Targeting Toll-like receptors on dendritic cells modifies the T H 2 response to peanut allergens in vitro Pierre Pochard, PhD, a,b * Brian Vickery, MD, a,b *à M. Cecilia Berin, PhD, c Alexander Grishin, PhD, c Hugh A. Sampson, MD, c Michael Caplan, MD, PhD, b and Kim Bottomly, PhD a § New Haven, Conn, and New York, NY Background: Delivery of allergens with bacterial adjuvants has been shown to be a successful immunotherapeutic strategy for food allergy treatment in animal models. How microbial signals, acting through the innate immune system, reshape ongoing allergic responses is poorly understood. Objective: To investigate the contribution of Toll-like receptors (TLRs) in the response to bacterial adjuvants, we designed an in vitro system to characterize the effect of heat-killed Escherichia coli vector (HKE) on peanut-induced responses of dendritic cells (DCs) and T cells. Methods: Wild-type or TLR signaling–deficient bone marrow– derived DCs were pulsed with crude peanut extract (CPE) alone (50 mg/mL) in the presence of HKE (10 6 /mL). DC maturation was analyzed by means of flow cytometry. Treated DCs were cocultured with carboxyfluorescein succinimidyl ester (CFSE)-labeled CD4 1 T cells from sensitized mice. Cytokine production from DCs and T cells was measured by using Bioplex assays. Results: Peanut-pulsed DCs induced the production of IL-4, IL-5, and IL-13, as well as IL-17 and IFN-g, from primed T cells. Adding HKE to CPE-pulsed DCs resulted in a significant decrease in T H 2 cytokine production associated with an increase in IFN-g levels and profound attenuation of T-cell proliferation. These effects were linked to HKE-induced TLR-dependent changes in DC reactivity to CPE, especially the production of polarizing cytokines, such as IL-12. Conclusions: TLR signals modulate peanut-induced DC maturation in vitro, leading to changes in the T-cell response to peanut. These TLR effects must be confirmed in vivo and might constitute another alternative for allergen immunotherapies. (J Allergy Clin Immunol 2010;126:92-7.) Key words: Peanut allergy, EMP-123, dendritic cell, Toll-like receptor, MyD88, IL-12 Food allergy is a growing problem in westernized countries. Peanut allergy is important because (1) accidental reactions can be severe, 1 (2) the prevalence has doubled among children and af- fects approximately 1% of the US population, and (3) it is rarely outgrown. 2 The only current treatment is strict dietary elimina- tion, but peanut-free environments are difficult to maintain. Be- cause inadvertent exposures are common, patients must scrutinize all foods and carry autoinjectable epinephrine at all times, 3 impairing their quality of life. 4 These data indicate a need for an effective therapy for allergic patients. Previous attempts have been either unsafe or impracti- cal. 5,6 To enhance the safety of potential immunotherapies, the major peanut allergens Ara h 1, Ara h 2, and Ara h 3 were engi- neered to disrupt immunodominant IgE-binding sites. 7-9 Deliver- ing these modified allergens with a bacterial adjuvant was studied. Using Listeria monocytogenes 10 and then Escherichia coli, 11 Li et al showed that administration of these engineered allergens to C3H/HeJ mice, combined with bacterial adjuvants, protected animals from anaphylaxis on challenge. This protection was associated with decreased histamine and IgE levels. The shift to- ward a T H 1 response was consistent with previous allergen-based immunotherapies that promote tolerance by reshaping T-cell responses. 12 Regulation of allergic responses can occur through activation of the innate immune system. Microbial products act on pattern- recognition receptors, including Toll-like receptors (TLRs), to initiate rapid host immune responses. Dendritic cells (DCs) are professional antigen-presenting cells that critically link innate and adaptive immunity. Detection of microbes initiates DC maturation characterized by phenotypic changes and cytokine release. In the gut commensal flora provide critical immune signals to maintain homeostasis, 13-15 and DCs actively participate in oral tolerance induction. 