1,25(OH)2VitD3 supplementation enhances suppression of grass pollen-induced allergic asthma by subcutaneous and sublingual immunotherapy in a mouse model

Allergen specific immunotherapy (AIT) can provide long-term alleviation of symptoms for allergic disease but is hampered by suboptimal efficiency. We and others have previously shown that 1,25(OH)2-VitaminD3 (VitD3) can improve therapeutic efficacy of AIT. However, it is unknown whether VitD3 supplementation has similar effects in sublingual and subcutaneous immunotherapy. Therefore, we aimed to test VitD3 supplementation in both grass pollen (GP) subcutaneous-IT (SCIT) and sublingual-IT (SLIT) in a mouse model for allergic airway inflammation. To this end, GP-sensitized BALB/c mice received GP-SCIT or GP-SLIT with or without 10 ng VitD3, followed by intranasal GP challenges and measurement of airway hyperresponsiveness (AHR) and inflammation. VitD3 supplementation of GP-SCIT resulted in enhanced induction of GP-specific (sp)-IgG2a and suppression of spIgE after challenge. In addition, eosinophil numbers were reduced and levels of IL10 and Amphiregulin were increased in lung tissue. In GP-SLIT, VitD3 supplementation resulted in enhanced sp-IgG2a levels in serum, enhanced suppression of eosinophils and increased IL10 levels in lung tissue, as well as suppression of AHR to methacholine. These data show that VitD3 increases efficacy of both SCIT and SLIT, by enhancing induction of blocking antibodies and suppression of airway inflammation, underscoring the relevance of proficient VitD3 levels for successful AIT.

properties through induction of tolerogenic dendritic cells (DCs) 9 . VitD3 has been shown to prevent maturation of DCs leading to down-regulation of costimulatory molecules (CD40, CD80, CD86) and enhanced IL10 production 10 , facilitating the generation of adaptive Treg cells 11 .
Recent clinical studies indicate that VitD3 supplementation had limited positive effects on HDM-SCIT treatment, with asthma symptom score as the only improvement compared to control HDM-SCIT treatment 12 . In contrast, VitD3 supplementation of GP-SLIT was reported to suppress nasal and asthmatic symptoms to the control GP-SLIT treated group 13 . The discrepancy between these studies might be due to differences in allergen used (HDM versus GP), duration of treatment (12 versus 5 months) or the route of application of the allergen vaccine. Therefore, a head-to-head comparison of VitD3 supplementation in SCIT versus SLIT will help to evaluate VitD3 as an adjuvant for AIT delivered through either subcutaneous or sublingual application.
We have previously studied SCIT and SLIT using grass pollen (GP) and directly compared the two treatment regimens 14 . Here, we employed this experimental model of side-by-side subcutaneous and sublingual AIT to directly compare the efficacy of VitD3 as an adjuvant between SCIT and SLIT treatments. Both experimental models in BALB/c mice contain two sensitizing intraperitoneal injections comprised of an alum-absorbed GP extract followed by 3 subcutaneous injections for GP-SCIT or 40 sublingual administrations for GP-SLIT with or without 10 ng VitD3. Subsequently, mice are challenged three times with intranasal GP and thereafter, AHR to methacholine is measured as well as serological responses, ear-swelling responses (ESR), eosinophilic inflammation in broncho-alveolar lavage fluid (BALF) and lung, and cytokines after re-stimulation of lung cells. We show that VitD3 supplementation augments induction of blocking antibody responses and leads to enhanced suppression of eosinophilic inflammation and increased production of IL10 in lung tissue in both SCIT and SLIT treatment, while an effect on AHR was observed in SLIT treatment only. These studies underscore the relevance of proficient VitD3 levels for successful AIT and support the potential use of VitD3 as an adjuvant to improve efficacy of both SCIT and SLIT in clinical practice.

