Modulation of Allergic Airway Inflammation by the Oral Pathogen Porphyromonas gingivalis
Accumulating evidence suggests that bacteria associated with periodontal disease may exert systemic immunomodulatory effects. Although the improvement in oral hygiene practices in recent decades correlates with the increased incidence of asthma in developed nations, it is not known whether diseases of the respiratory system might be influenced by the presence of oral pathogens. The present study sought to determine whether subcutaneous infection with the anaerobic oral pathogen Porphyromonas gingivalis exerts a regulatory effect on allergic airway inflammation. BALB/c mice sensitized and subsequently challenged with ovalbumin exhibited airway hyperresponsiveness to methacholine aerosol and increased airway inflammatory cell influx and Th2 cytokine (interleukin-4 [IL-4], IL-5, and IL-13) content relative to those in nonallergic controls. Airway inflammatory cell and cytokine contents were significantly reduced by establishment of a subcutaneous infection with P. gingivalis prior to allergen sensitization, whereas serum levels of ovalbumin-specific IgE and airway responsiveness were not altered. Conversely, subcutaneous infection initiated after allergen sensitization did not alter inflammatory end points but did reduce airway responsiveness in spite of increased serum IgE levels. These data provide the first direct evidence of a regulatory effect of an oral pathogen on allergic airway inflammation and responsiveness. Furthermore, a temporal importance of the establishment of infection relative to allergen sensitization is demonstrated for allergic outcomes.
INFECTION AND IMMUNITY, June 2010, p. 2488–2496 Vol. 78, No. 6
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Modulation of Allergic Airway Inﬂammation by the
Oral Pathogen Porphyromonas gingivalis
Jeffrey W. Card,
† Michelle A. Carey,
† James W. Voltz,
J. Alyce Bradbury,
Catherine D. Ferguson,
Eric A. Cohen,
Gordon P. Flake,
Daniel L. Morgan,
Samuel J. Arbes, Jr.,
David A. Barrow,
Silvana P. Barros,
and Darryl C. Zeldin
Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health,
Research Triangle Park, North Carolina,
and Department of Periodontology and Center for Oral and
Systemic Diseases, School of Dentistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
Received 10 November 2009/Returned for modiﬁcation 14 December 2009/Accepted 12 March 2010
Accumulating evidence suggests that bacteria associated with periodontal disease may exert systemic im-
munomodulatory effects. Although the improvement in oral hygiene practices in recent decades correlates with
the increased incidence of asthma in developed nations, it is not known whether diseases of the respiratory
system might be inﬂuenced by the presence of oral pathogens. The present study sought to determine whether
subcutaneous infection with the anaerobic oral pathogen Porphyromonas gingivalis exerts a regulatory effect on
allergic airway inﬂammation. BALB/c mice sensitized and subsequently challenged with ovalbumin exhibited
airway hyperresponsiveness to methacholine aerosol and increased airway inﬂammatory cell inﬂux and Th2
cytokine (interleukin-4 [IL-4], IL-5, and IL-13) content relative to those in nonallergic controls. Airway
inﬂammatory cell and cytokine contents were signiﬁcantly reduced by establishment of a subcutaneous
infection with P. gingivalis prior to allergen sensitization, whereas serum levels of ovalbumin-speciﬁc IgE and
airway responsiveness were not altered. Conversely, subcutaneous infection initiated after allergen sensitiza-
tion did not alter inﬂammatory end points but did reduce airway responsiveness in spite of increased serum
IgE levels. These data provide the ﬁrst direct evidence of a regulatory effect of an oral pathogen on allergic
airway inﬂammation and responsiveness. Furthermore, a temporal importance of the establishment of infec-
tion relative to allergen sensitization is demonstrated for allergic outcomes.
A causative relationship between decreased microbial expo-
sure and infection in recent decades and the concurrent in-
crease in asthma prevalence in developed countries has been
suggested and is thought to be attributable, at least in part, to
a phenomenon known as the hygiene hypothesis (30). Origi-
nally put forth by Strachan (32), the hypothesis proposes that
increased cleanliness of modern industrialized societies has
resulted in decreased exposure to bacterial, viral, and other
immunomodulatory organisms and their products, particularly
in early life, and that this has in turn resulted in a loss of
potentially protective effects of these exposures on the devel-
opment of allergic diseases. Accumulating clinical and exper-
imental evidence largely supports the hygiene hypothesis as it
relates to asthma, although a consensus has not been reached.
