Vol.2, No.1, 17-24 (2012) Open Journal of Immunology
http://dx.doi.org/10.4236/oji.2012.21003
Adjuvant effects of different TLR agonists on the
induction of allergen-specific Th2 responses
Matthias J. Duechs, James E. Brunt, Florian Gantner, Klaus J. Erb*
Respiratory Diseases Research, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany;
*Corresponding Author:
Received 5 December 2011; revised 20 January 2012; accepted 15 February 2012
ABSTRACT
Currently different Toll-like receptor (TLR) ago-
nists are tested in humans for their ability to
enhance the efficacy of specific immunotherapy
(SIT). Recent clinical data suggest that this may
be achieved by increasing allergen-specific Th1
responses. However, it is not clear which TLR
agonist is best suited to be used in combination
with SIT. We tested the ability of five TLR ago-
nists, LTA, poly(I:C), LPS, R848, and CpG-ODN,
activating TLR2, 3, 4, 7, and 9, to induce aller-
gen-specific Th1 and suppress allergen-specific
Th2 responses in a preclinical setting. Mice were
immunized by intraperitoneal injection of oval-
bumin (OVA)/Al(OH)3 together with different doses
(0.0025, 0.025, 0.25, and 2.5 mg/kg) of agonists
followed by two OVA aerosol challenges. The re-
sults of these experiments showed, tha t the sup-
pression of allergen-specific Th2 responses and
the induction of Th1 responses dependedon the
dose and the agonists used. All TLR agonists
increased allergen-specific IgG2a, and with the
exception of poly(I:C), reduced allergen-specific
IgE levels in the serum. Allergic cutaneous ana-
phylaxis was also suppressed in mice when LPS
or CpG was given together with OVA/ alum. The
strongest Th1 responses were induced by CpG
and poly(I:C), characterized by the presence of
IFN-g in the BAL and the highest OVA-specific
IgG2a levels in the serum. This study suggests
that the TLR9 agonist CpG-ODN and TLR4 ago-
nist LPS have the strongest suppressive effects
on the development of allergen-specific Th2 re-
sponses in mice and CpG-ODN ind uces the stron-
gest allergen-specific Th1 responses. Therefore
these t wo TLR ago nists may be good candidates
to combine with allergen in novel SIT formula-
tions in humans.
Keywords: Asthma; TLR-Agonists;
Innate-Inflammation; Inhibition
1. INTRODUCTION
Allergic immune responses in the lung to common en-
vironmental antigens lead to the development of atopic
asthma. The most important and widely used therapeutics
for asthma are long-acting β-agonists, inhaled or orally
applied steroids or leukotriene modifiers (Montelukast).
In addition, anti IgE therapy is also used to treat patients
with severe atopic asthma. None of these drugs show any
disease-modifying effects in asthma or other atopic dis-
eases [1,2] and need to be taken continuously. At present,
the only well-established disease-modifying treatment
available for allergy sufferers is the allergen-specific im-
munotherapy (SIT) [3]. This involves the subcutaneous
(SCIT) or sublingual (SLIT) application of increasing
doses of different allergens (usually standardised extracts
of the allergen) over a period of up to 3 - 5 years [4,5].
The mechanisms of how SIT mediates protective effects
are not entirely clear. Based on current literature, it ap-
pears to be associated with increased allergen-specific
IgG4 levels, induction of immune tolerance, or by im-
mune deviation of the allergen-specific Th2 responses
towards Th1 [6,7]. Although effective in mild allergic
responses, SIT shows only limited effects in treating pa-
tients with asthma [8]. In particular if the patient is aller-
gic to numerous allergens.
Current efforts to improve the efficacy of SIT and short-
ening the time of treatment are focussing on combining
allergen with adjuvants. Alum, MLP, virus-like particles
and CpG-ODN fused to allergen have been tested suc-
cessfully in the clinic [9,10]. Using other or stronger im-
mune-modulatory adjuvants may lead to a further in-
crease in the efficacy of SIT possibly also in patients suf-
fering from asthma.
