/ml
collagenase (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 were
minced and treated with 5 mg collagenase plus 50 U DNase (Sigma-
Aldrich) for 40 min at 37˚C. Mediastinal lymph node (MedLN) cells
were 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 with
total IgE in the bronchoalveolar lavage fluid (BALF) and Der p 1–specific
serum IgE. PC-specific ELISAs were conducted using high-binding enzyme
immunoassay/RIA plates (Costar) coated with 2 mg/ml PC-BSA (Biosearch
Tech) and detected with alkaline phosphatase–conjugated goat anti-mouse
IgM, 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 OD
values at 405 nm. Der p 1–specific IgE was determined by coating plates
with 2 mg/ml natural Der p 1 (Indoor Biotechnologies), and Ab was detected
using alkaline phosphatase–conjugated goat anti-mouse IgE (Southern Bio-
technology Associates). Total IgE levels were determined by ELISA by
coating plates with 2 mg/ml rat anti-mouse IgE (Southern Biotechnology;
clone 23G2), and standard curves were prepared with known concentrations of
IgE Abs (Southern Biotechnology; clone 0114-01). IgM bound to HDM
particles was calculated by incubating equal volumes of BALF with anti-Der p
1–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 known
quantities 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 SPECTROstar V Reader (BMG Labtech) at 405 nm.
ELISPOT assay
High-binding flat-bottom enzyme immunoassay/RIA plates (Costar) were
coated 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˚C
and 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 were
digested with a mixture of 1 mg/ml collagenase (Sigma-Aldrich) with 50 U
DNase in 5 mL HBSS (Life Technologies). Single-cell suspensions were
prepared in RPMI 1640 (Life Technologies) supplemented with 2% ultralow
IgG FBS (Life Technologies), and then 5 3 106
cells were added to one well
and diluted 2-fold in RPMI 1640 plus 2% low IgG FCS. Plates were then
incubated 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 alkaline
phosphatase (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 three
times with PBS plus 0.05% Tween and developed for 18 h at 4˚C in substrate
buffer (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 in
dH2O, 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, and
IgA Abs were purified on Sepharose 4B affinity columns coupled with rat
monoclonal anti-IgA. HDM particulates and pneumococcal bacterial vaccine
preparations were stained with purified mAbs BH8 (anti-PC IgM) and S107
(anti-PC IgA), followed by secondary goat anti-mouse FITC Abs against
respective isotypes (Southern Biotechnology). BALF, MedLN, and lung cells
were stained for flow cytometry using fluorochrome-conjugated Abs specific
for 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 from
BD Biosciences, and anti-IgM from eBioscience. Anti–CD11b-Alexa Fluor
488, TC68-Alexa Fluor 647, BH8-Alexa Fluor 647, and PC-BSA (Biosearch
Technologies)-Alexa Fluor 647 were conjugated in our laboratory, accord-
ing to manufacturer’s instructions. Leukocytes were identified using flow
cytometry 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; CD11b2
CD11c+
),
immature DCs (CD11b+
CD11c+
), neutrophils (CD11b+
Ly6G+
), IgE-bound
B cells (B220+
IgE+
), basophils (B2202
IgE+
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 plasmablasts
were detected by gating on viable B220low
IgM+
cells that also expressed
CD138. PC-specific B cells were identified as B cells (B220+
CD19+
) that
bound both PC-BSA-Alexa Fluor 647 and the anti-idiotype reagent TC68
Alexa Fluor 647. T cells (CD3+
CD4+
) from the MedLN were stained with
isotype control Ab or anti-GATA3 Ab in conjunction with the eBioscience
Foxp3 staining buffer kit (00-5523-00). Following restimulation, per-
meabilization, and fixation, MedLN T cells (CD3+
CD4+
) were stained with
isotype control Ab as well as for production of Th1 and Th2 cytokines listed
above. Macrophages (CD11b+
CD11c2
), DCs (CD11b2
CD11c+
), immature
DCs (CD11b+
CD11c+
), and alveolar macrophages (Siglec-F+
CD11c+
) were
also analyzed for uptake of Alexa Fluor 647–labeled HDM. All flow
cytometry analyses were performed on a FACSCalibur (BD Biosciences) or
LSR II (BD Biosciences) and analyzed using FlowJo software (Tree Star).