As reviewed recently (31), a variety of infections of a viral,
bacterial, and parasitic nature inﬂuence the host immune re-
sponse, such that regulation of the Th1-Th2 balance is modi-
ﬁed to promote Th1 responses and impede Th2 responses,
thereby reducing Th2-mediated allergic outcomes. However,
this is likely a simplistic view of the effects of infections on
immune system development and responses, and other factors,
including host genetic makeup and timing of exposures to the
infective agent relative to allergen exposure, undoubtedly con-
tribute to the overall allergic phenotype.
In addition to the inﬂuence of environmental exposure to
microbes, the potential regulation of allergic diseases by the
microﬂora of the host is receiving increased attention. Evi-
dence suggests that the composition of the gastrointestinal
microﬂora differs between individuals with and without allergy
(reviewed in reference 25), and disruption of the normal gut
microﬂora by antibiotic administration leads to allergic airway
responses following allergen challenge in mice not previously
sensitized to the allergen (24). Moreover, although similar
beneﬁts have not yet been demonstrated in humans, the oral
administration of probiotic bacteria was recently shown to de-
crease allergic airway inﬂammation in mice (8, 9).
As in the gut, the microenvironment of the oral cavity is
complex and comprises hundreds of bacterial species. Porphy-
romonas gingivalis, a Gram-negative opportunistic periodontal
pathogen, can initiate periodontal lesions in nonhuman pri-
mates when introduced into the periodontal microbiota (15)
and is a major etiological agent in severe forms of periodontal
disease such as chronic periodontitis (21). Interest in chronic
oral infections and their potential role in adverse systemic
health effects has been heightened by observations of positive
associations between serum concentrations of antibodies to
oral pathogens such as P. gingivalis and the incidence of car-
diovascular diseases and renal dysfunction (3, 19, 28, 29). Re-
cently, however, an inverse relationship between serum con-
centrations of antibodies to P. gingivalis and the prevalence of
asthma, wheeze, and hay fever was observed in a representa-
* Corresponding author. Mailing address: NIH/NIEHS, 111 T. W.
Alexander Drive, Building 101, Room A222, Research Triangle Park,
NC 27709. Phone: (919) 541-1169. Fax: (919) 541-4133. E-mail: zeldin
† J.W.C. and M.A.C. contributed equally to this work.
Published ahead of print on 22 March 2010.
tive sample of the population of the United States (2). Fur-
thermore, a signiﬁcant inverse association between periodon-
titis and the incidences of hay fever and allergy to house dust
mites was reported for a northeast German population, with a
borderline signiﬁcant inverse association between periodontitis
and asthma also observed (10). While limitations of these ob-
servational studies include potential recall bias pertaining to
asthma symptoms and the inability to directly assess cause and
effect, these ﬁndings nonetheless suggest a potential protective
effect of infection with oral pathogens such as P. gingivalis on
In order to examine the inﬂuence of oral pathogens on the
development of allergic airway disease under controlled experi-
mental conditions, the present study sought to determine whether
infection with P. gingivalis modiﬁed allergic outcomes in a murine
model of asthma. To accomplish this, a subcutaneous chamber
model was employed wherein mice were subjected to a local
infection with live P. gingivalis either before or after sensitization
to allergen, and the effects of this infection on subsequent re-
sponses to allergen challenge were assessed. The results indicate
that P. gingivalis infection exerts a modulatory effect on allergic
airway responses and that this effect is dependent on the timing of
infection relative to allergic sensitization.
MATERIALS AND METHODS
Animals and treatments. All studies were conducted in accordance with the
principles and procedures outlined in the National Institutes of Health Guide for
the Care and Use of Laboratory Animals (23a) and were approved by the Animal
Care and Use Committee of the National Institute of Environmental Health
Sciences and by the Animal Welfare Committee and the Institutional Animal
Care and Use Committee of the University of North Carolina at Chapel Hill.
Female BALB/c mice (6 weeks of age; Taconic Farms, Germantown, NY) were
used in an ovalbumin (OVA)-induced allergic airway inﬂammation model. Mice
were sensitized with an intraperitoneal injection of 20 g OVA (grade V; Sigma,
St. Louis, MO) in 0.2 ml aluminum hydroxide adjuvant (Alhydrogel; Accurate
Chemical, Westbury, NY) on two consecutive days. Subsequent exposure to
aerosolized OVA occurred on ﬁve consecutive days (1% aerosol in phosphate-
buffered saline [PBS] for 30 min per day), and assessment of respiratory me-
chanics and collection of bronchoalveolar lavage (BAL) ﬂuid and tissue samples
occurred 24 h following the ﬁnal aerosol exposure.
