Background: Two major distinct subsets of dendritic cells (DCs) are arranged to regulate immune responses: DEC-205+ DCs drive Th1 polarization and 33D1+ DCs establish Th2 dominancy. Th1 polarization can be achieved either by depletion of 33D1+ DCs with a 33D1-specific monoclonal antibody (mAb) or by activation of DEC-205+ DCs via intraperitoneal injection of α-galactosylceramide (α-GalCer). We studied the effect of 33D1+ DC depletion or DEC-205+ DC activation in vivo using an established mouse model of allergic rhinitis (AR). Methods: Mice were injected intraperitoneally with OVA plus alum and challenged 4 times with daily intranasal administration of OVA. Immediately after the last challenge, allergic symptoms such as sneezing and nasal rubbing as well as the number of cells in the bronchoalveolar lavage fluid (BALF) and nasal lavage fluid (NALF) were counted. The levels of serum OVA-specific IgG1, IgG2a, and IgE were also determined by ELISA. Results: The allergic symptom scores were significantly decreased in 33D1+ DC-depleted or DEC-205+ DC-activated AR mice. The levels of OVA-specific IgG1, IgG2a, and IgE, and the number of NALF cells, but not BALF cells, were reduced in 33D1+ DC-depleted but not in DEC-205+ DC-activated AR mice. Moreover, the activated DEC-205+ DCs suppressed histamine release from IgE-sensitized mast cells, probably through IL-12 secretion. Conclusions: The manipulation of innate DC subsets may provide a new therapeutic strategy for controlling various allergic diseases by reducing histamine release from IgE-sensitized mast cells by driving the immune response towards Th1 dominancy via activation of DEC-205+ DCs in vivo.

Dendritic cells (DCs) serve an important role by providing an appropriate internal immune balance between Th1 and Th2 responses [1,2,3]. In mice, this Th1/Th2 balance appears to be regulated primarily by two distinct DC subsets [4,5,6]: CD8α+ DEC-205 (CD205)+ DCs, which have the capacity to induce Th1 polarization, and 33D1 [DC-inhibitory receptor-2 (DCIR2)]+ [7] DCs, which elicit Th2 dominance. In acquired immunity, Th1 polarization will initiate cellular immune responses against foreign bodies to eradicate them, while Th2 dominancy will induce acquired humoral immunity to produce various antibodies, including IgE, against antigens for elimination.

Additionally, in innate immunity, a Th1 and Th2 balance can still be observed. Although various types of DC subpopulations have been described [3], Steinman and colleagues [5] have suggested that two DC subsets, 33D1+ DCs and DEC-205+ DCs, have innate propensities to differentially affect the Th1/Th2 balance in vivo. Indeed, we have previously shown that it is possible to deplete 33D1+ DCs and establish Th1 dominance by DEC-205+ DCs through the administration of a 33D1-specific monoclonal antibody (mAb) in vivo [8]. Based on these findings, we recently reported that the depletion of 33D1+ DCs in pregnant mice following syngeneic mating strongly induced spontaneous abortion during the perinatal period that was mediated through Th1 augmentation initiated by DEC-205+ DC dominance [9]. In contrast, Th2 dominance organized by the 33D1+ DC subset may be required to maintain a successful pregnancy. In fact, we have found that progesterone, a maternal hormone that maintains the pregnant state, has the capacity to stimulate and activate the 33D1+ DC subset. Moreover, it has been proposed that Th2 dominancy is also required to elicit an allergic status; many allergic symptoms worsen during pregnancy [10,11]. Thus, the alteration of Th2 to Th1 dominance in vivo appears to be a promising strategy to overcome a variety of allergic diseases.

In the present study, using the established allergic rhinitis (AR) mouse model, we examined the effects of depleting the 33D1+ DC subset either on the sensitization phase of an allergen or on the induction phase of allergic symptoms via allergen challenge in sensitized mice. We found that both the production of allergen-specific IgE and allergic responses to the allergen challenge were markedly inhibited by depletion of the 33D1+ DC subset before and during sensitization with the allergen. However, although allergic responses were apparently inhibited, allergen-specific IgE production was not suppressed by depletion of the 33D1+ DC subset after sensitization. These findings indicate that the 33D1+ DC subset should be required for both sensitization with an allergen to initiate IgE production and elicitation of antigen-specific allergic responses; thus, we can prevent antigen-specific IgE production by eliminating the 33D1+ DC subset before sensitization.

Moreover, our findings also suggest that we can manipulate allergic reactions by selectively activating the DEC-205+ DC subset, which may induce a Th1-dominant status. It has recently been reported that α- galactosylceramide (α-GalCer), a known glycolipid antigen that stimulates invariant natural killer T (iNKT) cells expressing invariant Vα14 TCRs in a CD1d-restricted manner [12], can specifically stimulate DEC-205+ DCs [13]. Based on these findings, we intraperitoneally administered α-GalCer to allergen-sensitized mice and found that we could reduce allergic reactions using this procedure.

Taken together, our findings suggest that we can manipulate allergic reactions to specific allergens not only by depleting the 33D1+ DC subset, but also by selectively activating the DEC-205+ DC subset through the administration of a glycolipid such as α-GalCer, even during the antigen-specific IgE-producing state. The findings shown here shed new light on a method that can be used to analyze the pathogenesis of allergy as well as allergy treatment.

Mice

Six- to 8-week-old female BALB/c (H-2d) mice were purchased from Charles River (Tokyo, Japan), maintained in microisolator cages under pathogen-free conditions, and fed autoclaved laboratory chow and water. At least 4 mice in each experimental cohort were examined. All of the animal experiments were performed according to the guidelines for the care and use of laboratory animals by the National Institutes of Health (NIH; Bethesda, Md., USA) and approved by the Review Board of Nippon Medical School (Tokyo, Japan).