Single-frequency forced oscillation technique for mechanical
ventilation
Mice were anesthetized with ketamine xylazine (100 mg/kg) and pan-
curonium bromide (0.8 mg/kg), and their tracheas were cannulated with an
18-G tube connected to the inspiratory and expiratory ports of a Flexivent
ventilator (SCIREQ), in which mice were ventilated at a rate of 160 breaths
2 ANTI-PC Abs REDUCE DUST MITE–INDUCED ALLERGIC DISEASE

3. 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 taken
from 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, after
xylene incubation, embedded in paraffin (Leica EG1150H). Six-micron lung
sections were cut (Leica RM2235), rehydrated, and stained with either H&E
(Sigma-Aldrich) or periodic acid–Schiff (Sigma-Aldrich) stain, according
to manufacturer’s instructions, before being dehydrated and mounted in a
xylene-based mounting media (poly-mount xylene). For cryosections, mouse
lungs were inflated with a solution of 50% OCT compound (Tissue-Tek) and
50% 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 unmilled
HDM species Der p (Greer) were cytocentrifuged onto slides and stained with
BH8-Alexa Fluor 647 or isotype control Ab. For BALF stains, identical
volumes of mouse BALF cell suspensions were cytocentrifuged (Shandon
Elliott) onto glass slides at 1000 rpm for 5 min before staining with modified
Wright’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 PBS
bronchoalveolar lavages were pooled from five C57BL/6 mice. Mouse lungs
were perfused and subjected to sterile collagenase digestion, followed
by lymphocyte enrichment with lymphocyte separation medium (Cellgro;
Sigma-Aldrich). BALF cells were seeded at 200 cells/well and lung digests
at 100,000 cells/well in 6-well plates and then rested for 12 h. BALF cells
from naive unmanipulated mice were primarily SiglecF+
CD11c+
alveolar
macrophages, whereas leukocyte preparations from lung digests contained
multiple pulmonary APCs: CD11b+
CD11c2
resident macrophages, CD11c+
CD11b+
DCs, and CD11c+
Siglec-F+
alveolar macrophages. The RAW 264.7
cell 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-HDM was incubated
with 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 seeded
BALF (alveolar macrophages) or lung digests (pulmonary APCs: resident
macrophages, DCs, and alveolar macrophages) and incubated for another
3 h. The percentage of alveolar macrophages, pulmonary APCs, or RAW
264.7 cells that had taken up Alexa Fluor 647–labeled HDM was determined
by flow cytometry in each case.
BALF cytokine analysis
The first milliliter of fluid extracted from mouse BALF was used to measure
the levels of 25 cytokines and chemokines using the Milliplex Map mouse
cytokine/chemokine magnetic bead panel kit (Millipore MCYTOMAG-
70K-PMX), according to the manufacturer’s instructions. The data were
analyzed using the Milliplex Analyst 5.1 software.
T cell restimulations
MedLN cells were separated by mechanical disruption and incubated
with 0.081 mM PMA, 1.34 mM ionomycin (eBioscience Cell Stimulation
Cocktail 00-4970), and 3.0 mg/ml brefeldin A (eBioscience 00-4506-51)
for 6 h. Following restimulation, cells from the MedLN were treated
with the BD Cellular Fixation/Permeabilization Kit (554714), according
to manufacturer’s instructions, before staining with Abs directed against
the 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 below
were performed with Prism 4.0 software (GraphPad). Comparison of three
or more groups was performed by a one-way ANOVA test, followed by
Tukey’s post hoc analysis. Data from only two groups were analyzed by
a 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.