Materials and Methods
Animals. BALB/cByJ mice (8-9 weeks-old) were purchased from Charles River (L' Arbresle, France) and bred in individually ventilated cages and fed a hypo-allergen GP-free diet (4 kcal/gr, 25% protein, 11% fat, 47% sugars, 5% fibers; AB Diets, Woerden, The Netherlands), which has a theoretical pre-manufacture level of 2,900 IU/kg Vitamin D3. Due to the high sensitivity of vitamin D3 to light, air, heat and humidity, the actual level of Vitamin D3 might alter during storage and usage. Induction of allergic asthma and treatment protocols. All mice received two intraperitoneal injections of 5,000 standardized quality (SQ) units (5kSQ = 8 μg allergen extract of GP (Phleum pratense, Phl p; ALK-Abelló, Hørsholm, Denmark) adsorbed to 1.6 mg Alum (Imject Alum, Pierce, USA) in 100 µL Phosphate-buffered Saline (PBS, Figs. 1A,B and 4A,B, S1A,B, and S4A,B). SCIT was performed by three 100 µL injections or SLIT was performed by 40 × 5 µL sublingual administrations, containing either saline or GP with or without 1α,25-dihydroxyvitaminD 3 (VitD3, Sigma-Aldrich, The Netherlands). Inhalation challenges were administered as droplets of 25kSQ GP in 25 μL PBS after light isoflurane anesthesia. After two days, airway responsiveness was determined, and serum samples, broncho-alveolar lavage fluid (BALF), and lung lobes were stored for further analyses (−80 °C) 7,15 . The ear swelling test: early phase hypersensitivity. Before and after SIT treatments, an ear-swelling test (EST) was performed to evaluate the early phase response to GP to test for allergic sensitization. Herein 10 μL of PBS is injected intradermal in the left ear as a control and 1kSQ of GP in 10 μL is injected in the right ear of mice under isoflurane/oxygen anesthesia 14,15 . After 2 h, ear thickness was measured using a digimatic force-micrometer at 0.5 N ( ± 0.15 N, Mitutoyo, Japan) and the net GP-induced swelling (Δ, in µm) was calculated by subtracting the thickness of the left ear from the right ear.
Airway responsiveness. By measuring airway resistance (R in cmH 2 O.s/mL) in response to intravenous administration of increasing doses of methacholine (Sigma-Aldrich) the airway responsiveness was assessed. Next, lung compliance (C in mL/H 2 O) was examined as a measure of the comparative stiffness of the lung. In short, anesthetized mice were tracheotomized, cannulated through the jugular vein, and attached to a small animal ventilator; the FlexiVent (SCIREQ, Canada), and ventilated (280 breaths/minute) with a tidal volume of 10 mL/kg, pressure limited at 300 mmH 2 O 15,16 . In response to increasing dosages of methacholine, the airway resistance was calculated from the pressure response to a 2-second pseudorandom pressure wave. In analyzing the peak resistance and peak compliance, all values with a coefficient of determination (COD)-value below 0.85 were excluded. Moreover, responsiveness was expressed as the effective dose (ED) of methacholine required to induce a resistance of 3 cmH 2 O.s/mL (ED 3 ).
Evaluating inflammation in BALF. Lungs were lavaged with 1 mL PBS containing 5% Bovine Serum Albumin (BSA, Sigma Aldrich, Zwijndrecht, The Netherlands) and a cocktail of protease inhibitors (Complete mini tablet; Roche, Germany), directly after AHR measurements. Subsequently, four lavages were performed with 1 mL non-supplemented PBS. After centrifugation (500 × g, 4 min), the cell-free supernatant of the first mL was stored as BALF (in duplo, −80 °C). The cells from the first mL were added to the cells from the 4 mL PBS lavages and counted using the Z2 coulter particle count and size analyzer (Beckman Coulter, Woerden, The Netherlands).

Measurement of GP-specific Immunoglobulins in serum.
Blood was collected in MiniCollect serum tubes (Greiner Bio-One, Alphen a/d Rijn, The Netherlands) at several time points via orbital puncture (pre-sera) and after the experiment via the vena cava inferior (post-sera, Figs. 1A and 4A) 14 .
For GP-spIgG1 and GP-spIgG2a, plates were coated using 10 μg/mL rough extract GP, blocked using 3%BSA in ELISA buffer, incubated with sera samples (1:300,000 for GP-spIgG1 and 1:100 for GP-spIgG2a), and labeled using biotinylated anti-mouse IgG1 or -IgG2a (1 μg/mL, BD Pharmingen). Concentrations were calculated according to the standard curve (using reference serum) and the results are expressed as arbitrary unit (AU)/mL. Biotinylation of GP extract was performed using EZ-link Sulfo-NHS-LC-Biotin according to the manufacturers operating instructions (Thermo Scientific) and using a Slide-A-Lyzer cassette (3.5 K MWCO, Thermo scientific) for purification by dialysis overnight.
Analysis of cytokine levels in lung tissue. The right superior lung lobe was used for measurement of total protein content and a cytokine profile. First, lungs were weighed, homogenized and dissolved in Luminex buffer (weight to volume ratio 1:5) and the total protein content was measured using a BCA protein assay according to manufacturer's protocol (Thermo Scientific, USA). Concentrations of IL4, IL5, IL-10, IL13, IL-17, IL33, IFNγ, Eotaxin (CCL11), TARC (CCL17), and MIP3α (CCL20) were measured using a multiplex Mouse Cytokine/Chemokine Magnetic Bead Panels (MILLIPLEX Map Kit; Merck Millipore, Germany) according to manufacturer's protocol. Plates were analyzed using a MAGPX1023 4002 with Luminex xMAP technology. Statistical analysis. All data are expressed as mean ± SEM. The Mann-Whitney U Test was used to analyze the results, and p < 0.05 was considered significant. Within the ELISA data, an AU-value which was more than three times the interquartile (IQ) range higher than the upper Q or more than three times the IQ range lower than the lower Q was considered to be an extreme outlier and was removed for further analysis. Within the AHR measurements, to compare the entire curve between groups, a generalized estimated equation (GEE) analysis was used, using SPSS Statistics 20.0.0.2 17 .