16,17 As part of a larger ongoing effort to understand the role of microbial adjuvants in immunotherapy, we designed an in vitro system to study how TLR signaling in DCs can modify the immune response to peanut. We show that TLR signaling pro- foundly changes the DC response to peanut, leading to lasting functional changes in the T H 2 cytokine response. METHODS Animals Four- to 6-week-old wild-type C57BL/6 mice were purchased from the National Cancer Institute (Bethesda, Md) or Jackson Laboratories From the Departments of a Immunobiology and b Physiology, School of Medicine, Yale University, New Haven, and c the Department of Pediatrics, Division of Allergy and Immunology, Mount Sinai School of Medicine, New York. *These authors contributed equally to this work. àBrian Vickery is currently affiliated with Duke University Medical Center, Durham, NC. §Kim Bottomly is currently affiliated with Wellesley College, Wellesley, Mass. Supported by National Institutes of Health/National Institute of Allergy and Infectious Diseases grant U19AI066738. Disclosure of potential conflict of interest: M. C. Berin receives research support from the National Institutes of Health, the Environmental Protection Agency, and the Food Allergy Initiative. A. Grishin receives research support from Allertein Therapeutics, LLC. H. A. Sampson is a consultant for and 4% shareholder in Allertein Pharmaceu- ticals, LLC; receives research support from the Food Allergy Initiative and the National Institutes of Health/National Institute of Allergy and Infectious Diseases; is a consultant and scientific advisor for the Food Allergy Initiative; has served as President of the American Academy of Allergy, Asthma & Immunology; and is 45% owner of Herbal Springs, LLC. M. Caplan is a consultant for Allertein Pharmaceuticals. K. Bottomly is a shareholder in Allertein Therapeutics, LLC, and has provided legal consultation/expert witness testimony in cases related to allergy. The rest of the authors have declared that they have no conflict of interest. Received for publication October 8, 2009; revised February 19, 2010; accepted for pub- lication April 6, 2010. Available online June 10, 2010. Reprint requests: Brian Vickery, MD, Duke University Medical Center, Box 2644, Durham, NC 27710. E-mail: [email protected]. 0091-6749/$36.00 Ó 2010 American Academy of Allergy, Asthma & Immunology doi:10.1016/j.jaci.2010.04.003 92
Transcript
Targeting Toll-like receptors on dendritic cells modifies theTH2 response to peanut allergens in vitro
Pierre Pochard, PhD,a,b* Brian Vickery, MD,a,b*� M. Cecilia Berin, PhD,c Alexander Grishin, PhD,c Hugh A. Sampson, MD,c
Michael Caplan, MD, PhD,b and Kim Bottomly, PhDa§ New Haven, Conn, and New York, NY
Background: Delivery of allergens with bacterial adjuvants hasbeen shown to be a successful immunotherapeutic strategy forfood allergy treatment in animal models. How microbial signals,acting through the innate immune system, reshape ongoingallergic responses is poorly understood.Objective: To investigate the contribution of Toll-like receptors(TLRs) in the response to bacterial adjuvants, we designed an invitro system to characterize the effect of heat-killed Escherichiacoli vector (HKE) on peanut-induced responses of dendritic cells(DCs) and T cells.Methods: Wild-type or TLR signaling–deficient bone marrow–derived DCs were pulsed with crude peanut extract (CPE) alone(50 mg/mL) in the presence of HKE (106/mL). DC maturation wasanalyzed by means of flow cytometry. Treated DCs were coculturedwith carboxyfluorescein succinimidyl ester (CFSE)-labeled CD41
T cells from sensitized mice. Cytokine production from DCs and Tcells was measured by using Bioplex assays.Results: Peanut-pulsed DCs induced the production of IL-4,IL-5, and IL-13, as well as IL-17 and IFN-g, from primedT cells. Adding HKE to CPE-pulsed DCs resulted in asignificant decrease in TH2 cytokine production associated withan increase in IFN-g levels and profound attenuation of T-cellproliferation. These effects were linked to HKE-inducedTLR-dependent changes in DC reactivity to CPE, especially theproduction of polarizing cytokines, such as IL-12.Conclusions: TLR signals modulate peanut-induced DCmaturation in vitro, leading to changes in the T-cell response to
From the Departments of aImmunobiology and bPhysiology, School of Medicine, Yale
University, New Haven, and cthe Department of Pediatrics, Division of Allergy and
Immunology, Mount Sinai School of Medicine, New York.