Results
Neonatal exposure to PC-bearing R36A reduces infiltration of
cells into the BALF and lungs following adult exposure to
HDM
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 on
a panel of bacteria. In this work, we found that PC-specific IgM and
IgA Abs bound small milled and sonicated HDM particles in addition
to intact HDM (Fig. 1C–E). Because the bacterially induced anti-PC
Abs reacted with a significant human allergen, we asked whether
early-life microbial PC exposure could reduce the development of
HDM-induced allergic disease (Fig. 1F). Three-day-old individual
C57BL/6 littermate mouse pups were immunized with R36A, a PC-
bearing unencapsulated pneumococcus strain (Fig. 1A); JY2190, an
unencapsulated PC-deficient pneumococcal mutant (Fig. 1B); or
treated with PBS alone. In this study, we used littermate mice to
avoid the variability in immune responses resulting from differing
commensal microbiota among separately caged mice. In addition to
these 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 HDM
on day 0 and then challenged i.t. with HDM particulates daily on
days 7–11 to induce allergic disease (Fig. 1F). Mice were eutha-
nized 14 d following initial sensitization (day 0), and all analyses
were 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, and mast cells into
both the bronchoalveolar space and the pulmonary parenchyma
(Figs. 1G–L, 2A, 2B). BALF from mice immunized with PC-bearing
R36A as neonates and from the T15 KI mice had half as many
T cells, eosinophils, neutrophils, APCs, mast cells, and basophils
infiltrating the bronchoalveolar space following HDM exposure as
adults compared with mice immunized with JY2190 as neonates or
treated with PBS alone (Fig. 1G, 1H). The numbers of alveolar
macrophages collected from all groups of mice were statistically
similar, as they are normally resident cells of the lung, and are not
known to be mobilized in large numbers after HDM exposure
(Fig. 1H). Wright stains of BALF cytospins confirmed that all groups
of mice had similar numbers of alveolar macrophages per field
(Fig. 1I–L). However, fewer neutrophils, eosinophils, and lymphocytes
were detected in the BALF of C57BL/6 mice immunized with R36A
as neonates and the T15 KI mice compared with mice exposed to
JY2190 or PBS as neonates (Fig. 1I–L). Thus, mice immunized with
PC-bearing R36A as neonates and the T15 KI mice had a significantly
decreased infiltration of allergy-associated cells into their bron-
choalveolar space following HDM exposure compared with C57BL/6
mice 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 HDM
sensitization and challenge, we enumerated inflammatory and al-
lergy-associated cells in the pulmonary parenchyma. Both C57BL/6
mice immunized with R36A as neonates and the T15 KI mice
had 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 PBS
early in life (Fig. 2A, 2B). The infiltration of allergy-associated cells
was validated by fluorescence microscopy of cryosections of lungs
from mice exposed to HDM (Fig. 2C–F). C57BL/6 mice vaccinated
The Journal of Immunology 3

4. with R36A as neonates and the T15 KI mice had decreased eo-
sinophilic infiltration surrounding their bronchioles compared with
mice immunized with JY2190 or treated with PBS early in life
(Fig. 2C–F). Because initiation of HDM-induced allergic disease
depends on the activation of DCs in the lung (23), we assessed APC
activation by quantifying alveolar and tissue-resident DC and
macrophage expression of CD86, a MHC-II costimulatory mole-
cule. Tissue-resident APCs, but not alveolar macrophages, in the
pulmonary parenchyma of C57BL/6 mice immunized with R36A
and the T15 KI mice expressed significantly less CD86 compared
with C57BL/6 mice immunized with JY2190 or treated with PBS as
neonates (Fig. 2G, 2H). Therefore, exposure to PC-bearing R36A as
neonates and the presence of anti-PC Abs in T15 KI mice each
significantly inhibited lung-resident APC activation and diminished
the infiltration of allergy-associated cells into the pulmonary pa-
renchyma following adult exposure to HDM compared with
C57BL/6 mice exposed to PC-deficient JY2190 or PBS early in life.
Neonatal exposure to PC-bearing R36A diminishes mediastinal
lymph node T cell activation, IgE secretion, AHR, and allergic
disease development
Upon natural inhalation of allergen, scavenging APCs migrate from
the pulmonary parenchyma and present allergen to naive T cells in
draining lymph nodes to prime a Th2-polarized response (24). To
assess HDM priming of this Th2 response, we collected the me-
diastinal lymph node (MedLN) following HDM sensitization and
challenge. 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 KI
mice each possessed MedLNs that were half the size and weight
of those collected from C57BL/6 mice immunized with JY2190
or treated with PBS as neonates (Fig. 3A, 3B). As expected, the
smaller size of MedLNs corresponded with lower lymph node
cellularity, 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 cells
expressed significantly decreased levels of CD44, a marker of Ag
experience (Fig. 3E–G). Thus, in C57BL/6 mice exposed to PC-
bearing R36A as neonates, and in T15 KI mice, T cell activation in
the MedLNs is inhibited following HDM exposure compared with
C57BL/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 the
amount of total IgE from the BALF and Der p 1–specific IgE in
the serum. Mice immunized with PC-bearing R36A as neonates
and the T15 KI mice each had significantly decreased total IgE
levels in their lung (Fig. 4A) and Der p 1–specific IgE in the serum
(Fig. 4B) compared with C57BL/6 mice exposed to PC-deficient
JY2190 or PBS early in life.