The aim of our study was to combine allergen with
five of the most widely studied and used TLR agonist as
adjuvants to investigate which combination suppresses
the development of allergic airway responses most effec-
tively. We used a model of murine allergic airway disease
and immunised mice with ovalbumin (OVA)/alum plus
different amounts of the following TLR-agonists: CpG-
ODN (TLR-9), LPS (TLR-4), LTA (TLR-2), poly(I:C)

M. J. Duechs et al. / Open Journal of Immunology 2 (2012) 17-24
18
(TLR-3) and R848 (TLR-7) and then analysed which
type of response was initiated in the lung and serum after
allergen-challenge. We found that all of the agonists in-
duced OVA-specific Th1 responses. However, the strength
of the suppressive effects on the allergic response and
pro-inflammatory responses depended on the TLR ago-
nist used and the concentration applied.
2. MATERIALS AND METHODS
2.1. Mice
Female Balb/c mice were purchased from Charles
River (Sulzfeld, Germany). Animals were maintained un-
der conventional conditions in an isolation facility. At the
onset of the experiments, animals were between 8 and 12
weeks of age. All experiments were performed according
to the guidelines of the local and government authorities
for the care and use of experimental animals.
2.2. TLR Agonists
For activation of murine TLR2, TLR3, TLR4, TLR7
and TLR9 the respective agonists were used; lipoteichonic
acid from Staphylococcus aureus; LTA-SA, synthetic
analogue of double stranded RNA; poly(I:C), lipopoly-
saccharide from E.coli K12; LPS-EK, small synthetic
antiviral imidazoquinoline compound; R848, and syn-
thetic oligodeoxynucleotides containing unmethylated CpG
dinucleotides; ODN1826. All TLR agonist were purchased
from InvivoGen, San Diego, USA.
2.3. Treatment Protocols
Balb/c mice were sensitized intraperitoneally (i.p.)
with 20 µg OVA (Serva, Heidelberg, Germany), adsorbed
to Al(OH)3 (Pierce, Rockford, USA) in 0.9% NaCl in a
total volume of 200 µl per animal on days 1, 14 and 21.
Negative controls received saline and Al(OH)3 only. On
day 26 and 27 mice were challenged with 1% OVA aero-
sol for 20 min. Negative controls received vehicle corre-
spondingly. 0.0025, 0.025, 0.25, and 2.5 mg/kg of the
respective TLR-agonist were administered intraperito-
neally on day 0, 14, and 21 together with OVA/Al(OH)3.
Mice were sacrificed 24 hours after the last challenge.
2.4. Bronchoalveolar Lavage
At day 28, 24 h after the last OVA challenge, animals
were sacrificed, trachea were cannulated and a bronchoal-
veolar lavage (BAL) was performed as described previ-
ously [11].
2.5. Detection of Cytokines
Cytokines and chemokines in the BAL fluid were de-
termined with mouse 22 plex cytokine/chemokine multi-
plex assays (LINCOplex, Millipore, St. Charles or Mul-
tiplex, Meso Scale Discovery, Gaithersburg, USA) ac-
cording to the manufactures instructions.
2.6. Allergen Specific Immunoglobulin
Measurement
Blood samples were collected 23 h after the last chal-
lenge. Samples were incubated for 30 min at room tem-
perature and then centrifuged at 14.000 rpm for 20 min.
Supernatant was collected and frozen. OVA-specific IgE
and IgG2a were measured using standard ELISA tech-
nique. 100 ug/ml of OVA (Serva, Heidelberg, Germany)
was used to coat the plates. Serial dilutions of the differ-
ent samples where then incubated with the OVA coated
plates. The following antibodies were then used to detect
the antibodies binding to the OVA; biotin rat anti-mouse
IgE (BD Biosciences, Erembodegem, Belgium), IgE an-
tibody standard (Serotec Oxford, England), anti-OVA
chicken (Dianova, Asker, Norway), and anti-mouse IgG2a
biotin (BD Biosciences, Erembodegem, Belgium).