To assess the inﬂuence of infection with P. gingivalis on the development of
OVA-induced pulmonary allergic responses, mice were exposed to live P. gingi-
valis before or after sensitization to OVA, using a previously described subcu-
taneous chamber model system (12, 22, 23). Brieﬂy, a single cylindrical coiled-
spring chamber made of stainless steel was surgically implanted in the
dorsolumbar region of each mouse. Two weeks later, mice were immunized by
intrachamber injection of 0.1 ml of suspension containing 10
gingivalis organisms. Two weeks following this, chambers were injected with 0.1
ml of suspension containing 10
live P. gingivalis cells in order to establish a
localized infection. Injection of chambers with live bacteria occurred either
before or after sensitization to OVA in order to assess the inﬂuence of P.
gingivalis infection on allergic airway responses to subsequent allergen exposure.
Some mice were not sensitized or challenged with OVA but were exposed to
heat-killed and live bacteria (infection only), some were not exposed to any
bacteria but were sensitized and challenged with OVA (allergic group), and some
were exposed only to heat-killed bacteria either before or after OVA sensitiza-
tion. Details of the experimental groups and timing of the various procedures are
provided in Fig. 1.
Preparation of bacterial suspensions. A stock of P. gingivalis strain A7436 was
stored in Wilkins Chalgren anaerobic broth (WC broth; DSMZ, Braunschweig,
Germany) containing 10% skim milk at ⫺80°C. Bacteria were cultivated in WC
broth at 37°C in an anaerobic chamber (Coy Laboratory Products Inc., Ann
Arbor, MI) with 5% H
, 10% CO
, and 85% N
. Bacterial suspensions were
FIG. 1. Experimental groups and timeline of study. HK, heat killed; P.g., P. gingivalis; OVA, ovalbumin. Six-week-old female BALB/c mice were
cage acclimated for 1 week prior to chamber implantation surgery and thus were approximately 7 weeks old at “day 0.”
OL. 78, 2010 ORAL BACTERIA AND ALLERGIC LUNG INFLAMMATION 2489
prepared from primary cultures at the log phase of growth. Bacterial concentra-
tion was evaluated by spectrophotometry (Cecil Instruments Ltd., Cambridge,
United Kingdom), with a measured optical density at 600 nm of 1.0 correspond-
ing to 10
bacteria/ml, and adjusted to the desired treatment concentration by
dilution with broth.
Analysis of lung function and airway responsiveness. Respiratory mechanics
and airway responsiveness to aerosolized methacholine were determined on day
54 of study (24 h following the last exposure to aerosolized OVA), using invasive
analysis with a FlexiVent mechanical ventilator system (SCIREQ, Montreal,
Canada) as described previously (5). Total respiratory system resistance (R) was
determined at baseline and in response to increasing concentrations of aerosol-
ized methacholine (0 to 25 mg/ml in PBS), and the provocative concentration of
methacholine aerosol resulting in a 200% increase (PC200) over the baseline
value of R was calculated.
Tissue collection and sample analysis. Immediately following lung function
assessment, mice were removed from the ventilator and a blood sample was
drawn from the abdominal aorta. Serum was subsequently extracted, frozen, and
stored at ⫺80°C. BAL was performed with two 1.0-ml aliquots of Hanks’ bal-
anced salt solution; recovery was ⬎75% for each mouse. Recovered BAL ﬂuid
was processed and analyzed for total and differential cell counts by routine
methods, and aliquots of cell-free BAL ﬂuid were frozen and stored at ⫺80°C.
The right bronchus was ligated, and right lungs were removed, frozen, and stored
at ⫺80°C. The left lungs were then inﬂated with 0.4 ml paraformaldehyde that
was injected slowly over approximately 10 s via a blunt needle that was inserted
into the trachea. The tracheas were immediately tied off with sutures, and the
lungs were submersed in 4% paraformaldehyde and used for preparation of
slides for histopathological evaluation.
Histopathological evaluation of inﬂammation was performed on sections of
left lung that were stained with hematoxylin and eosin. The evaluation was
conducted by a pathologist in a blinded manner such that the treatment group
and animal information were not known. An inﬂammatory score was calculated
for each sample, based on microscopic assessment of the extent and severity of
inﬂammation. Brieﬂy, perivascular, peribronchial, and pleural inﬂammation lev-
els were each assigned a score of 0 to 3. Perivascular inﬂammation was scored as
follows: 0, none; 1, occasional inﬂamed vessels or mild inﬂammation only; 2,
frequent inﬂamed vessels, but ⬍50% of vessels inﬂamed, or ⬎50% of vessels
inﬂamed, but with predominantly mild inﬂammation; and 3, ⬎50% of vessels
showing moderate or marked inﬂammation. Peribronchial inﬂammation was
scored as follows: 0, none; 1, occasional inﬂamed bronchi; 2, frequent inﬂamed
bronchi, but ⬍50% of bronchi inﬂamed, or ⬎50% of bronchi inﬂamed, but with
many showing very mild inﬂammation; 3, ⬎50% of bronchi showed mild to
moderate or marked inﬂammation. Pleural inﬂammation was scored as follows:
0, none; 1, mild and patchy inﬂammation; 2, inﬂammation was moderate in
intensity and frequent in distribution; 3, either moderate and frequent inﬂam-
mation with the presence of polypoid foci, the presence of foci with marked
pleuritis, or diffuse involvement of the pleura. The total score (range of 0 to 9),
representing the sum of the individual scores, was calculated for each animal and
averaged for each experimental group.