OVA-Induced Sneezing or Nasal Rubbing in Mice

Mice were injected intraperitoneally with 20 µg OVA (Sigma-Aldrich; St Louis, Mo., USA) emulsified with 2.25 mg alum [Al(OH)3] (Pierce; Rockford, Ill., USA) in 200 µl PBS on days 0, 7, 14, and 21 of the experiment. The mice were challenged 4 times by daily intranasal administration of 600 µg OVA dissolved in 20 µl PBS on days 35-38 without anesthesia. Allergic responses were monitored immediately following the last challenge, and sneezing and nasal rubbing were counted for 5 min.

Isolation of Bronchoalveolar Lavage Fluid and Nasal Lavage Fluid

Six hours after the last nasal challenge, mice were euthanized by intraperitoneal injection of an overdose of pentobarbital sodium solution. Bronchoalveolar lavage fluid (BALF) was collected via three injections of 0.5-ml aliquots of PBS (total volume of 1.5 ml) using a tracheal cannula. PBS (160 µl) was instilled via tracheotomy into the nasal cavity by using a micropipette, and nasal lavage fluid (NALF) was collected. The cells were centrifuged at 300 g for 5 min and resuspended in PBS for analysis. The total numbers of cells in the BALF or NALF were counted using a hemocytometer. Slides were then prepared using a cytocentrifuge (StatSpin; Beckman Coulter, Brea, Calif., USA), stained with May-Grünwald-Giemsa staining, and the number of eosinophils were evaluated by observing at least 300 cells.

In vivo Depletion of 33D1+ DCs and Activation of DEC-205+ DCs

In vivo depletion of 33D1+ DCs in mice was performed as described previously [8]. In brief, the culture supernatant of 33D1 hybridoma cells (ATCC, Manassas, Va., USA) was collected, and a 33D1 mAb (rat IgM) against DCIR2 was purified from the supernatant by ion-exchange chromatography using diethylaminoethyl (DEAE)-cellulose (DE52; Whatman, Maidstone, UK). Mice were injected intraperitoneally with 0.5 mg purified 33D1 mAb or control rat IgM (Jackson Immuno Research Laboratories, West Grove, Pa., USA) as described in the experimental protocols for each figure.

In vivo activation of DEC-205+ DCs was performed via the injection of α-GalCer (KRN7000; Funakoshi Co., Ltd., Tokyo, Japan). Mice were injected intraperitoneally with 2 μg α-GalCer in 100 μl distilled water on day 32 as described in the legend for figure 4 and on days -1, 6, 13, 20, 27, and 34 as described in the legend for figure 5.

Determination of the Amount of Anti-OVA Antibodies and IL-12p40 in Sera

The production of OVA-specific IgG1 and IgE antibodies, anti-OVA-specific IgG2a antibodies, and IL-12p40 was measured with an anti-OVA EIA Kit (Cayman Chemical Company, Ann Arbor, Mich., USA), anti-OVA IgG2A Antibody Assay Kit (Chondrex Inc., Redmond, Wash., USA), or IL-12p40 detection Kit (R&D Systems, Minneapolis, Minn., USA), respectively, using blood samples collected 6 h after the last nasal challenge. The results are presented as arbitrary units.

Flow Cytometric Analysis

Flow cytometric analyses were performed to determine the surface molecule expression of the cells using a FACSCanto II six-color cytometer (Becton Dickinson Immunochemical Systems, Mountain View, Calif., USA). Half a million cells were pelleted and resuspended in 100 µl PBS containing 1% FCS and 0.1% sodium azide. Fluorescent dye-labeled mAbs were added to the pellet and incubated for 30 min at 4°C. The cells were then washed twice and resuspended in PBS. The following antibodies were used: FITC-labeled anti-mouse CD11c (clone N418; BioLegend, San Diego, Calif., USA), PE or PE/Cy7-labeled anti-mouse CD205 (DEC-205) (clone NLDC-145; BioLegend), APC-labeled anti-mouse DC marker (33D1) (clone 33D1; BioLegend), PE-labeled anti-mouse CD1d (clone 1B1; BioLegend), APC/Cy7-labeled anti-mouse MHC class II (MHC-II) (clone M5/114.15.2; BioLegend), PE-labeled anti-mouse CD8α (clone 53-6.7; BioLegend), PE-labeled anti-mouse CD80 (clone 16-10A1; BioLegend), PE-labeled anti-mouse CD86 (clone GL1; BioLegend), PE-labeled anti-mouse CD40 (clone 3/23; BioLegend), PE-labeled anti-mouse PD-L1 (clone 10F.9G2; BioLegend), and PE-labeled anti-mouse α-GalCer:CD1d complex (clone L363). Dead cells were gated out by forward and side scatter based on propidium iodide uptake. 10,000 events were acquired for each sample and analyzed using FlowJo software (Tree Star Inc., Ashland, Oreg., USA).

Histamine Detection

Bone marrow (BM) cells of BALB/c mice (2 × 106 cells/well) were cultured in RPMI-1640 medium (Sigma-Aldrich, St. Louis, Mo., USA), supplemented with 10% heat-inactivated FCS, 2 mML-glutamine (Sigma-Aldrich), 10 mM HEPES, 100 µM nonessential amino acids (Invitrogen Life Technologies, Carlsbad, Calif., USA), 10 mM sodium pyruvate (Sigma-Aldrich), 100 U/ml penicillin (Invitrogen Life Technologies), 100 µg/ml streptomycin (Invitrogen Life Technologies), and 10 ng/ml IL-3 (PeproTech, Rocky Hill, N.J., USA) in a humidified incubator at 37°C and 5% CO2. After 3-4 weeks of culture, the obtained c-kit+ BM-mast cells were adjusted to >95% pure mast cells. BM-mast cells were incubated at a density of 2 × 105 cells with anti-DNP mouse IgE mAb (240 ng/ml; clone SPE7; Sigma-Aldrich) at 37°C overnight with either IL-10 (20 ng/ml; PeproTech) or IL-12 (20 ng/ml; R&D Systems), in the presence of 2 × 105 cells of BM-derived DCs (BM-DCs) or α-GalCer (20 ng/ml)-stimulated BM-DCs, 33D1-depleted BM-DCs. After being washed 3 times with RPMI-1640, the treated cells were further incubated for 60 min with 10 ng/ml DNP-BSA (Sigma-Aldrich) at 37°C. Quantitative determination of histamine in the cell-free supernatants was performed using a histamine enzyme immunoassay kit (SPI-BIO, Bretonneux, France).