Th2-associated cytokines that induce IgE production are also
significant contributors to the AHR development, production of
mucus in bronchioles, and recruitment of allergy-mediating cells
around bronchioles (26). To assess AHR development, following
HDM exposure, mice were mechanically ventilated and a forced
oscillation technique was used to measure total resistance of the
respiratory system after vaporized doses of methacholine (Fig. 4C).
A B C
D 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

5. 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 received
HDM (Fig. 4C). These C57BL/6 mice had significantly lower air-
way resistance prior to methacholine exposure and following the
highest dose (50 mg/ml) of methacholine compared with mice
treated 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 in
bronchioles 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 in
number compared with C57BL/6 mice exposed to JY2190 or PBS
as neonates (Fig. 4D, 4F). H&E-stained lung sections of naive
mice not treated with HDM were free of inflammatory infiltrates
(results not shown). The extensive leukocytic infiltrates associated
with the bronchioles of PBS- or JY2190-immunized HDM-
exposed C57BL/6 mice (Fig. 4E, 4G) were not observed around
the bronchioles of mice first treated with R36A as neonates or of
the T15 KI mice (Fig. 4I, 4K). These results demonstrate that
neonatal 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 with
mice treated with PC-deficient JY2190 or PBS as neonates.
IgM-secreting PC-specific B cells dominate the local immune
response to HDM among mice immunized with PC-bearing
R36A as neonates
Although mice immunized with R36A as neonates and the T15 KI
mice have reduced numbers of allergy-associated cells infiltrating
their lungs, they had approximately twice as many B cells in their
pulmonary parenchyma following HDM exposure compared with
mice first treated with JY2190 or PBS as neonates (Fig. 5A). More
than 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 immunized
with R36A as neonates and in T15 KI mice were higher compared
with those in C57BL/6 mice immunized with JY2190 as neonates
(Fig. 5C, 5D). ELISPOT analysis demonstrated that C57BL/6 mice
immunized with R36A as neonates and T15 KI mice have more
than 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 were
stained for IgM (green) and laminin (gray) (Fig. 5I–L). C57BL/6
mice immunized with R36A early in life and the T15 KI mice both
had higher numbers of IgM+
cells proximal to their bronchioles
compared with mice immunized with JY2190 (Fig. 5H–L). Thus,
mice immunized with PC-bearing R36A as neonates and the T15 KI
mice had an increased frequency of IgM-expressing B cells in their
lungs located proximal to their bronchioles compared with mice
treated 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 repeated
HDM exposure revealed that mice immunized with R36A early in
life had ∼10-fold more PC-specific IgM-secreting cells in their
lungs compared with mice immunized with JY2190 as neonates
(Fig. 6A, 6B). By contrast, none of the groups of mice analyzed had
detectable numbers of PC-specific IgA-secreting cells in their lungs
(data not shown). Using flow cytometry, PC-specific B cells were
identified as B cells that bound both PC-BSA and TC68, an Ab
reagent 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 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 , 0.05, **p , 0.01, and ***p , 0.001.
The Journal of Immunology 5

6. neonates and the T15 KI mice had an increased percentage of PC-
specific B cells in their lung compared with mice immunized with
JY2190 or PBS as neonates (Fig. 6D). These PC-specific B cells
were stained for CD5 and CD138 expression to determine their B1a
or 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 further
determine the phenotype and distribution of these PC-specific
B cells following HDM exposure, we stained lung cryosections
with 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 CD44high
CD4+
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 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 , 0.05, **p , 0.01, and ***p , 0.001.