*These authors contributed equally to this work.
�Brian Vickery is currently affiliated with Duke University Medical Center, Durham,
NC.
§Kim Bottomly is currently affiliated with Wellesley College, Wellesley, Mass.
Supported by National Institutes of Health/National Institute of Allergy and Infectious
Diseases grant U19AI066738.
Disclosure of potential conflict of interest: M. C. Berin receives research support from the
National Institutes of Health, the Environmental Protection Agency, and the Food
Allergy Initiative. A. Grishin receives research support from Allertein Therapeutics,
LLC. H. A. Sampson is a consultant for and 4% shareholder in Allertein Pharmaceu-
ticals, LLC; receives research support from the Food Allergy Initiative and the
National Institutes of Health/National Institute of Allergy and Infectious Diseases; is a
consultant and scientific advisor for the Food Allergy Initiative; has served as President
of the American Academy of Allergy, Asthma & Immunology; and is 45% owner of
Herbal Springs, LLC. M. Caplan is a consultant for Allertein Pharmaceuticals.
K. Bottomly is a shareholder in Allertein Therapeutics, LLC, and has provided legal
consultation/expert witness testimony in cases related to allergy. The rest of the authors
have declared that they have no conflict of interest.
Received for publication October 8, 2009; revised February 19, 2010; accepted for pub-
lication April 6, 2010.
Available online June 10, 2010.
Reprint requests: Brian Vickery, MD, Duke University Medical Center, Box 2644,
� 2010 American Academy of Allergy, Asthma & Immunology
doi:10.1016/j.jaci.2010.04.003
92
peanut. These TLR effects must be confirmed in vivo and mightconstitute another alternative for allergen immunotherapies.(J Allergy Clin Immunol 2010;126:92-7.)
Food allergy is a growing problem in westernized countries.Peanut allergy is important because (1) accidental reactions canbe severe,1 (2) the prevalence has doubled among children and af-fects approximately 1% of the US population, and (3) it is rarelyoutgrown.2 The only current treatment is strict dietary elimina-tion, but peanut-free environments are difficult to maintain. Be-cause inadvertent exposures are common, patients mustscrutinize all foods and carry autoinjectable epinephrine at alltimes,3 impairing their quality of life.4
These data indicate a need for an effective therapy for allergicpatients. Previous attempts have been either unsafe or impracti-cal.5,6 To enhance the safety of potential immunotherapies, themajor peanut allergens Ara h 1, Ara h 2, and Ara h 3 were engi-neered to disrupt immunodominant IgE-binding sites.7-9 Deliver-ing these modified allergens with a bacterial adjuvant was studied.Using Listeria monocytogenes10 and then Escherichia coli,11 Liet al showed that administration of these engineered allergensto C3H/HeJ mice, combined with bacterial adjuvants, protectedanimals from anaphylaxis on challenge. This protection wasassociated with decreased histamine and IgE levels. The shift to-ward a TH1 response was consistent with previous allergen-basedimmunotherapies that promote tolerance by reshaping T-cellresponses.12
Regulation of allergic responses can occur through activationof the innate immune system. Microbial products act on pattern-recognition receptors, including Toll-like receptors (TLRs), toinitiate rapid host immune responses. Dendritic cells (DCs) areprofessional antigen-presenting cells that critically link innateand adaptive immunity. Detection of microbes initiates DCmaturation characterized by phenotypic changes and cytokinerelease. In the gut commensal flora provide critical immunesignals to maintain homeostasis,13-15 and DCs actively participatein oral tolerance induction.16,17
As part of a larger ongoing effort to understand the role ofmicrobial adjuvants in immunotherapy, we designed an in vitrosystem to study how TLR signaling in DCs can modify theimmune response to peanut. We show that TLR signaling pro-foundly changes the DC response to peanut, leading to lastingfunctional changes in the TH2 cytokine response.