Statistical Analysis

The results were analyzed using Student's t test and Tukey's multiple comparison test, and the results are presented as the mean value ± SD. Differences at p < 0.05 were considered statistically significant.

Establishment of an AR Mouse Model

To examine the role of DCs in experimental AR in mice, we first confirmed the characteristics of our established AR mouse model based on previous findings [14]. BALB/c mice were sensitized via intraperitoneal injection of 20 µg OVA mixed with 2.25 mg of the adjuvant alum 4 times on days 0, 7, 14, and 21. The AR mice were then challenged 4 times by daily intranasal administration of 600 µg OVA in PBS on days 35-38 (online suppl. fig. 1a; see www.karger.com/doi/10.1159/000443237 for all online suppl. material). Allergic symptoms were estimated based on the number of observations of sneezing and nasal rubbing during the 5 min immediately after the final nasal challenge with OVA. Sneezing and nasal rubbing after the final challenge were much higher in AR mice than in control mice that were sensitized with alum alone (online suppl. fig. 1b, c). Six hours after the final challenge, the mice were euthanized and the sera, BALF, and NALF were isolated for characterization.

The levels of serum OVA-specific IgG1, IgG2a, and IgE were all higher in AR mice than in control mice (online suppl. fig. 1d-f). To examine whether the symptoms of asthma could be induced via sensitization with OVA plus alum in AR mice, the number of cells in the BALF was estimated. Although there were no apparent differences between AR and control mice in terms of BALF cells (online suppl. fig. 1g), the number of cells in the NALF was significantly higher in AR mice than in control mice (online suppl. fig. 1h). Moreover, it should be noted that the number of eosinophils was always far higher in the NALF, but not in the BALF, of AR compared with control untreated mice (online suppl. fig. 1i), suggesting that the AR mice were certainly sensitized with the allergen OVA and that the nasal route played a major role in inducing allergic reactions in AR mice. These results indicate that an experimental AR mouse model exhibiting various IgE-mediated allergic symptoms could be successfully achieved via intranasal sensitization with OVA plus the adjuvant alum in BALB/c mice.

Characterization of DC Subsets in the Spleen of Normal BALB/c Mice

DCs play a pivotal role in providing an appropriate internal balance between the Th1 and Th2 immune responses. As demonstrated in the left panel of figure 1a, murine DCs could be obtained as with the CD11c+MHC-II+ subset in the spleen (0.776%). These CD11c+MHC-II+ DCs could be divided mainly into three nonoverlapping populations: DEC-205+, 33D1+, and DEC-205- 33D1- (double negative) DCs (middle panel of fig. 1a). It should be noted that DEC-205+ 33D1+ (double positive) DCs were hardly detected (middle panel of fig. 1a). Moreover, the majority of the DEC-205+ DCs were CD8α+ (right panel of fig. 1a). In addition, the expression of CD1d was examined in each DC subset and found in approximately 80% of DEC-205+ DCs, 50% of 33D1+ DCs, and 20% of DEC-205- 33D1- DCs (fig. 1b). The Th1/Th2 balance appears to be regulated primarily by those two distinct DC subsets, DEC-205+ DCs, which have the capacity to establish Th1 polarization, and 33D1+ DCs, which induce Th2 dominance [4,6,15], and may assist in inducing an allergic status.

Fig. 1

Characterization of DC subsets in the spleen of normal BALB/c mice. a CD11c+MHC-II+ cells in the spleen (0.776%) of normal BALB/c mice were considered to be DCs (indicated by the square in the left panel). The gated CD11c+MHC-II+ DCs were further stained with 33D1-specific mAb, DEC-205-specific mAb, and CD8α mAb, and analyzed by flow cytometry (right panels). b CD1d molecule-expression in each DC subset was also examined and the percentage of CD1d molecule-expressed DCs among each subset (left panels) and their percentages are indicated (right panel). Gray histograms indicate the isotype-matched negative control. The results shown are representative of 3 independent experiments using 4 mice per group.

Fig. 1

Characterization of DC subsets in the spleen of normal BALB/c mice. a CD11c+MHC-II+ cells in the spleen (0.776%) of normal BALB/c mice were considered to be DCs (indicated by the square in the left panel). The gated CD11c+MHC-II+ DCs were further stained with 33D1-specific mAb, DEC-205-specific mAb, and CD8α mAb, and analyzed by flow cytometry (right panels). b CD1d molecule-expression in each DC subset was also examined and the percentage of CD1d molecule-expressed DCs among each subset (left panels) and their percentages are indicated (right panel). Gray histograms indicate the isotype-matched negative control. The results shown are representative of 3 independent experiments using 4 mice per group.