2.7. Active Cutaneous Anaphy laxis
For measurement of active cutaneous anaphylaxis sen-
sitized and challenged mice received an intra venous (i.v.)
application of 200 µl 1% Evans blue. Mice were then
anaesthetised with isoflurane (3% - 4% in pressurized air)
and 5 µl of PBS with 5 µg OVA was applied intradermal
(i.d.) into the right ear. The negative controls received 5
µl of PBS i.d. in the left ear. 28 minutes after injection
mice were sacrificed and from the treated areas of both
ears a tissue sample with a diameter of 8 mm was
punched. For dye extraction tissue samples were incu-
bated with 300 µl formamide at 65˚C for 24 h at 450 rpm
on a shaker. After dye extraction concentration was mea-
sured with a wavelength of 620 nm using a photometer.
2.8. Histology
Lungs were fixed in 4% formalin for 24 h and after-
wards embedded in paraffin wax. Lungs slices with a
thickness of 2 - 3 µm were stained using standard histo-
logical protocols. Haematoxylin and eosin (H&E) re-
agent (Merck, Darmstadt, Germany) were used for ana-
lysis of inflammatory infiltrates, periodic acid-Schiff (PAS)
reagents (Sigma-Aldrich GmbH, Steinheim, Germany)
for goblet cells and mucus production. Intensity of in-
flammation and mucus was scored by two independent
observers (0 = no inflammation or mucus, 1 = slight in-
flammation or mucus, 2 = moderate inflammation or mu-
cus, 3 = strong inflammation or mucus).
2.9. Statistical Analysis
Statistical differences between different groups were
evaluated by One-way ANOVA. One-way analysis of
variance together with the Dunett post test was used for

M. J. Duechs et al. / Open Journal of Immunology 2 (2012) 17-24
19
comparisons between groups. A p value of less than 0.05
was considered significant.
the different ligands were applied together with OVA/alum
on day 0, 14 and 21. Mice were challenged with OVA on
day 26 and 27. On day 28 mice were sacrificed and BAL
was collected (Figure 1(a)). We then analyzed the effects
on the development of allergen-specific Th2-responses in
the lung. Figure 1(b) shows that LPS and CpG-ODN
3. RESULTS
To determine the effects the different TLR-agonists
had on the development of OVA-specific Th2 responses
Figure 1. CpG, LPS and the highest concentration of LTA reduces allergen induced eosinophilia in
the lung. (a) Treatment protocol; mice received an intra peritoneal co-administration of OVA and
TLR agonist using different concentrations on day 0, 14 and 21. The mice were then exposed to
nebulized OVA on day 26 and 27; (b) Total amounts of macrophages, neutrophils and eosinophils in
BAL were measured 24 h after the last OVA exposure. Cell counts are presented as mean ± SEM of 8
mice/group. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the OVA group.
OPEN A CCESS

M. J. Duechs et al. / Open Journal of Immunology 2 (2012) 17-24
20
significantly and dose dependently reduced the develop-
ment of airway eosinophilia, whereas LTA reduced eosi-
nophilia only in the highest concentration and R848 and
poly(I:C) showing no inhibition. Interestingly, poly(I:C)
at the highest dose increased eosinophil numbers.
Increased neutrophil numbers were detected when
LTA, LPS, poly(I:C) or CpG-ODN were applied together
with OVA (Figure 1(b)). In control experiments mice
were treated i.p. with the combinations of PBS/TLR-
agonists or OVA/TLR-agonists. Both groups of mice
were then challenged with either PBS or OVA. We found
that when the mice were challenged with PBS no in-
crease in neutrophils were detected in any of the groups.
When the mice were challenged with OVA, only the
OVA/TLR-agonist treated group and not the PBS/TLR-
agonist group showed an increase in neutrophils (data
not shown). This clearly suggests that the observed neu-
trophilia was OVA specific and due to an altered immune
response towards OVA since it was not observed in the
OVA only treated mice.