Levels of interleukin-4 (IL-4), IL-5, IL-13, and granulocyte-macrophage col-
ony-stimulating factor (GM-CSF) in BAL ﬂuid and serum were determined with
a Bio-Plex mouse cytokine kit (Bio-Rad, Hercules, CA), using ﬂuorescently
labeled microsphere beads and a Bio-Plex suspension array system (Bio-Rad)
according to the manufacturer’s instructions. Total serum IgE content was quan-
tiﬁed by enzyme-linked immunosorbent assay (ELISA; BD Biosciences, San
Diego, CA) according to the manufacturer’s instructions. OVA-speciﬁc IgE in
serum was quantiﬁed with the same ELISA kit according to the manufacturer’s
instructions, except for the following two modiﬁcations: plates were coated
overnight with OVA (10 g/ml PBS; 100 l/well), and the standard curve was
created using an OVA-speciﬁc IgE standard from AbD Serotec (Raleigh, NC).
Statistical analysis. All data are expressed as group means ⫾ standard errors
of the means and were pooled from two independent experiments conducted
approximately 5 months apart. Statistical comparisons were performed by one-
way analysis of variance (ANOVA) followed by Newman-Keuls post hoc tests,
using GraphPad Prism software (GraphPad Software Inc., San Diego, CA). In all
instances, statistical signiﬁcance was denoted when the P value was ⬍0.05.
Allergen-induced airway hyperresponsiveness is decreased
when P. gingivalis infection is established after sensitization to
allergen. Invasive determination of R was performed to assess
whether a localized P. gingivalis infection inﬂuenced the devel-
opment of airway hyperresponsiveness to cholinergic stimula-
tion, a cardinal feature of asthma and experimental allergic
airway disease. No differences in baseline R values were ob-
served among the various treatment groups (Fig. 2A). Allergic
mice demonstrated increased airway responsiveness to metha-
choline aerosol compared to nonallergic mice (infected with P.
gingivalis only), as evidenced by higher R values (Fig. 2A). The
lower calculated PC200 value for R in allergic mice, indicative
of increased sensitivity to methacholine, conﬁrmed this obser-
vation (Fig. 2B). The effect of P. gingivalis infection on airway
responsiveness was dependent on the timing of the infection
relative to allergic sensitization. No effect was observed when
P. gingivalis infection was established before OVA sensitization
occurred, but infection initiated after OVA sensitization re-
sulted in reduced airway responsiveness to methacholine aero-
FIG. 2. Airway responsiveness to methacholine aerosol in allergic mice is reduced by establishment of infection with P. gingivalis after, but not
prior to, sensitization to allergen. (A) Increased airway responsiveness in allergic mice was blunted when infection with P. gingivalis occurred after,
but not prior to, sensitization to OVA.
, P ⬍ 0.05 versus infection only (n ⫽ 7 to 13 per group). (B) Calculated PC200 values for methacholine
(y axis) were lowest for the allergic group. Infection with P. gingivalis prior to sensitization to OVA did not alter these values, but infection
established after sensitization increased the PC200 value for allergic mice.
, P ⬍ 0.05 versus infection-only group and group receiving infection
after allergic sensitization (n ⫽ 7 to 13 per group).
2490 CARD ET AL. I
sol (Fig. 2A) and a corresponding increase in the PC200 value
for R (Fig. 2B) relative to those for allergic mice not exposed
to P. gingivalis.
Allergic airway inﬂammation is decreased when P. gingivalis
infection is established before sensitization to allergen. Sec-
tions of lung from the different experimental groups were pre-
pared and stained with hematoxylin and eosin in order to allow
for semiquantitative histopathological assessment of the inﬂu-
ence of infection on allergic inﬂammation (representative sec-
tions are shown in Fig. 3A). As expected, the histopathological
inﬂammatory score was higher for the allergic group than for
the infection-only group (Fig. 3B). Establishment of P. gingi-
valis infection prior to allergic sensitization resulted in a mod-
erate but signiﬁcantly decreased inﬂammatory score for this
group compared with that for the allergic group, whereas in-
fection established after sensitization did not (Fig. 3B). Suba-
nalysis of the data revealed that the decreased overall score for
the group infected with P. gingivalis prior to allergic sensitiza-
tion was the result of a decrease in the pleural inﬂammation
score for this group (1.25 out of 3) compared to that for the
allergic group (1.79 out of 3) (P ⬍ 0.05). No signiﬁcant differ-
ences were observed among the groups in terms of perivascular
or peribronchiolar inﬂammation scores (data not shown).