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In vivo Depletion of 33D1+ DCs by Intraperitoneal Injection with Anti-33D1 mAb in Normal BALB/c Mice

We recently reported that 33D1+ DCs can be depleted in vivo by treating C57BL/6 mice with an anti-33D1 mAb and that the Th1/Th2 balance can be controlled by the manipulation of DC subsets [8,9]. To observe the effects of 33D1+ DC depletion in AR mice, we first confirmed whether 33D1+ DCs could also be depleted in normal BALB/c mice using this procedure. Normal BALB/c mice were injected with 0.5 mg purified anti-33D1 mAb i.p. for 3 consecutive days, and the percentages of 33D1+ DCs and DEC-205+ DCs in the spleen were analyzed by flow cytometry 1 day, 1 week, and 2 weeks after the final injection (online suppl. fig. 2a). As shown in online supplementary figure 2b, 33D1+ DCs in the spleen were almost completely depleted from 1 day to 1 week after the final injection, but a small number of cells reappeared after 2 weeks. However, the percentages of the DEC-205+ DC subset were somewhat increased in anti-33D1 mAb-treated mice (online suppl. fig. 2b). Therefore, to deplete or suppress the numbers of 33D1+ DCs in the spleens of BALB/c mice continuously for approximately 6 weeks, the mice were injected with the anti-33D1 mAb on 3 successive days and given weekly booster injections with the same mAb 5 times (online suppl. fig. 2c). The percentages of 33D1+ DCs and DEC-205+ DCs in the spleen cells of the treated mice were analyzed by flow cytometry. The 33D1+ DCs in the spleen were completely depleted for approximately 6 weeks, while the percentage of DEC-205+ DCs remained almost unchanged (online suppl. fig. 2d). These findings indicate that we could continuously eradicate 33D1+ DCs from normal BALB/c mice for approximately 6 weeks by weekly injections with an anti-33D1 mAb after the initial administration. As shown in the figure 1, DEC-205+ 33D1+ (double positive) DCs could not be detected among the splenic cells of AR model mice. Thus, DEC-205+ DCs were not affected by the treatment with anti-33D1 mAb.

The Effects of 33D1+ DC Depletion before OVA plus Alum Sensitization in the AR Mouse Model

As shown in the experimental protocol presented in figure 2a, BALB/c mice were injected intraperitoneally with 0.5 mg purified anti-33D1 mAb for 3 successive days followed by weekly booster injections with the same mAb for 5 weeks. As in normal untreated mice, spleen cells were prepared 4 days after the last mAb injection (38 days after the first sensitization with OVA plus alum), and the percentages of both 33D1+ DCs and DEC-205+ DCs were analyzed by flow cytometry. As previously shown in online supplementary figure 2d, although 33D1+ DCs were almost completely depleted from the spleens of untreated normal BALB/c mice, a small percentage of the 33D1+ DC subset always remained when OVA plus adjuvant alum was administered in AR mice (fig. 2b), suggesting that the adjuvant alum and/or OVA sensitization may possess the required potency to maintain 33D1+ DC subset numbers in vivo.

Fig. 2

Depletion of 33D1+ DCs in vivo via the injection of anti-33D1 mAb in AR mice. a Mice were intraperitoneally (i.p.) injected with 0.5 mg purified anti-33D1 mAb on 3 consecutive days (days -3, -2, and -1) and boosted with 0.5 mg the same mAb once per week 5 times (days 6, 13, 20, 27, and 34). These mice were also sensitized by an intraperitoneal injection of OVA plus the adjuvant alum on days 0, 7, 14, and 21. Two weeks after the final sensitization, the mice were challenged 4 times by daily intranasal administration of OVA on days 35-38. b Six hours after the last challenge, spleen cells were prepared from the mice, and thepercentages of 33D1+ DCs and DEC-205+ DCs were analyzed by flow cytometry. c The effects of 33D1+ DC depletion by anti-33D1 mAb on OVA plus adjuvant alum sensitization of AR mice were examined. Sneezing and nasal rubbing (d, e), anti-OVA-specific immunoglobulins (f-h), and the total numbers of cells in BALF (i) and NALF (j) were determined. The results shown are representative of 3 independent experiments using 4 mice per group. Statistical analyses were performed using Tukey's multiple comparison test, and * p < 0.05 and ** p < 0.01 were considered statistically significant.

Fig. 2

Depletion of 33D1+ DCs in vivo via the injection of anti-33D1 mAb in AR mice. a Mice were intraperitoneally (i.p.) injected with 0.5 mg purified anti-33D1 mAb on 3 consecutive days (days -3, -2, and -1) and boosted with 0.5 mg the same mAb once per week 5 times (days 6, 13, 20, 27, and 34). These mice were also sensitized by an intraperitoneal injection of OVA plus the adjuvant alum on days 0, 7, 14, and 21. Two weeks after the final sensitization, the mice were challenged 4 times by daily intranasal administration of OVA on days 35-38. b Six hours after the last challenge, spleen cells were prepared from the mice, and thepercentages of 33D1+ DCs and DEC-205+ DCs were analyzed by flow cytometry. c The effects of 33D1+ DC depletion by anti-33D1 mAb on OVA plus adjuvant alum sensitization of AR mice were examined. Sneezing and nasal rubbing (d, e), anti-OVA-specific immunoglobulins (f-h), and the total numbers of cells in BALF (i) and NALF (j) were determined. The results shown are representative of 3 independent experiments using 4 mice per group. Statistical analyses were performed using Tukey's multiple comparison test, and * p < 0.05 and ** p < 0.01 were considered statistically significant.

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Then, we examined whether 33D1+ DCs could be depleted via the described procedure in mice treated with either alum alone or OVA alone. As shown in figure 2c, although 33D1+ DCs were almost completely eradicated from the spleens of untreated BALB/c mice (left upper middle panel, 0.06%), a small number of the 33D1+ DC subset remained following the injection of adjuvant alum into the mice (right upper middle panel, 0.85%). These results suggest that the adjuvant alum might have the ability to maintain the percentage of 33D1+ DCs in the spleens of AR mice, even with frequent injections of anti-33D1 mAb.