PBSJY2190R36AT15 KI
0
100
200
300
*
***
PBSJY2190R36AT15 KI
0.0
0.5
1.0
1.5
ng/mLTotalIgE
*
**
0 10 20 30 40 50
1
2
3
4
Vaporized Methacholine (mM)
Rrs(cmH2O.s/mL)
**
D
E
F
G
H
I
J
K
C
100 m
50 m
50 m
PBS R36A T15 KIJY2190
Vaporized Methacholine (mg/mL)
Rrs(cmH2O.s/mL)
PBS
JY2190
R36A
T15 KI
No HDM
TotalIgE(ng/mL)
PBS JY2190 R36A T15 KI PBS JY2190 R36A T15 KI
Derp1-IgE(ng/mL)
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

7. and anti-laminin (gray) (Fig. 6G–J). IgM+
CD138+
cells were de-
tected in lung sections from mice treated with JY2190 or PBS
as 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 frequency
than those identified in PBS-treated mice (Fig. 6G) or those im-
munized with JY2190 as neonates (Fig. 6H). Mice immunized
with R36A as neonates also had some clusters of non-PC–binding
IgM-expressing cells in their lungs (data not shown). Thus, mice
immunized with PC-bearing R36A as neonates and the T15 KI
mice had an increased frequency of CD138+
IgM-expressing PC-
specific cells in their lungs following exposure to HDM compared
with mice immunized with PC-deficient JY2190 or treated with
PBS 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 mice
were undetectable (data not shown). However, following repeated
HDM exposure, mice immunized with R36A as neonates secreted
higher amounts of PC-specific IgM and IgG3 Ab, but not IgA, into
their bronchoalveolar spaces compared with those immunized
with JY2190 (Fig. 6C and data not shown). Thus, in mice exposed
to PC-bearing R36A early in life and in T15 KI mice, there was an
increased secretion of PC-specific Abs into the bronchoalveolar
space compared with mice treated with PC-deficient JY2190 or
PBS as neonates.
Anti-PC IgM Abs decrease ex vivo uptake of HDM by resident
APCs and the in vivo trafficking of HDM to the MedLN and
priming of a Th2 response
In the presence of complement, anti-PC IgM Abs are efficient at
promoting 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
F
G 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

8. (ISO) were administered i.t. to mice along with Alexa Fluor 647–
labeled HDM. Uptake of HDM by APCs in the lung was measured
by flow cytometry at 24 h. Purified anti-PC IgM mAb (BH8) de-
creased the interaction of HDM with alveolar macrophages, resident
DCs, and macrophages in the lung by about half compared with
mice treated with HDM and isotype control (ISO) or HDM alone
(Fig. 7C). Alveolar macrophages collected from the BALF of mice
treated with HDM plus anti-PC IgM Ab also expressed decreased
amounts of CD86 compared with mice treated with HDM plus
isotype control Ab (Fig. 7C). Collectively, these results demonstrate
that anti-PC IgM interferes with the uptake of HDM by APCs
in 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 the
BALF and quantified the amount of IgM bound to these particles.
Significantly more IgM was bound to HDM from the BALF of
C57BL/6 mice immunized with R36A and from T15 KI mice than
was 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–labeled
HDM. Labeling of HDM with Alexa Fluor 647 did not affect the
outcome of HDM-induced allergic disease (data not shown). We
observed a decreased percentage of alveolar macrophages, and
lower numbers of resident DCs, macrophages, and neutrophils in
the lungs of C57BL/6 mice immunized with R36A and the T15 KI
mice 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 KI
0
3
6
50
100
150
200
*
**
PBSJY2190R36AT15 KI
0
10
20
30
ng/mLanti-PCIgM
**
*
NS
CD138
F B220+
PBS
JY2190
R36A
T15 KI
PBS JY2190 R36A T15 KI
ng/mLanti-PCIgM
%ofMax
C
Spotsper5x106Cells
PBS JY2190 R36A T15 KI
A PBS JY2190
R36A T15 KI
B
T15 KI
JG H IPBS JY2190 R36A IgM
V
V
Br
Br
V
D
E
PC-BSA
TC68
PBS JY2190 R36A T15 KI
TC68+PC-BSA+
PercentofBCells
PBS
JY2190
R36A
T15 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 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 and **p , 0.01.