Histological analysis of the main bronchus area and
lung tissue surrounding it supported our previous find-
ings in the BAL, that no reduction of inflammatory cell
influx could be detected in poly(I:C) and R848 treated
animals (Figure 2(a)). In mice that received LTA we saw
a slight decrease in inflammatory cells in the lung. A
significant reduction in OVA-induced cellular inflamma-
tion and goblet cell metaplasia was seen when the ago-
nists LPS and CpG were applied together with OVA
(Figures 2( a) and (b)).
Figure 3(a) shows that with the exception of R848, all
TLR agonist significantly inhibited the levels of IL-5
detected in the airways. CpG, poly(I:C) and LPS also
significantly reduced IL-4, whereas LTA did not. The
inhibitory effect on IL-4 and IL-5 was strongest in the
LPS and CpG treated animals. When poly(I:C) or CpG-
ODN were applied, increased amounts of IFN-g and IL-6
were detected in the BAL.
Allergen-specific IgE and IgG2a was measured in the
serum of treated mice and normalized to levels of mice
treated only with OVA. In the experiments each TLR
agonists reduced the level of OVA-specific IgE, with the
exception poly(I:C) in each case significantly (Figure
3(b)). The strongest reduction was seen in animals
treated with CpG followed by R848 treated animals.
OVA-specific IgG2a was significantly increased in all
TLR agonist treated animals, albeit to different degrees.
To test if TLR application was able to prevent the de-
velopment of cutaneous anaphylaxis, we repeated the
experiment discussed above and tested active cutaneous
anaphylaxis in the PBS, OVA and OVA/TLR agonist
treated animals. The application of LPS and CpG com-
pletely and significantly inhibited the development of
allergen-induced anaphylactic reactions in the ear (Fig-
ure 2(c)).
4. DISCUSSION
TLR-agonists are not only being developed for the
treatment of allergic disorders alone but also to be used
in combination with allergen during SIT [9,12-14]. For
this reason we were interested which effects the different
TLR-agonists had on the development of OVA-specific
Th2 responses and if suppression is associated with in-
creased allergen specific Th1 response. To address this
question the TLR agonists LTA, poly(I:C), LPS, R848
and CpG-ODN were applied together with OVA/alum.
We found that only the application of LPS and CpG-
ODN significantly and dose dependent reduced the de-
velopment of airway eosinophilia, an effect we have pre-
viously seen when using BCG, heat killed BCG or PPD
in combination with OVA/alum [15]. At the highest
CpG-ODN dose used, the suppressive effect was close to
100%. All TLR-agonists reduced OVA-specific IgE lev-
els and increased OVA-specific IgG2a levels, with CpG-
ODN and poly(I:C) increasing the OVA-specific IgG2a
levels more than 100 fold. These two groups were also
the only ones where IFN-g could also be detected in the
BAL, indicative of enhanced Th1 responses. Increased
neutrophil numbers were detected when LTA, LPS,
poly(I:C) or CpG-ODN when applied together with OVA.
This clearly suggests that the observed neutrophilia was
OVA specific and due to an altered immune response to-
wards OVA, since this was not observed in the OVA only
treated mice. Taken together, the data clearly suggests
that the application of the different TLR-agonists reduced
allergic Th2 responses (CpG-ODN >> LPS > LTA ≥
poly(I:C) > R848) and increased an allergen-spe- cific
Th1 response (CpG-ODN > poly(I:C) >> LPS > LTA >
R848).
First reports regarding the potential of TLR9 agonist to
prevent development of allergic reactions in mice have
already been published over 10 years ago [16]. Other
reports using different allergens like birch pollen or
house-dust mite further confirmed the strong potential of
TLR9 to induce strong immune deviation from Th2 to
Th1 responses which was seen in this study as well [17-
19].