Quantiﬁcation of inﬂammatory cell inﬂux and cytokine con-
tent in BAL ﬂuid was also performed. The total number of
cells recovered from the airways of allergic mice was increased
relative to that recovered from mice only infected with P.
gingivalis (Fig. 4A), and the majority of these (⬃62%) were
eosinophils. Infection with P. gingivalis before sensitization to
allergen resulted in an ⬃50% reduction in the number of total
FIG. 3. Histopathological evidence of airway inﬂammation is reduced in mice infected with P. gingivalis prior to but not after sensitization to
allergen. (A) Representative histological sections (stained with hematoxylin and eosin) demonstrating areas of eosinophilic and lymphocytic
inﬂammation in perivascular and peribronchiolar regions (depicted by arrows). These regions were less numerous and intense in mice infected
before allergic sensitization. (B) Calculated histopathological scores revealed decreased inﬂammation in allergic mice infected with P. gingivalis
prior to sensitization to OVA.
, P ⬍ 0.05 versus allergic group and group receiving infection after allergic sensitization (n ⫽ 9 to 16 per group).
OL. 78, 2010 ORAL BACTERIA AND ALLERGIC LUNG INFLAMMATION 2491
cells and an ⬃67% reduction in the number of eosinophils
recovered from allergic mice (P ⬍ 0.05 for both) (Fig. 4A).
Moreover, the percentage of recovered cells that were eosin-
ophils (⬃34%) was considerably decreased in this group. Con-
versely, infection with P. gingivalis subsequent to allergic sen-
sitization did not signiﬁcantly alter the number of total cells or
eosinophils recovered (Fig. 4A) or the percentage of eosino-
phils in the total cell population (⬃63%) relative to that found
for allergic mice not infected with P. gingivalis. Levels of the
Th2 cytokines IL-4, IL-5, IL-13, and GM-CSF were increased
in BAL ﬂuid of allergic mice relative to those in mice that were
only infected with P. gingivalis (Fig. 4B). P. gingivalis infection
established prior to OVA sensitization resulted in decreased
airway levels of IL-4, IL-5, IL-13, and GM-CSF relative to
those found in allergic mice not exposed to bacteria, whereas
infection established after sensitization did not alter these lev-
els (Fig. 4B). BAL ﬂuid levels of other inﬂammatory cytokines
(IL-6, IL-12, IL-1␤, and IFN-␥) were at or near the lower limit
of detection and did not differ among the groups (data not
shown). In contrast to what was observed in BAL ﬂuid sam-
ples, no consistent pattern of effect resulting from P. gingivalis
infection was observed for serum cytokines. Serum IL-4 levels
were not different among the groups, and IL-5, IL-13, and
GM-CSF levels were generally increased in all groups relative
to those in the infection-only group (data not shown).
Infection with P. gingivalis alone resulted in an increased
trend in serum total IgE levels compared to those observed in
allergic mice (Fig. 4C). Infection established before OVA sen-
sitization resulted in a signiﬁcantly increased serum total IgE
level compared to that observed for the allergic group, whereas
infection established after OVA sensitization resulted in the
highest level of serum total IgE among all the groups (Fig. 4C).
The serum OVA-speciﬁc IgE level was also highest when in-
fection was established after OVA sensitization, whereas in-
fection established before sensitization did not alter the
OVA-speciﬁc IgE level compared to that observed for the
allergic group (Fig. 4D). As expected, no OVA-speciﬁc IgE
was detected in mice that were only infected with P. gingi-
valis (Fig. 4D).
Cumulatively, these data indicate that allergic airway inﬂam-
mation was reduced by the establishment of a P. gingivalis
infection prior to, but not after, allergen sensitization and that
alterations in airway cytokine and serum IgE levels may have
been involved in these effects.
FIG. 4. BAL ﬂuid inﬂammatory parameters and serum IgE levels in mice infected with P. gingivalis prior to or after sensitization to allergen.
(A) Total cell and eosinophil populations in the airways of allergic mice were reduced when infection with P. gingivalis was established prior to
sensitization to OVA.
, P ⬍ 0.05 versus allergic group and group receiving infection after allergic sensitization (n ⫽ 9 to 16 per group). (B) BAL
ﬂuid cytokine content in allergic mice was reduced when infection with P. gingivalis was established prior to sensitization to OVA.