Next, we examined the effects of 33D1+ DC depletion in AR mice before sensitization with alum plus OVA using the protocol shown in figure 2a. Sneezing and nasal rubbing after nasal challenge with OVA were examined to compare the untreated control, AR, and 33D1+ DC-depleted AR mice. Both sneezing (fig. 2d) and nasal rubbing (fig. 2e) were significantly decreased in 33D1+ DC-depleted AR mice compared with untreated AR mice. Additionally, the levels of serum OVA-specific IgG1 (fig. 2f), IgG2a (fig. 2g), and IgE (fig. 2h) were lower in 33D1+ DC-depleted AR mice than in untreated AR mice. Moreover, it should be noted that, although the number of BALF cells in 33D1+ DC-depleted AR mice remained almost unchanged compared with that in untreated AR mice (fig. 2i), the 33D1+ DC-depleted AR mice displayed a significant reduction in the number of NALF cells (fig. 2j). The number of eosinophils in NALF was again markedly higher in AR mice stimulated with OVA than in control mice (data not shown). Therefore, depletion of the 33D1+ DC subset in vivo during antigenic sensitization with OVA plus alum inhibits not only various allergic symptoms such as sneezing and nasal rubbing, but also the production of allergen-specific immunoglobulins in the serum. These results indicate that the 33D1+ DC subset plays an important role in the induction of allergic responses through sensitization with OVA to produce specific antibodies.

The Effects of 33D1+ DC Depletion in OVA-Sensitized AR Mice

Next, we examined the effects of depleting the 33D1+ DC subset in the spleen on allergic reactions in OVA-sensitized AR mice. The AR mice were prepared by injecting OVA plus the adjuvant alum as shown in figure 2, and the 33D1+ DC subset in the spleens of the sensitized mice was depleted with anti-33D1 mAb immediately prior to nasal challenge (fig. 3a). Six hours after the last nasal challenge on day 38, we used flow cytometry to test whether the 33D1+ DC subset was depleted from the spleens of sensitized mice. As shown in figure 3b, the percentage of 33D1+ DCs in anti-33D1 mAb-treated AR mice (right upper panel; 0.05%) was significantly lower than that in untreated AR mice (middle upper panel; 3.23%) or control untreated mice sensitized with alum alone (left upper panel; 2.17%). As expected, the percentage of the 33D1+ DC subset increased slightly on day 41 (data not shown) in anti-33D1 mAb-treated AR mice, indicating the slow but gradual recovery of this subset even after strong depletion in AR mice. As also shown in the lower panels of figure 3b, the percentage of the DEC-205+ DC subset displayed a relative increase in AR mice treated with anti-33D1 mAb (right lower panel; 1.12%). Under these conditions in which a relatively high percentage of the DEC-205+ DC subset was detected in sensitized AR mice whose 33D1+ DCs were depleted, sneezing and nasal rubbing after nasal challenge were significantly inhibited compared with the untreated AR mice (fig. 3c, d). However, the production of serum OVA-specific IgG1, IgG2a, and IgE remained almost unchanged between 33D1+ DC-depleted and untreated AR mice (fig. 3e-g). Moreover, while the numbers of BALF cells were again almost the same in these two groups (fig. 3h), the numbers of NALF cells in 33D1+ DC-depleted AR mice were lower than those in untreated AR mice (fig. 3i). Taken together, although the magnitude of the effect of 33D1+ DC depletion in OVA-sensitized AR mice was smaller than that observed via continuous depletion throughout the sensitization period, the presence of the 33D1+ DC subset appears to assist in the induction of allergic responses to OVA nasal challenge.

Fig. 3

Effects of 33D1+ DC depletion on allergic symptoms in OVA-sensitized mice. a Mice were injected intraperitoneally (i.p.) with 20 µg OVA mixed with 2.25 mg alum in 200 µl PBS on days 0, 7, 14, and 21. The 33D1+ DCs were depleted by intraperitoneal injection with anti-33D1 mAb on days 32-34. Following the depletion of 33D1+ DCs, the mice were challenged with daily intranasal OVA solution on days 35-38. b Six hours after the last challenge on day 38, spleens were isolated from the mice and the percentages of 33D1+ DCs and DEC-205+ DCs were analyzed by flow cytometry. After depletion of the 33D1+ DCs, the mice were challenged with daily intranasal OVA solution on days 35-38. Sneezing (c), nasal rubbing (d), anti-OVA-specific immunoglobulins (e-g), and total numbers of cells in BALF (h) and NALF (i) were counted. The results presented are representative of 3 independent experiments using 4 mice per group. Statistical analyses were performed using Tukey's multiple comparison test, and * p < 0.05 and ** p < 0.01 were considered statistically significant.

Fig. 3

Effects of 33D1+ DC depletion on allergic symptoms in OVA-sensitized mice. a Mice were injected intraperitoneally (i.p.) with 20 µg OVA mixed with 2.25 mg alum in 200 µl PBS on days 0, 7, 14, and 21. The 33D1+ DCs were depleted by intraperitoneal injection with anti-33D1 mAb on days 32-34. Following the depletion of 33D1+ DCs, the mice were challenged with daily intranasal OVA solution on days 35-38. b Six hours after the last challenge on day 38, spleens were isolated from the mice and the percentages of 33D1+ DCs and DEC-205+ DCs were analyzed by flow cytometry. After depletion of the 33D1+ DCs, the mice were challenged with daily intranasal OVA solution on days 35-38. Sneezing (c), nasal rubbing (d), anti-OVA-specific immunoglobulins (e-g), and total numbers of cells in BALF (h) and NALF (i) were counted. The results presented are representative of 3 independent experiments using 4 mice per group. Statistical analyses were performed using Tukey's multiple comparison test, and * p < 0.05 and ** p < 0.01 were considered statistically significant.