How activation of TLR affects asthma is not entirely
clear and published results have not always been consis-
tent. Redecke et al. for example published that TLR2
ligands bias the adaptive immune response toward a Th2
phenotype and lead to aggravation of asthma [20]. In
contrast, Velasco et al. reported that both TLR2 and
TLR4 agonists showed efficacy in preventing aller-
gen-induced pulmonary responses [21]. In a study con-
ducted by Sel et al. both, the activation of TLR3 and
TLR7 shortly before allergen sensitization were reported
to prevent all features of experimental asthma including

M. J. Duechs et al. / Open Journal of Immunology 2 (2012) 17-24 21
Figure 2. LPS and CpG inhibits allergen-induced cell influx and mucus production in the lung. Lung tissues were obtained from
naïve mice treated with PBS, mice sensitized and exposed to OVA and mice treated with OVA plus TLR agonists (2.5 mg/kg). Tis-
sues were stained with (a) haematoxylin and eosin or by (b) periodic acid Schiff staining, and examined by light microscopy. Scale
bar = 100 µm. Shown are representative examples. The amount of inflammation and goblet cell metaplasia was quantified by
scoring stained sections of main bronchus area with n = 8/group. (0 = no inflammation or mucus, 1 = slight inflammation or mu-
cus, 2 = moderate inflammation or mucus, 3 = strong inflammation or mucus). *p < 0.05, **p < 0.01, ***p < 0.001, compared
with the OVA group.

M. J. Duechs et al. / Open Journal of Immunology 2 (2012) 17-24
22
Figure 3. Cytokine production in the lung, serum Immunogloblin levels, and cutaneous anaphylaxis in TLR agonist treated
mice. (a) Level of IL-4, IL-5, IL-6 and IFN-g in the BALF determined 24 hours after the last challenge and (b) measurement
of allergen specific IgE and IgG2a in serum; (c) Allergen dependent active cutaneous anaphylaxis was measured 24 hours af-
ter the last challenge and assessed by vascular leakage. Cytokine, immunoglobulin levels and optical density values are pre-
sented as mean ± SEM of 8 mice/group. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the OVA group.

M. J. Duechs et al. / Open Journal of Immunology 2 (2012) 17-24
23
airway hyperresponsiveness and allergic airway inflam-
mation [22]. Why did we not see these effects? We can
currently not explain the divergent results, however these
might be due to the difference in concentrations applied,
as Sel et al. use a 10 times higher dose of poly(I:C) and a
2.5 times higher dose of R848. Our finding that CPG-
ODN are very potent inhibitors of the development of
allergen-specific Th2 responses is in line with previously
published reports [16-19].
OPEN A CCESS
A very interesting finding of our study was that the
induction of strong allergen-specific Th1 responses did
not always correlate with a strong reduction in Th2 re-
sponses. Poly(I:C) had the second strongest Th1-induc-
ing effect, but did not reduce OVA-specific IgE levels in
the serum, goblet cell metaplasia in the lung or cutaneous
anaphylaxis. Another surprising effect was that only LPS
and CpG were able to significantly reduce all Th2 pa-
rameters measured. This clearly suggests that only parts
of the allergen-specific Th2 response could be modulated
by R848, poly(I:C) and LTA and that the effect varied
from agonist to agonist. For example, LTA reduced air-
way eosinophilia, IL-5, and IgE levels but not IL-4, gob-
let cell metaplasia or cutaneous anaphylaxis. In contrast
R848 did not reduce airway eosinophilia, IL-4 or IL-5
levels but reduced IgE levels. We have no explanation
why some parameters are affected and some not. How-
ever, it is clear that it depends on the TLR agonist used.
Taken together our results clearly show that LPS and
CpG have the strongest suppressive effects on the de-
velopment of numerous allergen-specific Th2 responses
in mice. TLR9 and TLR4 agonists have already been
used as adjuvants in clinical SIT trials [9,23] and our
results support the further clinical testing of these ago-
nists. Interestingly, the strong induction of allergen-spe-
cific Th1 responses did not always correlate with a strong
reduction in the allergic response, suggesting that this
measure of an effective SIT response may not translate in
a reduction in the allergic response.
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