, P ⬍ 0.05 versus
allergic group and group receiving infection after allergic sensitization (n ⫽ 9 to 16 per group). (C) Serum IgE levels were higher in the
infection-only group than in the allergic group, but this was not statistically signiﬁcant. Infection initiated either before or after sensitization to
OVA increased levels relative to those for the allergic group, with infection initiated after sensitization resulting in the highest levels.
, P ⬍ 0.05
versus allergic group;
, P ⬍ 0.05 versus all other groups (n ⫽ 9 to 15 per group). (D) OVA-speciﬁc IgE levels in serum were highest when
infection was initiated after sensitization to OVA, whereas infection initiated prior to sensitization to OVA did not alter the level compared to that
in the allergic group.
, P ⬍ 0.05 versus infection-only group;
, P ⬍ 0.05 versus all other groups (n ⫽ 10 to 15 per group).
2492 CARD ET AL. I
Immunization with heat-killed P. gingivalis alone does not
alter allergic airway inﬂammation or hyperresponsiveness. To
determine if the measured allergic outcomes were inﬂuenced
by the immunization procedure with heat-killed bacteria, sep-
arate groups of mice were immunized by introducing heat-
killed P. gingivalis into the subcutaneous chambers but were
not subsequently challenged with live bacteria. Relative to that
for the allergic group not treated with any bacteria (PC200
value for R of 13.0 ⫾ 2.0 mg/ml) (Fig. 2B), the airway hyper-
responsiveness to methacholine was not signiﬁcantly altered in
mice only immunized with heat-killed bacteria either before or
after sensitization to OVA (PC200 values for R of 17.5 ⫾ 1.7
and 17.8 ⫾ 1.5 mg/ml, respectively; P ⬎ 0.05 for both). Airway
inﬂammatory cell and cytokine contents also did not differ in
mice only immunized with heat-killed bacteria either before or
after sensitization to OVA (Fig. 5A and B) relative to those for
allergic mice not treated with any bacteria (Fig. 4A and B).
Thus, the immunization procedure with heat-killed bacteria
did not in and of itself exert a regulatory effect on the observed
While considerable attention has been given to the inﬂuence
of environmental exposures to bacterial products such as lipo-
polysaccharide (LPS) on asthma pathogenesis (14, 33), much
less is known regarding the possible inﬂuence of bacteria and
bacterial products derived from the host microﬂora on allergic
airway disease. The gastrointestinal microﬂora has been pro-
posed to exert regulatory effects on immune system develop-
ment and, in particular, to affect allergic respiratory diseases
(8, 9, 13, 24–26). To our knowledge, however, the potential
inﬂuence of oral bacteria in an experimental model of allergic
airway disease has not been reported. The purpose of this
study was to determine whether allergic airway inﬂammation is
modiﬁed by infection with the periodontal pathogen P. gingi-
valis in an established murine model of asthma. The results
indicate that P. gingivalis exerted differential regulatory effects
on allergic airway inﬂammation that were dependent on the
timing of the establishment of infection relative to allergic
Several studies have identiﬁed a positive association be-
tween serum levels of antibodies to P. gingivalis and cardiovas-
cular disease risk in humans (3, 28, 29), although recent ob-
servations of inverse relationships between serum P. gingivalis
antibody levels and asthma prevalence and between the inci-
dences of periodontitis and allergic respiratory diseases (2, 10)
suggest that not all extra-oral effects of this bacterium may be
deleterious. Furthermore, the improvement in oral hygiene in
recent decades coincides with the increase in asthma preva-
lence in developed nations (1, 16), suggestive of a potentially
causative relationship. A major limitation of these observa-
tions, however, is the inability to discern whether the presence
of oral bacteria, and of P. gingivalis in particular, promotes/
inhibits the development of the diseases in question, or vice
versa. The present study thus evaluated the inﬂuence of P.
gingivalis infection initiated prior to or after allergic sensitiza-
tion under controlled experimental conditions. With this ap-
proach, we found that P. gingivalis infection established before
allergen sensitization had the most striking effects on airway
inﬂammation, with reduced airway levels of IL-4, IL-5, IL-13,
and GM-CSF, decreased histological inﬂammation, and de-
creased airway eosinophilia. Neither OVA-speciﬁc IgE in se-
rum nor airway hyperresponsiveness was decreased in this set-
ting. Airway hyperresponsiveness was decreased, however,
when P. gingivalis infection was established after allergic sen-
sitization occurred, although inﬂammation was unaffected and
OVA-speciﬁc IgE levels in serum were increased. These ﬁnd-
ings highlight a complex regulatory scheme for allergic airway
inﬂammation and functional alterations that is affected in a
temporally important fashion by P. gingivalis infection.