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Effects of α-GalCer Administration on Allergic Reactions in AR Mice

As mentioned before, the Th1/Th2 balance appears to be regulated primarily by two distinct DC subsets, DEC-205+ DCs and 33D1+ DCs, and allergic reactions appear to be initiated and maintained via Th2 polarization by 33D1+ DCs. It was recently reported that a known target of NKT cells, specifically the CD1d-restricted glycolipid antigen α-GalCer, can selectively activate DEC-205+ DCs to shift the Th1/Th2 balance towards Th1 dominance [13]. Based on the experimental protocols shown in figure 4a, we used a single intraperitoneal injection with 2 µg/mouse of α-GalCer to determine whether activation of DEC-205+ DCs could be achieved in OVA-sensitized AR mice. Although the percentage of 33D1+ DCs remained almost unchanged following the administration of α-GalCer, as shown in the upper panels of figure 4b, there was a significant increase in the number of DEC-205+ DCs (lower panels of fig. 4b). The percentage of DEC-205+ DCs was markedly increased in OVA-sensitized AR mice that received an intraperitoneal injection of 2 µg α-GalCer, although the percentage of 33D1+ DCs was slightly decreased (fig. 4c). As expected, allergic symptoms such as sneezing and nasal rubbing were both significantly reduced in OVA-challenged AR mice inoculated intraperitoneally once with 2 µg/mouse of α-GalCer (fig. 4d, 4e). It should be noted that the sensitized mice still produced OVA-specific antibodies such as IgG1 (fig. 4f), IgG2a (fig. 4g), and IgE (fig. 4h). In addition, while the number of BALF cells remained almost unchanged (fig. 4i), the number of NALF cells was significantly reduced in the challenged mice that received α-GalCer (fig. 4j). These findings suggest that the selective activation of DEC-205+ DCs in OVA-sensitized mice resulting in the production of OVA-specific IgG1, IgG2a, and IgE suppressed local allergic reactions following a nasal challenge with OVA, but did not cause a decrease in antibody production.

Fig. 4

Effects of DEC-205+ DC activation by α-GalCer in OVA-sensitized mice. a For the sensitization, mice were injected intraperitoneally (i.p.) with 20 µg OVA mixed with 2.25 mg alum in 200 µl PBS on days 0, 7, 14, and 21. DEC-205+ DCs were activated by a single intraperitoneal injection with 2 µg/ml α-GalCer on day 32. After activation of the DEC-205+ DCs, the mice were challenged with daily intranasal (i.p.) OVA solution on days 35-38. b, c Six hours after the last challenge (38 days), spleens were isolated from the mice and the percentages of 33D1+ DCs and DEC-205+ DCs were analyzed by flow cytometry. Sneezing (d) and nasal rubbing (e), anti-OVA-specific immunoglobulins (f-h), and total numbers of cells in BALF (i) and NALF (j) were determined. The results shown are representative of 3 independent experiments using 4 mice per group. Statistical analyses were performed using Tukey's multiple comparison test, and * p < 0.05 and ** p < 0.01 were considered statistically significant.

Fig. 4

Effects of DEC-205+ DC activation by α-GalCer in OVA-sensitized mice. a For the sensitization, mice were injected intraperitoneally (i.p.) with 20 µg OVA mixed with 2.25 mg alum in 200 µl PBS on days 0, 7, 14, and 21. DEC-205+ DCs were activated by a single intraperitoneal injection with 2 µg/ml α-GalCer on day 32. After activation of the DEC-205+ DCs, the mice were challenged with daily intranasal (i.p.) OVA solution on days 35-38. b, c Six hours after the last challenge (38 days), spleens were isolated from the mice and the percentages of 33D1+ DCs and DEC-205+ DCs were analyzed by flow cytometry. Sneezing (d) and nasal rubbing (e), anti-OVA-specific immunoglobulins (f-h), and total numbers of cells in BALF (i) and NALF (j) were determined. The results shown are representative of 3 independent experiments using 4 mice per group. Statistical analyses were performed using Tukey's multiple comparison test, and * p < 0.05 and ** p < 0.01 were considered statistically significant.

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Effects of α-GalCer Preadministration on Allergic Sensitization

Next, to examine the possibility that preadministration of α-GalCer could inhibit allergic sensitization with OVA plus alum adjuvant as indicated in figure 5a, BALB/c mice were injected intraperitoneally with 2 µg/mouse of α-GalCer 6 times at weekly intervals before, during, and after sensitization to determine whether activated DEC-205+ DCs could inhibit OVA-sensitization of AR mice in vivo (fig. 5a). Although the percentage of 33D1+ DCs was slightly decreased by the 6 injections of α-GalCer, as shown in the upper panels of figure 5b, there was a significant increase in the number of DEC-205+ DCs (lower panels of fig. 5b). Additionally, the percentage of DEC-205+ DCs increased markedly when OVA-sensitized AR mice received an intraperitoneal injection of 2 µg α-GalCer, although the percentage of 33D1+ DCs decreased slightly (fig. 5c). As expected, allergic symptoms such as sneezing and nasal rubbing were significantly suppressed in the OVA-challenged AR mice that received 6 injections of α-GalCer at a concentration of 2 mg/mouse (fig. 5d, e). It should be noted that the α-GalCer-treated sensitized mice produced OVA-specific IgG1 and IgE antibodies (fig. 5f, h), but also exhibited significantly reduced IgG2a production (fig. 5g). In addition, although the number of BALF cells remained almost unchanged (fig. 5i), the number of NALF cells was again significantly reduced in the challenged mice that received α-GalCer (fig. 5j). In summary, although DEC-205+ DC activation due to α-GalCer administration in OVA-sensitized AR mice did not affect OVA-specific IgE production, DEC-205+ DC activation prior to sensitization suppressed the production of IgG2a as well as various allergic symptoms.

Fig. 5

Effects of DEC-205+ DCs activation by α-GalCer on OVA-sensitization in AR mice. a To examine the possibility that the preadministration of α-GalCer could inhibit allergic sensitization with OVA plus alum, BALB/c mice were injected intraperitoneally (i.p.) with 2 µg/mouse of α-GalCer 6 times at weekly intervals before, during, and after sensitization to determine whether the activation of DEC-205+ DCs could inhibit OVA sensitization in AR mice. b, c Six hours after the last challenge (38 days), spleens were isolated from the mice and the percentages of 33D1+ DCs and DEC-205+ DCs were analyzed by flow cytometry. Sneezing (d) and nasal rubbing (e), anti-OVA-specific immunoglobulins (f-h), and total numbers of cells in BALF (i) and NALF (j) were determined. The results shown are representative of 3 independent experiments using 4 mice per group. Statistical analyses were performed using Tukey's multiple comparison test, and * p < 0.05 and ** p < 0.01 were considered statistically significant.