METHODS
AnimalsFour- to 6-week-old wild-type C57BL/6 mice were purchased from
the National Cancer Institute (Bethesda, Md) or Jackson Laboratories
was inoculated and placed at 378C in a shaking incubator overnight. Bacteria
were washed, collected in PBS supplemented with 20% glycerol, and stored at
288C. Bacterial concentration was determined by plating serial dilutions.
E coli was heat inactivated for 30 minutes at 658C, and viability was checked
by means of culture.
Bone marrow–derived dendritic cell cultureAt day 6, bone marrow–derived dendritic cells (BMDCs) were gently
collected, washed, and resuspended in complete medium without GM-CSF at
a final concentration of 5 3 105 cells/mL (2 mL). Cells were incubated for
24 hours with either CPE (at an optimized concentration of 50 mg/mL),
heat-killed Escherichia coli vector (HKE; ratio of 1 bacterium per BMDC),
or both stimuli.
The following ultrapure TLR ligands were used for stimulation of
individual BMDC TLRs: LPS and CpG oligonucleotide, both at 500 ng/mL
(InvivoGen, San Diego, Calif).
Coculture with primed T cellsFirst system. Stimulated BMDCs (1 3 105) were cultured with
1 3 106 primed T cells for 3 days (250 mL) in 48-well culture plates. For
the neutralizing experiments, LEAF anti–IL-12/23 P40 antibody (clone
C17.8), LEAF anti–IFN-g antibody (clone H22), or LEAF isotype control
antibody was used and maintained at 20 mg/mL during the whole time of
the culture (Biolegend, San Diego, Calif).
Second system. CPE-pulsed BMDCs (1.5 3 105) were cultured
with 1.5 3 106 primed T cells for 6 days (500 mL) in 48-well culture plates.
T cells were then isolated, washed, counted (to obtain a 1:10 ratio), and cocul-
tured with 1 3 105 CPE-pulsed BMDCs for 3 more days.
BMDC surface marker analysisCollected BMDCs were incubated for 10 minutes with FcgR blocking
antibody and 30 minutes at 48C with fluorescein isothiocyanate–conjugated
CD80 or IAb (equivalent to human MHC class II), phycoerythrin-conjugated
CD86 or CD40, and allophycocyanin-conjugated CD11c (BD Biosciences).
Matching isotype controls were used. After thorough washing, cells were fixed
with 1% paraformaldehyde solution and analyzed with a FACSCalibur
(BD Biosciences). Results were expressed as mean fluorescence intensity.
Cytokine assaySupernatants from BMDC cultures were collected after 24 hours. IL-6,
IL-10, IL-12p70, TNF-a, and IL-1b levels were measured. Supernatants from
cocultures were analyzed for the presence of IL-4, IL-5, IL-13, IL-10, IFN-g,
and IL-17 at day 3. All cytokine analyses were performed with a multiplex kit
according to the manufacturer’s instructions (Upstate, Temecula, Calif) and
analyzed on a Bioplex system (Bio-Rad Laboratories, Hercules, Calif).
Proliferation assayFor proliferation, CD41 T cells were resuspended in PBS (106 cells/mL) and
incubated with carboxyfluorescein succinimidyl ester (CFSE) (10 mmol/L)
at 378C for 10 minutes. The reaction was stopped by adding FCS, and extensive
washes were performed. After culture, CD41 T cells were labeled with APC-
conjugated CD3 (BD Biosciences), and proliferation was assessed by means
of flow cytometry.
Statistical analysisStatistical analysis was performed with Student t tests (Prism for Macin-
tosh; GraphPad Software, La Jolla, Calif). P values of .05 or less were consid-
ered significant.