The mechanistic basis for the reduction of allergic inﬂam-
matory responses in the lung due to P. gingivalis infection
established prior to, but not after, allergic sensitization is un-
clear, but it does not appear to involve alterations in serum
levels of total or OVA-speciﬁc IgE. Infection with P. gingivalis
alone resulted in a higher total IgE level in serum than that
observed in allergic mice, suggestive of a signiﬁcant immuno-
modulatory effect of this bacterium. Supportive of this concept
are data demonstrating that administration of LPS derived
from P. gingivalis to newborn BALB/c mice results in increased
serum IgE levels upon maturity, whereas administration of
LPS derived from Actinobacillus actinomycetemcomitans or
FIG. 5. Allergic airway inﬂammation is not altered by heat-killed P. gingivalis. BAL ﬂuid cell counts (A) and cytokine levels (B) were not altered
by intrachamber injection of heat-killed P. gingivalis either before or after sensitization to OVA (compare data to those in Fig. 3C and D) (n ⫽
4 to 7 per group).
OL. 78, 2010 ORAL BACTERIA AND ALLERGIC LUNG INFLAMMATION 2493
Escherichia coli does not (17). Moreover, P. gingivalis-derived
LPS stimulates the release of tumor necrosis factor alpha from
macrophages from Toll-like receptor 4 mutant C3H/HeJ mice
(18), suggestive of an alternative Toll-like receptor or other
signaling pathway for LPS derived from P. gingivalis. IgE is a
recognized contributor to allergic airway inﬂammation and
hyperresponsiveness, and asthma therapies based on targeting
of IgE are now in clinical use (34). Nonetheless, serum IgE and
experimental allergic outcomes are not always positively cor-
related (36, 37), and serum IgE levels did not appear to cor-
relate well with the inﬂammatory and functional outcomes
observed in the treatment groups studied here. For example,
mice infected with P. gingivalis prior to allergic sensitization
had reduced airway inﬂammatory cells and cytokines but a
⬎4-fold increase in serum total IgE and unaltered OVA-spe-
ciﬁc IgE compared to levels in allergic mice not infected with
P. gingivalis. Similarly, mice infected with P. gingivalis after
allergic sensitization had airway inﬂammatory cell and cyto-
kine contents comparable to those in allergic mice not infected
with P. gingivalis, despite having ⬎10-fold and nearly 3-fold
higher total and OVA-speciﬁc IgE levels in serum, respec-
tively. The fact that mice infected with P. gingivalis alone had
higher (albeit not statistically signiﬁcant) total IgE levels in
serum than did allergic mice suggests that the presence of
infection was a signiﬁcant contributor to the outcomes ob-
served, likely due in part to immunomodulatory effects re-
ﬂected by the changes in total IgE.
In the subcutaneous chamber model used herein, immuni-
zation of mice with heat-killed P. gingivalis prior to exposure to
live bacteria allowed for colonization of the chamber by the
live organisms, avoidance of host clearance, and establishment
of an infection (12). Immunization with heat-killed bacteria is
designed to emulate the effects of chronic infection rather than
acute infection. In humans, these organisms are commensal,
but they are foreign to the mouse. Thus, if this strain of P.
gingivalis is provided as a challenge in a mouse that has not
been immunized with heat-killed bacteria, bacteria can dissem-
inate from the chamber more broadly to cause abscess forma-
tion, acute sepsis, and death (12). Importantly, we were able to
demonstrate that the immunization procedure itself did not
inﬂuence allergic airway outcomes, as mice only immunized
with heat-killed P. gingivalis, either before or after allergic
sensitization, demonstrated a phenotype similar to those of the
allergic group. A necessity for live organisms has been ob-
served for the modulating effect of probiotics on experimental
allergic airway inﬂammation (9). It is unclear, however,
whether our observation of attenuated inﬂammation as a result
of establishment of infection prior to allergic sensitization was
dependent on the presence of live bacteria within the chamber
at the time sensitization occurred. This was the case in the
present study by virtue of the experimental design. Indeed,
sensitization to allergen occurred 7 days after establishment of
infection, well within the 14-day period in which live bacteria
can be cultured from chamber ﬂuid (12) and, based on previ-
ous experimental data using the chamber model, a time by
which bacteria had likely entered the systemic circulation (22).