Fig. 5

Effects of DEC-205+ DCs activation by α-GalCer on OVA-sensitization in AR mice. a To examine the possibility that the preadministration of α-GalCer could inhibit allergic sensitization with OVA plus alum, BALB/c mice were injected intraperitoneally (i.p.) with 2 µg/mouse of α-GalCer 6 times at weekly intervals before, during, and after sensitization to determine whether the activation of DEC-205+ DCs could inhibit OVA sensitization in AR mice. b, c Six hours after the last challenge (38 days), spleens were isolated from the mice and the percentages of 33D1+ DCs and DEC-205+ DCs were analyzed by flow cytometry. Sneezing (d) and nasal rubbing (e), anti-OVA-specific immunoglobulins (f-h), and total numbers of cells in BALF (i) and NALF (j) were determined. The results shown are representative of 3 independent experiments using 4 mice per group. Statistical analyses were performed using Tukey's multiple comparison test, and * p < 0.05 and ** p < 0.01 were considered statistically significant.

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Kinetics of DC Subset Activation and Costimulatory Molecule Expression in DC Subsets in Response to α-GalCer Stimulation

To confirm whether DEC-205+ DCs were activated in vivo by the intraperitoneal administration of 2 µg/mouse of α-GalCer, we investigated the kinetics of the percentage of 33D1+ DCs and DEC-205+ DCs. The percentages of CD11c+ DC subsets were determined for 48 h after α-GalCer injection. As shown in online supplementary figure 3a, although the percentage of 33D1+ DCs gradually decreased, the percentage of DEC-205+ DCs apparently increased among CD11c+ cells in the spleens of treated BALB/c mice. In addition, the expression of various costimulatory molecules required for T-cell activation, such as CD80, CD86, and CD40, increased on DEC-205+ DCs compared with 33D1+ and DEC-205- 33D1- DCs, while that of PD-L1 was not significantly augmented among the CD11c+ DC subsets stimulated with 2 µg/mouse of α-GalCer (online suppl. fig 3b). Moreover, the expression of the α-GalCer:CD1d complex on the DCs of α-GalCer inoculated mice was specifically augmented on the DEC-205+, but not on the 33D1+ or DEC-205- 33D1- DCs (online suppl. fig. 3c). Therefore, α-GalCer stimulation appeared to predominantly activate the DEC-205+ DCs among the CD11c+ DC subsets.

Effect of Activated DEC-205+ DCs on Histamine Release from IgE-Sensitized Mast Cells

Finally, although we have shown here that allergen-specific IgE production was not suppressed by 33D1+ DC subset depletion after sensitization, the treatment still seemed to inhibit the symptoms of AR. This effect may have been due to the ability of the treatment to inhibit the release of histamine from antigen-sensitized mast cells. To examine this possibility, we stimulated IgE-specific antigen (DNP)-sensitized mast cells in vitro with antigen (DNP-BSA) overnight at 37°C and measured the amount of histamine released in the presence of DC-related cytokines or syngeneic DCs or their subsets. As demonstrated here, the amount of released histamine was slightly reduced by the addition of 20 ng/ml IL-12, but significantly augmented by the addition of 20 ng/ml IL-10 or in the presence of syngeneic untreated DCs (fig. 6a). However, the amount of histamine was markedly inhibited to levels below the unstimulated control in the presence of 33D1+ DC-depleted DCs or α-GalCer-stimulated DCs, or both (fig. 6b). Also, the amount of IL-12 secreted from mice was markedly augmented either by depletion of 33D1+ DCs with anti-33D1 mAb injection or by activation of DEC-205+ DCs with α-GalCer administration in vivo (fig. 6c). These findings strongly suggest that the manipulation of innate DC subsets may provide a new therapeutic strategy for controlling various allergic diseases by reducing the release of histamine from IgE-sensitized mast cells, thus driving the immune response towards Th1 dominance via activation of DEC-205+ DCs in vivo.

Fig. 6

Effects of DEC-205+ DC activation on histamine release from IgE-sensitized mast cells. a Histamine release from BM-mast cells cocultured with either IL-10, IL-12, or DCs was analyzed by ELISA. b Histamine release from BM-mast cells cocultured with DCs, DCs and α-GalCer, 33D1-depleted DCs, or 33D1-depleted DCs and α-GalCer was analyzed by ELISA. c The amount of IL-12p40 secretion in the sera was also determined by ELISA.

Fig. 6

Effects of DEC-205+ DC activation on histamine release from IgE-sensitized mast cells. a Histamine release from BM-mast cells cocultured with either IL-10, IL-12, or DCs was analyzed by ELISA. b Histamine release from BM-mast cells cocultured with DCs, DCs and α-GalCer, 33D1-depleted DCs, or 33D1-depleted DCs and α-GalCer was analyzed by ELISA. c The amount of IL-12p40 secretion in the sera was also determined by ELISA.

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In the present study, based on a recent report [14], we showed that we could successfully establish a BALB/c-based AR mouse model exhibiting antigen-specific IgE-mediated allergic symptoms. Using the AR mice, we demonstrated that various allergic symptoms could be markedly suppressed either by the depletion of a DC subset expressing 33D1 molecules [8] or by the activation of another distinct DC subset expressing DEC-205 molecules [13]. These findings suggest that we can control various antigen-specific allergic diseases such as allergic rhinitis, sneezing, and bronchial asthma by decreasing NALF eosinophils [16] through the manipulation of DC subsets, even in an allergen-challenged state with relatively high levels of allergen-specific IgE. This phenomenon may be caused by a shift in the internal Th1/Th2 balance towards a Th1 dominant state, given that such a Th1/Th2 balance appears to be regulated primarily by two distinct DC subsets, DEC-205+ DCs and 33D1+ DCs, and allergic reactions appear to be initiated and maintained via Th2 polarization mediated primarily by 33D1+ DCs.