RESULTS
CPE induces a mixed T-cell immune responseMice were immunized twice with CPE and alum to study the
immune response to peanut. CD41 T cells from spleens were co-cultured with CPE-stimulated BMDCs. CPE induced a mixed im-mune response characterized by significant production of IL-4,IL-5, IL-13, IL-17, and IFN-g and an intensive T-cell proliferation(Fig 1 and see Fig E1 in this article’s Online Repository at www.jacionline.org). This effect was preserved in cocultures by usingBMDCs deficient in TLR4 and MyD88/TRIF (Fig 2 and see FigE1), suggesting that the immunogenicity of peanut proteins isTLR independent.
DC treatment with HKE alters T-cell immune
responses to peanutMicrobial signals from commensal flora or adjuvants can
influence the response to food antigens, and therefore we assessed
FIG 1. DC treatment with HKE alters T-cell immune responses to peanut.
CD41 T cells were cocultured (ratio 10:1) with CPE-pulsed BMDCs stimu-
lated with CPE (50 mg/mL), HKE (1 bacterium per BMDC), or both for 72
hours. A, Concentrations in IL-4, IL-5, IL-13, and IFN-g were determined.
Concentrations represent means 6 SEMs (n 5 5). *P < .05. B, After 5
days, proliferation of CD31 cells was analyzed by means of flow cytometry.
One representative experiment of 5 is shown. CFSE, Carboxyfluorescein
succinimidyl ester; CM, complete media.
FIG 2. HKE alters peanut response by triggering TLR signaling in DCs. Wild-
type (WT) CD41 T cells were cocultured (ratio 10:1) with CPE-pulsed BMDCs
(WT vs MyD88/TRIF-KO) stimulated with CPE (50 mg/mL), HKE (1 bacterium
per BMDC), or both for 72 hours. Concentrations of IL-4, IL-13, and IFN-g
were determined. Concentrations represent means 6 SEMs (n 5 5). After 5
days, proliferation of CD31 cells was analyzed by means of flow cytometry.
Percentages of proliferative cells are indicated by means 6 SEMs (n 5 5). Sta-
tistical differences are indicated as follows: **P < .01. CM, Complete media.
J ALLERGY CLIN IMMUNOL
JULY 2010
94 POCHARD ET AL
the effect of E coli on the peanut response. To prevent outgrowth,we used HKE with CPE in our in vitro coculture system. AddingHKE and CPE to BMDCs significantly decreased TH2 cytokineproduction and increased production of IFN-g (Fig 1). Thischange was associated with inhibited T-cell proliferation (Fig 1).To determine whether this bacterial effect was mediated byTLRs, we performed cocultures using BMDCs from MyD88/TRIF double-deficient animals. The absence of MyD88/TRIFsignaling in DCs reversed the HKE-induced inhibition of TH2cytokine production, restoring it to the level induced by CPEalone. IFN-g was also suppressed. In addition, the inhibition ofproliferation by HKE was completely reversed in the absenceof MyD88/TRIF (Fig 2 and see Fig E2 in this article’s OnlineRepository at www.jacionline.org).
Taken together, these results indicate that DC TLR signaling, inresponse to microbial stimulation, modifies recall responses topeanut proteins, resulting in a shift toward a TH1 response. Thismodification is MyD88/TRIF dependent.
E coli–induced inhibition of TH2 polarization and
peanut-specific T-cell proliferation is long-lastingTargeting TLR signaling on BMDCs attenuated the prolifera-
tion and TH2 cytokine responses of peanut-specific T cells. To in-vestigate whether this effect was long-lasting, we isolatedcocultured T cells that had been initially stimulated with DCs ex-posed to CPE in the presence of HKE and recultured these T cellswith DCs exposed to CPE alone. T cells incubated initially withDCs exposed to both CPE and HKE demonstrated impaired pro-duction of TH2 cytokines, whereas the release of IFN-g (Fig 3, A)
was unimpaired, compared with that seen in T cells initially incu-bated with DCs exposed to CPE in the absence of HKE. Theseresults correlated with the proliferation of these cells. Peanut-primed T cells initially exposed to DCs stimulated with bothHKE and CPE proliferated less (5.05%) than CPE-pulsed BMDCs(44.1%) when re-exposed to DCs stimulated with CPE alone(Fig 3, B). In conclusion, the addition of TLR signaling to DCsled to sustained suppression of T-cell recall responses to peanut.