8 ANTI-PC Abs REDUCE DUST MITE–INDUCED ALLERGIC DISEASE

9. 4 times lower percentage of HDM-bearing DCs in the MedLN
compared with DCs isolated from the MedLN of C57BL/6 mice
immunized with JY2190 as neonates (Fig. 7G, 7I). To determine
whether early exposure to R36A influences the degree of Th2
priming in adults, we quantified the number of MedLN T cells
expressing 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
H
D
M
onlyH
D
M
+ISOH
D
M
+BH
8
M
edia
0.0
0.5
1.0
1.5
CD86MFI(x103
)
***
Alv Mac Pulm APCsMac cell line
0
5
10
20
40
60
80
HDM-ISO
HDM-10 BH8
HDM-20 BH8
*
**
*
**
*
**HDM only
0
20
40
60
80
IgM(ng/mL)CoatedHDM
***
***C D
Alv Mac DC Neuts
0.0
0.2
0.4
1
2
3
4
5
HDMPosCells(x104
)
*
****
*
0.0
0.1
0.2
0.3
1
2
3
4
***
***
0
1
2
3
***
**
GATA3+TCells(x104)
PBS R36A T15 KIJY2190
IgM(ng/mL)CoatedHDM
E
F G H
I
MedLN MedLN
HDMPositiveDCs(x104)
BALF
PBS
JY2190
R36A
T15 KI
HDM
BH8
ISO
HDM
CD11c
CD86
BA HDMPositiveCells(%)
%ofMax
CD86MFI(x103)
Alv Mac Pulm APCs Mac cell line
4
3
2
1
1 HDM
2 HDM+ISO
3 HDM+BH8
4 Media
Alv Mac DCs Macs
0
2
4
5
10
15
20
HDMPositiveCells(%)
*
***
*
0
1
2
3
4
CD86MFI(x103
)
**
Alv Mac Alv Mac
0
20
40
60
80
100
HDMPositive(%)
**
*
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

10. with JY2190 or treated with PBS as neonates (Fig. 7H). These
results demonstrate that mice immunized with PC-bearing R36A as
well as the T15 KI mice have decreased HDM trafficking by DCs
and a significantly inhibited generation of a Th2 response in the
MedLN compared with mice treated with PC-deficient JY2190 or
PBS as neonates.
Early microbial exposure does not influence the Th1 or Th2
T cell subset balance
It has been hypothesized that early microbial exposures generate
a robust Th1 T cell response capable of suppressing the overt
development of Th2 responses (3, 4). BALF collected from naive
unmanipulated mice prior to HDM exposure did not contain
detectable levels of cytokines or chemokines (data not shown).
Following HDM exposure, the C57BL/6 mice immunized with
either pneumococcal strain as neonates had statistically similar
levels of Th1-associated cytokines, such as IL-2, IL-12, and IFN-g,
in the airways (Fig. 8A). There was also no statistical difference in
the frequency of IL-2–, IL-12–, or IFN-g–producing MedLN
T cells (Fig. 8D). However, the lungs of C57BL/6 mice treated
with R36A as neonates and the T15 KI mice contained signifi-
cantly lower concentrations of Th2-associated cytokines, such as
IL-4, IL-5, IL-6, IL-13, and IL-9 compared with mice exposed to
JY2190 or PBS as neonates (Fig. 8B). These protected mice also
had a significantly decreased frequency of IL-4–, IL-6–, and IL-
13–producing T cells in their MedLN (Fig. 8E). Therefore, mice
immunized with PC-bearing R36A as neonates secrete signifi-
cantly decreased Th2-associated cytokines in their airways and
had a significantly reduced frequency of Th2 cytokine-producing
cells 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 those
immunized with JY2190 or with R36A, as well as T15 KI mice
demonstrated 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 as
neonates, is not sufficient to significantly decrease the production
of 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 of
cells secreting Th2-associated cytokines or impaired production
of chemokines that recruit these cells, we quantified the level of
chemokines produced in the lung that are potentially capable of
recruiting Th2-associated cells into the lung. C57BL/6 mice im-
munized with R36A as neonates and the T15 KI mice secreted
decreased concentrations of CXCL1, CXCL2, RANTES, and
CCL4 compared with C57BL/6 mice exposed to JY2190 or PBS
as neonates (Fig. 8C). Therefore, neonatal immunization with PC-
bearing R36A leads to decreased chemokine secretion into mouse
lungs compared with immunization with PC-deficient JY2190.
These observations suggest that immunization with PC-containing
formulations can decrease the influx of allergy-associated cells
into the lung.