It is noteworthy that the IgE-dependent allergic status appears to worsen during pregnancy [10,11], during which the immune balance is shifted towards Th2 dominance, while the allergic status improves spontaneously after delivery. Indeed, we have recently reported that the number of 33D1+ DCs increases to maintain Th2 dominance during pregnancy [9], while such dominance appears to be completely abrogated after delivery. Thus, the internal immune balance is shifted towards a Th1 response to improve the allergic status. Moreover, it has been reported that DEC-205 targeting should be explored as a vaccination approach against symptomatic primary EBV infection and against EBV-associated malignancies [17], suggesting that the depletion of 33D1+ DCs or the activation of DEC-205+ DCs may prevent the progression of virus-associated disorders. In contrast, Price et al. [18] recently showed that DCIR2+ DCs (33D1+ DCs) are capable of inducing antigen-specific tolerance during ongoing autoimmunity, indicating that the abrogation of 33D1+ DC potency may elicit or worsen autoimmune disorders. Therefore, the internal Th1/Th2 balance appears to be regulated primarily by the number and activity of two distinct DC subsets. Mast cells have been considered to be the most critical cells in the induction of an allergic status; they express Fc-receptors to capture antigen-specific IgE. When an allergen is captured by specific IgE on sensitized mast cells, these cells promptly secrete a variety of chemical mediators such as histamine and serotonin to initiate allergic symptoms. Such IgE-dependent, allergen-activated mast cells also release leukotriene C4 and prostaglandin D2, which play important roles in the late phase of allergic reactions in asthma. In asthma, overproduction of prostaglandin D2 and leukotriene C4 results in an increase in the levels of Th2 cytokines and a decrease in Th1 cytokine expression, accompanied by the enhanced accumulation of eosinophils and lymphocytes in the lung [19,20]. However, the precise mechanisms underlying the effect of Th1 dominance induced by the depletion of 33D1+ DCs or the activation of DEC-205+ DCs on the abrogation of an IgE-dependent allergic status remain to be investigated [21].

Moreover, when Th1 dominance is established not only by depleting 33D1+ DCs but also by activating DEC-205+ DCs with α-GalCer prior to immunization with OVA antigen plus adjuvant alum, the production of antigen-specific IgE can also be inhibited. These results strongly indicate that the sensitization of antigen-specific IgE-producing B-2 cells [22] in the acquired arm appears to require the help of 33D1+ DCs in the innate arm. Therefore, the depletion or suppression of innate 33D1+ DCs before sensitization with antigen and alum adjuvant may interfere with their interactions, and thus inhibit IgE antibody production. However, the precise interactions between antigen-presenting 33D1+ DCs and antigen-specific IgE-producing B-2 cells during the production of IgE remain to be elucidated.

In addition, although 33D1+ DCs were almost completely depleted from the spleens of untreated normal BALB/c mice (fig. 2c), a small portion of the 33D1+ DC subset always remained when OVA plus the adjuvant alum was administered to AR mice (fig. 2b). Thus, we speculated that the adjuvant alum might have the ability to maintain the percentage of 33D1+ DCs in the spleens of AR mice, even with frequent injections of anti-33D1 mAb. It has been reported that the adjuvant alum enhances the magnitude and duration of the expression of peptide/MHC-II complexes on the surface of DCs with an accompanying increase in MHC-II expression [23], and that it induces a persistent Th2 response to boost antibody production via B-2 cell-dependent humoral immunity [24]. Thus, although the precise mechanism underlying that resistance remains to be elucidated, the alum appears to possess the capacity to stimulate 33D1+ DCs to maintain Th2-type immunity.

In the present study, we also described the effects of a known glycolipid, α-GalCer, on the enhancement of DEC-205+ DCs that stimulate Th1-type immunity. The glycolipid α-GalCer usually induces iNKT cells bearing a unique invariant T-cell receptor, specifically Vα14 in mice and Vα24 in humans, in a CD1d-restricted manner [25]. It has been reported that iNKT cells play important immunoregulatory functions in allergen-induced airway hyperresponsiveness and inflammation [21,26]. Indeed, activated iNKT cells in regional lymph nodes induce antiallergic effects through the production of IL-21 or IFN- γ [27]. Thus, using an α-GalCer-pulsed CD1d tetramer, we assessed the presence of iNKT cells in α-GalCer-inoculated AR mice. Through our careful examination, we were able to detect the apparent enhancement of Vα14+ NKT cells in the spleens of α-GalCer-inoculated AR mice, although we could not discern any measurable Vα14+ NKT cells in the spleens of 33D1+ DC-depleted mice.

The findings obtained in the present study also strongly indicate that the release of histamine from IgE-sensitized mast cells was enhanced in the presence of Th2-promoting DCs through IL-10 secretion. This result is consistent with recent findings demonstrating that the interaction between DCs and IgE-activated mast cells augment histamine release in a response to proinflammatory cytokines such as IL-10 [28,29], suggesting that disruption of the Th2-promoting DC-mast cell interaction may constitute an effective strategy to treat ongoing allergic diseases. Taken together, we suggest that various allergic symptoms and their related diseases may be controlled either by the depletion or suppression of a 33D1+ DC subset, or by the activation of a DEC-205+ subset rather than the use of antihistamines or anti-inflammatory drugs, even in an antigen-specific IgE-producing state. These findings will facilitate the identification of strategies to control various allergic diseases.

This study was supported in part by grants from the Ministry of Education, Science, Sport, and Culture; the Ministry of Health and Labor and Welfare, Japan (25461715 to H.T.); the Japanese Health Sciences Foundation and the Promotion and Mutual Aid Corporation for Private Schools of Japan; and the MEXT-supported Program for the Strategic Research Foundation at Private Universities, Japan.

The authors have no conflicting financial interests.

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