HKE modifies the DC response to peanutTo investigate how DC recognition of microbial components
could alter the T-cell response to peanut, we stimulated DCs withHKE and CPE to look for a change in DC maturation. CPEinduced TLR-independent maturation of BMDCs, which wascharacterized by the upregulation of the class II molecule IAb andcostimulatory molecules CD80 and CD86 but little CD40 (see FigE3 in this article’s Online Repository at www.jacionline.org).Along with phenotypic changes, peanut stimulation induced pro-duction of proinflammatory and polarizing cytokines. Comparedwith LPS and CpG, which induce production of TNF-a, IL-10,IL-6, and IL-12, CPE-pulsed DCs released TNF-a and IL-10but little IL-12 and IL-6 (Fig 4 and see Fig E4 in this article’s On-line Repository at www.jacionline.org). In summary, peanut anti-gens induced TLR-independent DC maturation and a uniquecytokine production profile.
The addition of HKE to CPE stimulation enhanced theexpression of CD80, CD86, IAb, and CD40 on DCs (see FigE3). These phenotypic changes were accompanied by a signifi-cant increase in the production of the TH1-polarizing cytokineIL-12. IL-6 and TNF-a levels were also increased. Interestingly,IL-10 production was synergistically enhanced by both stimuli(Fig 4). These effects were not observed when TLR- orMYD88/TRIF-deficient BMDCs were used, suggesting thatTLR pathways are necessary for this synergy.
In conclusion, TLR signaling enhances the peanut-induced DCmaturational program and has a particular effect on the release ofpolarizing cytokines, such as IL-10 and IL-12.
or a combination of both stimuli for 24 hours. Supernatants were analyzed
by means of ELISA for production of IL-10 and IL-12. Concentrations repre-
sent means 6 SEMs (n 5 4). **P <_ .01. CM, Complete media.
FIG 3. HKE has a long-lasting effect on peanut response. CD41 T cells were
cocultured (ratio 10:1) with BMDCs stimulated with CPE (50 mg/mL), HKE (1
bacterium per BMDC), or both. After 7 days, they were washed, counted,
and cocultured with CPE-pulsed BMDCs for 72 hours. A, Supernatants
were analyzed for the presence of IL-5, IL-13, IL-17, and IFN-g. *P < .05,
**P < .01. B, Proliferation of CD31 cells was determined by means of flow
cytometry. One representative experiment of 5 is shown. CFSE, Carboxy-
fluorescein succinimidyl ester; CM, complete media.
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POCHARD ET AL 95
TLR4 stimulation enhances IL-10 production by
CPE-stimulated BMDCs, whereas TLR9 enhances
IL-12 productionTo determine the contribution of individual TLRs in the E coli–
induced modification of DC responses to peanut, we added LPS orCpG to CPE and measured DC cytokine production. TLR4 signal-ing after stimulation with LPS and CPE triggered synergistic pro-duction of IL-10 (Fig 5). Interestingly, CpG signaling through
TLR9 had a similar effect on IL-10 production, suggesting thatthis effect is mediated by common TLR pathways. It is notewor-thy that CpG signaling through TLR9 induced synergistic produc-tion of IL-12 when added to CPE. These effects were not observedin DCs deficient in the relevant TLR (Fig 5).
Taken together, these observations strongly suggest that theE coli–induced modification of DC responses to peanut occursthrough the differential triggering of TLRs. The enhancementof the IL-10 and IL-12 production might play a key role in alteringT-cell responses.