Discussion
There is a clear association between the rise in incidences of atopic
conditions 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 subsets
has long been proposed (3, 4, 33), empirical evidence to support this
claim has been confounding. Less effort has been made to under-
stand how the developing neonatal immune system is affected by
early exposure to microbes. We, and others, have demonstrated that
neonatal 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 cells
initiate multiple manifestations of allergic pathologies, and are thus
clearly 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-g
0.000
0.005
0.010
0.015
0.2
0.4
0.6
0.8
5
IL-4 IL-6 IL-13
0.0
0.5
1.0
1.5
2.0
2.5
CellsintheMLN(x105)
JY2190
R36A
B6
T15
N.D. N.D. N.D.
***
*
**
IL-2 IL-12p70 IFNg
0
1
2
3
4
5
CytokinesintheBALF(pg/mL)
IL-4 IL-5 IL-6 IL-13 IL-9
0
1
2
3
20
40
60
CytokinesintheBALF(pg/mL)
* ***
* * *
**
*
CXCL1CXCL2RANTESCCL4
0
2
4
10
20
30
CytokinesintheBALF(pg/mL)
*
**
***
*
***
*
A B C
IL-2 IL-12p70 IFN- IL-4 IL-5 IL-6 IL-13 IL-9 CXCL1 CXCL2 RANTES CCL4
BALFCytokines(pg/mL)
BALFCytokines(pg/mL)
BALFCytokines(pg/mL)
PBS
JY2190
R36A
T15 KI
CellsintheMedLN(x105)
CellsintheMedLN(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

11. Ab products, can exert an opposing effect to this accepted patho-
logical function by impeding the development of allergic disease in
response 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 in
the pulmonary parenchyma, and higher concentrations of secreted
Abs in their lung following exposure to HDM compared with mice
exposed to PC-deficient pneumococcus. When passively admin-
istered or endogenously produced, these anti-PC IgM Abs reduced
the interaction of HDM with APCs in the lung. Increased anti-PC
IgM Ab titers were also associated with decreased trafficking of
the allergen to the draining lymph node and significantly reduced
a Th2 T cell response. Because Th2-associated cytokines induce the
pathology and inflammation associated with allergic disease (36),
we hypothesize that the diminished priming of a Th2 response in
the MedLN was directly responsible for the observed decrease in
IgE production and the recruitment of allergy-associated inflam-
matory mediators into the lung. This effect was accompanied by
a moderate development of airway resistance, inhibited production
of 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 expressed
lower levels of cell surface markers indicative of inflammation and
activation. Thus, a single immunization of neonatal mice with PC-
bearing pneumococcus 3 d after birth had a potent and long-lasting
impact on susceptibility to HDM-induced allergic inflammation in
adult mice (Fig. 8F).
A tenet of the hygiene hypothesis is that microbial exposure dur-
ing development tempers the initiation of allergic diseases later in
adult life via a global redirection of Th2 to Th1 responses (1, 4, 37).
However, C57BL/6 mice immunized with PC-deficient JY2190
pneumococcus as neonates developed manifestations of allergic
disease, upon HDM challenge, similar to those seen in mice treated
with PBS. Additionally, the limited development of allergic disease
observed in the T15 KI mice that had received no deliberate
bacterial exposure, which could alter the Th1/Th2 balance,
also strongly supports a protective contribution of bacterial-induced
specific Ab in this model of HDM-induced allergic disease. Based
on these findings, we infer that neonatal exposure to PC-bearing
pneumococcus 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 cells
and Abs play a pivotal role in the protection against allergic airway
disease development, we immunized 3-d-old B cell–deficient
mMT mice with PC-bearing pneumococcus R36A, or PC-deficient
JY2190, or PBS alone (Supplemental Fig. 2). At 8 wk of age,
these mice were challenged with HDM. mMT mice challenged
with HDM developed similar allergic manifestations whether they
had been treated with PBS, or immunized with JY2190 or R36A
as neonates. Compared with our observations in similarly treated
C57BL/6 mice, these results demonstrate that R36A-triggered pro-
tection against HDM-induced allergic disease does not occur in the
absence of B cells (Supplemental Fig. 2).