It would therefore be interesting to determine whether estab-
lishment of infection at a time point much earlier than that
utilized here (e.g., weeks to months prior to allergic sensitiza-
tion) would elicit the same blunting effect on allergic inﬂam-
Although beyond the scope of the present study, it also
would be beneﬁcial to ascertain whether establishment of an
oral P. gingivalis infection, a protocol recently shown to accel-
erate experimental atherosclerosis (20), elicits the same regu-
latory inﬂuence on allergic airway inﬂammation as that we
observed with a subcutaneous infection. P. gingivalis, however,
does not easily or reproducibly colonize in the oral cavity of the
mouse, and it is therefore difﬁcult to uniformly provide a sim-
ilar infectious dose by this route. The effects we sought to
emulate were those seen under conditions where the oral or-
ganism disseminates systemically, and this is much more effec-
tively reproduced using the chamber model. For example, the
organism is detectible in the liver at low levels following chal-
lenge in the chamber model, but variably so with the oral
infection model. Furthermore, although P. gingivalis is an oral
pathogen, it normally colonizes the subgingival environment
and is not subject to traditional mucosal immune surveillance.
The subgingival bioﬁlm is bathed in serum via the crevicular
ﬂuid, not saliva, and is exposed to serum antibody and not
secretory antibody. The host response to these invasive oral
pathogens is a systemic humoral response more akin to the
response to a cutaneous challenge. This is in contrast to oral
organisms which inhabit the tooth surface above the gum line,
such as Streptococcus mutans, which do not elicit a systemic
acquired immune response, but rather a mucosal immune re-
sponse. Thus, the chamber model bypasses the need for oral
disease, which causes ulceration of the subgingival environ-
ment and systemic invasion of the organism to trigger the
systemic humoral response. Challenge within the chamber re-
producibly provides a low-level systemic challenge which mim-
ics the presence of naturally occurring oral infection and en-
ables one to reduce the variability in the animal response by
providing a more controlled challenge dosage.
The apparent discrepancy between measures of allergic air-
way inﬂammation and airway responsiveness in the different
treatment groups is intriguing but not without precedent, and
it suggests a differential regulation of these processes in the
allergic airway. A disconnect between measures of allergic
inﬂammation and airway responsiveness to cholinergic stimu-
lation has been documented in several other studies (4, 6, 7, 11,
27, 35), reinforcing the notion that inﬂammatory outcomes and
functional alterations are not always strongly correlated. What
may be viewed as surprising in our study was the observation of
a lack of airway hyperresponsiveness in the group receiving
infection after OVA sensitization despite the presence of sub-
stantial allergic airway inﬂammation, as measured by BAL
ﬂuid cells and cytokines, lung histopathology, and serum IgE
levels. Although great strides have been made in our under-
standing of the mechanisms of allergic airway disease, the
identiﬁcation of an immunological biomarker that correlates
with airway hyperresponsiveness has remained elusive. This is
due to the tremendous complexity of the disease, as evidenced
by the growing variety of factors reported to differentially reg-
ulate inﬂammatory and functional outcomes in animal models
and humans. Indeed, potential candidates reported to underlie
the discordance between allergic airway inﬂammation and hy-
perresponsiveness include signaling molecules (e.g., NF-B)
(27), enzymes (e.g., cyclooxygenase-2) (11), receptors (e.g.,
2494 CARD ET AL. INFECT.IMMUN.
estrogen receptor alpha, D6 chemokine receptor) (6, 35),
and autonomic dysfunction (7). These and other reported
candidates provide a large number of potential mechanisms
and pathways through which P. gingivalis infection may dif-
ferentially regulate the various aspects of allergic airway
To the best of our knowledge, this is the ﬁrst report describ-
ing a direct effect of an oral pathogen on allergic airway dis-
ease. While it is recognized that the route of exposure to P.
gingivalis that was employed in this study does not reﬂect the
normal route of exposure to this organism, we maintain that
the results of this study reveal its potentially important regu-
latory inﬂuence on allergic airway responses. Delineating the
mechanisms responsible for the modulatory effect(s) of P. gin-
givalis infection on the allergic airway inﬂammatory and func-
tional processes observed in this study and more fully elucidat-
ing their temporal characteristics and importance are the
subjects of ongoing investigations that we anticipate will pro-
vide further insight into the regulatory role of oral bacteria in
allergic airway disease.
This research was supported by the Intramural Research Program of
the NIH, National Institute of Environmental Health Sciences (grant
no. Z01 ES025043 to D.C.Z.), by a grant (Mp1-RR-00046) from the
University of North Carolina General Clinical Research Center (S.O.),
and by a Senior Research Training Fellowship from the American
Lung Association of North Carolina (J.W.C.). This research was con-
ducted in part at the National Institute of Environmental Health Sci-
ences Inhalation Facility under contract to Alion Science and Tech-
We are grateful to Sandy Ward for help with cell differential count-
ing and to Jermaine Fuller and Patrick Galloway for the animal surgery
and microbiological work related to the chamber infection model.
G.P.F. conducted the histopathological evaluations.
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Editor: B. A. McCormick
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