E coli partially inhibits the peanut-induced immune
response through the production of IL-12To investigate the role of polarizing cytokines on the effect of
HKE, we performed cocultures using cells from animals deficientin IL-10. As presented in Fig E5 (available in this article’s OnlineRepository at www.jacionline.org), the absence of IL-10 did notaffect the effect of HKE. We next performed cocultures usingantibodies against IL-12/IL-23 p40 and IFN-g. The use of these2 antibodies profoundly inhibited the detection of IFN-g in the su-pernatant, whereas the IL-10 production was unchanged (data notshown), and an isotype control antibody had no effect. As shownin Fig 6, neutralizing IL-12 production partially inhibited the ef-fect of HKE on TH2 cytokines, restoring them to comparablelevels observed with CPE alone for IL-4 and IL-13 or to a higherlevel in the case of IL-5. This effect was associated with a signif-icant increase in proliferation when compared with CD4 T-cellproliferation after exposure to CPE and HKE but to a lower levelthan observed in cells exposed to peanut alone. Using a neutraliz-ing antibody for IFN-g, we were able to make very similar obser-vations for CD4 T-cell proliferation and for IL-5 production.Interestingly, IFN-g did not seem to have much effect on IL-4and IL-13 production because neutralization did not significantlychange the effect of HKE. These results strongly support the
FIG 6. E coli partially inhibits the peanut-induced immune response
through IL-12 production. CD41 T cells were cocultured (ratio 10:1) with
CPE-pulsed BMDCs stimulated with CPE (50 mg/mL), HKE (1 bacterium
per BMDC), or both for 72 hours. BMDCs were preincubated with either
an anti–IL-12 antibody or isotype control at 20 mg/mL. The anti–IFN-g anti-
body (20 mg/mL) was added with T cells. Antibody concentrations were
maintained during the whole time of the coculture. Concentrations of IL-
4, IL-5, and IL-13 were determined, as well as concentrations of IFN-g and
IL-10. Concentrations represent means 6 SEMs. **P <_ .01). After 5 days,
proliferation of CD31 cells was analyzed by means of flow cytometry.
Four combined experiments involving at least 5 mice are shown.
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96 POCHARD ET AL
concept that HKE-induced production of IL-12 has direct and in-direct (through the release of IFN-g) effects on TH2 cytokine pro-duction and T-cell proliferation.
In conclusion, bacterial components, acting through DC TLRsignaling, change the production of polarizing cytokines, durablyshift the peanut-induced TH2 response toward a TH1 profile, andmodify the CD41 T-cell proliferative response.
DISCUSSIONThe prevalence of food allergies has significantly increased in
westernized countries recently, and no treatment is available.Peanut allergy is rarely outgrown, and accidental exposures arecommon and severe. Relatively little is known about the immu-nology of peanut proteins. Although our model does not reproducethe complexity of the gastrointestinal immune system of a livinganimal, our in vitro system has enabled us to make a number ofnovel observations about the cellular and molecular processes in-volved in the DC response to peanut and bacterial adjuvants.
In our in vitro system peanut proteins induced a mixed TH1,TH2, and TH17 recall response. We show that peanut was ableto induce TLR-independent DC activation. Shreffler et al18 haveshown that dendritic cell–specific intercellular adhesion molecule3–grabbing nonintegrin (DC-SIGN) was involved in the recogni-tion of glycosylated Ara h 1 by human DCs, leading to TH2 prim-ing. Homologues of DC-SIGN have been recently described andmight play a role in this CPE-mediated effect.19
To assess how TLR signaling affects the T-cell immuneresponse to peanut, we stimulated DCs with peanut and HKEand cocultured them with primed T cells. Addition of E coli topeanut resulted in skewing the response toward TH1 by decreas-ing TH2 cytokine production, as well as increasing IFN-g levels.HKE also affected T-cell proliferation, which was largely in-hibited. Using MyD88/TRIF-double deficient DCs, we showedthat these changes were attributable to TLRs. Interestingly, we
found that the suppression of proliferation and TH2 cytokineproduction mediated by E coli is sustained when T cells werere-exposed to peanut alone.