Our studies also demonstrate that the decreased development of
HDM-induced allergy by early bacterial exposure required the PC
epitope to be expressed by both the challenging bacteria and the
allergen. The T15 KI mice and C57BL/6 mice treated with pneu-
mococcal strains JY2190 or R36A were not protected from allergic
disease resulting from repeated exposure to the PC-deficient timothy
grass pollen allergen (data not shown). We also investigated the sig-
nificance of timing of microbial exposure. Adult mice immunized
with either strain of pneumococcus exhibited similar development of
HDM-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). We
further determined whether anti-PC IgM Abs were sufficient to
dampen the development of HDM-induced allergic disease. Mice
administered anti-PC IgM Abs 1 h prior to sensitization and every
challenge with HDM displayed significantly decreased development
of HDM-induced allergic disease compared with mice administered
isotype control Ab (Supplemental Fig. 4). Thus, our combined ob-
servations of 1) the protective effects elicited by neonatal exposure to
PC-bearing pneumococcus but not PC-negative bacteria in B cell–
sufficient mice; 2) the optimal protection in T15 KI mice that have
not been deliberately immunized with PC-containing bacteria; 3) the
lack of protection observed in mMT mice; and 4) the protection
afforded by passive administration of highly purified IgM anti-PC
mAbs argue strongly for the central role for B cells and IgM Abs in
protection against the development of allergic airway disease in this
HDM 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 with
HDM particles can potentially ligate these PC-recognizing innate
receptors, CD36 and platelet-activating factor receptor may pro-
vide entry points for phagocytosis on cells that express these
proteins. Subsequent processing could then prepare the Der p 1
allergenic cargo for presentation, resulting in the activation of Th2
T cells. Although numerous pulmonary cells bearing PC-ligating
receptors are potentially involved in the initiation and development
of allergic disease, DCs are specifically essential in the development
of HDM-induced allergic disease (40). Thus, interference of HDM
allergen uptake by the DCs may be one way that anti-PC Abs
disrupt a critical step in the induction of allergic disease. Thus, we
hypothesize that Abs, by impeding the interaction of PC-decorated
HDM 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 developed
countries (41). Despite the recent introduction of neonatal vacci-
nation with Prevnar 13 and decreased infection rates of the
serotype-specific pneumococcal strains covered by this vaccine
(42), serotype replacement is prevalent (43), with the consequence
that pneumococcal infections still occur, even in developed
countries. Neonatal responses to purified polysaccharides are poor,
but, in children colonized or infected with pneumococcus, some
mount high Ab responses to PC, even at 2–3 y of age, whereas
others do not (44). Whether childhood pneumococcal infections
correlate 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 serotypes
compared with asthmatics, and when PBMCs from these non-
asthmatics were cultured with HDM, they released less IL-5 than
those from asthmatics (46). Many other organisms such as Neisseria
sp, Haemophilus influenzae, Pseudomonas aeruginosa, and hel-
minths also contain PC epitopes (39). Helminth infections are in-
versely correlated with allergic disease incidence (47). This inverse
correlation has mostly been attributed to the potent induction of
regulatory T cells; however, alternative mechanisms such as an Ab
response to PC-associated glycoprotein, ES-62, could also be
modulating 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

12. Although our PC-specific B cells do not express CD5, B1a cells lose
their CD5 expression as they become plasma cells (50); therefore,
we cannot exclude the possibility that B1a cells induced or expanded
by our PC-based vaccine modulate the development of HDM-
induced allergic disease. Currently, there is no Food and Drug
Administration–approved PC-based vaccine or cure for allergies or
asthma, and management of most atopic diseases involves treat-
ment of symptoms. Immunotherapy has successfully induced allergen
tolerance; however, this anti-inflammatory response is restricted to
the highly variable peptides and extracts administered during im-
munotherapy. Immunotherapy generates blocking Abs that can po-
tentially inhibit allergen-induced mediator release from mast cells
and basophils or Ag presentation to T cells (51). However, the
mechanism by which these Abs reduce allergic disease development
is limited to the Ag specificity of IgE Abs, and would most likely
only function after IgE generation and progression of allergic dis-
ease. Because we have shown that the PC-specific B cells generated
by neonatal exposure to PC-containing bacteria have the potential to
inhibit HDM-induced allergic disease development, we suggest that
a regimen of suitable vaccine exposure, informed microbiota ma-
nipulation, or probiotic use in at-risk children could be used to
prevent the development or further progression of respiratory aller-
gies and atopic diseases later in life.
Acknowledgments
We 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.
Disclosures
The authors have no financial conflicts of interest.
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