This TLR-mediated reshaping of the immune response topeanut appears to occur through the enhancement of TH1responses. IFN-g production occurs in a signal transducer and ac-tivator of transcription 4–dependent manner after the binding ofIL-12 to its receptor. This IL-12–driven TH1 response was de-scribed to downregulate TH2 responses. IL-12 has been shownto play a key role in both the prevention20,21 and treatment21 offood allergy in murine models. In our study adding bacteria toCPE resulted in increasing IL-12, followed by IFN-g, production.We were specifically interested in the possible role of unmethy-lated CpG motifs in this response because it is well establishedthat TLR9 signaling induces an increase in IL-12 production.We report that adding CpG to peanut extract produces IL-12 syn-ergistically, suggesting that the bacteria-induced TH1 responsewas attributed to TLR9. Indeed, the effect of HKE was partiallydependent on IL-12 in our system. This is consistent with the re-sults obtained in mice with peanut allergy successfully treatedwith HKE-expressing mutated recombinant peanut allergens(EMP-123)11 and CpG.13,22 Because IL-12 and IFN-g regulateeach other, we performed experiments to investigate whetherthe IL-12 effect was mediated through IFN-g. Neutralizing thiscytokine only restored IL-5 production and partially restoredCD4 T-cell proliferation, suggesting a direct effect of IL-12 onIL-4 and IL-13 production. These results cannot totally excludethe role of other cytokines, such as IL-18. Our data show thatTLR activation leads to TH1 skewing, which is able to reduceTH2 responses. Future immunotherapeutic strategies to redirectthe TH2 response to peanut or other allergens might target TH1enhancement through the use of microbial compounds. Indeed,clinical trials with TLR9 agonists as adjuvants for aeroallergenimmunotherapy have produced encouraging preliminary re-sults,23,24 but subsequent trials have been halted because of alack of efficacy.
We looked at other polarizing cytokines and observed thatadding E coli to peanut proteins synergistically enhanced IL-10production through TLR4 signaling. Gringhuis et al25 demon-strated that DC-SIGN modulates TLR4 function, leading toincreased IL-10 production. It is tempting to speculate thatthrough DC-SIGN homologues or other lectins, CPE in the pres-ence of TLR agonists will lead to increased IL-10 production.Using DCs and T cells that cannot produce this cytokine, we dem-onstrated that the inhibitory effect of HKE on the peanut responsewas not driven by IL-10 in our system. However, this observationdoes not exclude totally that IL-10, through its immunoregulatoryproperties, might play a role in vivo.
DC maturation was also associated with IL-6 production. IL-6induces IL-4,26 inhibits TH1 differentiation and regulatory T-cellactivity by blocking forkhead box protein 3,27 and plays a role inTH2 differentiation in the lungs of asthmatic patients.28 Indeed,primed CD41 T cells restimulated with CPE produce IL-4,IL-5, and IL-13. These data suggest that IL-6 might participatein the development of TH2 responses to peanut.
In conclusion, peanut antigens stimulate a TLR-independentproinflammatory and proallergic DC maturational program.Peanut-stimulated DCs activate primed T cells, inducing prolif-eration and mixed TH1/TH2/TH17 cytokine responses. DC TLRsignaling in response to microbial products induces IL-12 produc-tion and modifies the recall response to peanut antigens by
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robustly and durably suppressing T-cell proliferation and TH2 cy-tokine production. The ability of TLR signals to induce the syn-ergistic production of IL-12, as well as a shift toward a TH1responder phenotype, suggests that costimulation of innate recep-tors might be an important approach to immunotherapeutic strat-egies for food allergy. We are currently conducting further studiesto analyze the role of the innate immune system in the response tobacterial adjuvants in vivo.
Key messages
d Peanut proteins uniquely activate DCs independent ofTLR pathways.
d Peanut-induced DC signals can be altered by the presenceof TLR ligands, which redirect T-cell responses.
d These concepts might be useful in the design and applica-tion of immunotherapeutic strategies toward peanutallergy.
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