VitD3 supplementation enhances specific IgG2a responses induced by GP-SCIT. Previously, we
developed an experimental mouse model for GP-SCIT and GP-SLIT using a single allergen extract, effectively allowing side-by-side comparison 14 . Here, we aimed to study the efficacy of 10 ng VitD3 supplementation in GP-SCIT and GP-SLIT for suppression of asthmatic manifestations upon GP challenges (Figs. 1A,B and S1A,B). We first evaluated the effect on VitD3 on GP-SCIT treatment. To assess GP-SCIT induced immunoglobulin responses, we measured total IgE, GP-spIgE, GP-spIgG1, and GP-spIgG2a in sera taken before SCIT (white, Pre1), before allergen challenges (grey, Pre2), and after challenges (black, Post, Figs. 1C-F and S1C-F). As previously observed, GP-SCIT injections induced increases in total and GP-specific IgE, as well as increased levels of sp-IgG1 and sp-IgG2a. Upon subsequent GP challenges, GP-SCIT groups displayed a blunted IgE response compared to untreated controls (Figs. 1C,D and S1C,D). Supplementation of GP-SCIT with VitD3 did not alter IgE or IgG1 responses, but induced a strongly increased sp-IgG2a response (Figs. 1C-F and S1C-F).
Clinical efficacy of SIT is associated with the blocking capacity of the spIgGs, while symptom score in allergic asthma is inversely correlated to the ratio of spIgG over spIgE, indicating the relevance of IgG response during SIT 16 . We used the ratios of GP-spIgG1/GP-spIgE and GP-spIgG2a/GP-spIgE as a measure of blocking capacity after GP-SCIT. These ratios were unaffected by VitD3 supplementation (Figs. 1G,H and S1G,H). In addition, we were unable to detect a significant reduction in the fold increase of GP-spIgE levels induced by challenges (GP-spIgE Post/ Pre2) in the SCIT groups, whereas in the VitD3 supplemented GP-SCIT group (100D), we did observe a significant reduction in GP-spIgE as compared to the VitD3 supplemented positive controls (Fig. 1I). Finally, we measured GP-spIgA in BALF and sera taken after challenges (Post) and found that GP-SCIT injections (2020) 10:8960 | https://doi.org/10.1038/s41598-020-65946-6 www.nature.com/scientificreports www.nature.com/scientificreports/ induced increases of GP-spIgA levels in both sera and bronchoalveolar lavages, although VitD was unable to alter those responses (Fig. S1J,K).

VitD3 supplementation does not enhance suppression of ear swelling or AHR to methacholine.
To evaluate the effects of VitD3 supplementation of GP-SCIT on clinically relevant parameters of our experimental model of airway inflammation, we performed an ear swelling test (EST) by intradermal GP injection before and after SCIT treatment in GP-sensitized mice. GP-SCIT did not induce significant suppression of ear swelling after GP challenge, irrespective of VitD3 supplementation ( Figs. 2A and S2).
Next, we measured airway hyperresponsiveness (AHR) in response to increasing dosages of methacholine and calculated the dose of methacholine required to induce a resistance of 3 cmH2O.s/mL (ED3; Figs. 2B and S2B). We did not observe a significant increase of the ED3 after GP-SCIT treatment with or without VitD3 supplementation. Next, we compared airway resistance across the entire methacholine dose-response curve and found that GP-SCIT treatment significantly reduced airway resistance compared to the Sham-treated control groups (Figs. 2C and S2C). No effect of VitD3 was observed on this suppression of AHR by GP-SCIT. However, the VitD3 supplemented GP-SCIT resulted in increased compliance in these invasive lung function measurements as compared to the VitD3 supplemented Sham-treated control group (PCD, Figs. 2D and S2D).
To appraise effects of AIT on Th2 driven inflammation, we assessed cytokine levels in lung cell suspensions restimulated ex vivo with GP extract (Figs. 2E and S2E). Here, we observed that GP-SCIT-treated mice had significantly reduced IL13 production after ex vivo GP stimulation of lung cells, which was a trend only in the GP-SCIT treated group, but reached significance in the VitD3 supplemented GP-SCIT group.
Suppression of eosinophilic responses after VitD3 supplemented GP-SCIT. To assess suppression of airway inflammation by GP-SCIT, we compared eosinophil numbers in BAL and lung, and cytokine levels in lung tissue homogenates (Figs. 3A-E, and S3). We observed a reduction of lung tissue eosinophil numbers after GP-SCIT treatment (Figs. 3A-C and S3A-C), with the lowest numbers in the VitD3 supplemented group. To compare the effect of VitD3 supplementation on GP-SCIT, we calculated fold reduction in eosinophils of GP-SCIT treated groups with and without VitD3 supplementation relative to their respective Sham-treated groups. Here, we observed an enhancement of the suppression in eosinophil numbers in lung tissue after GP-SCIT by VitD3 supplementation (Figs. 3D and S3D).
Next, we analyzed cytokine levels in lung homogenates after challenges and observed that levels of the type-2 cytokines IL4, IL5 and IL13 were not affected by GP-SCIT treatment (Figs. 3E and S3E). Although no induction of IL10 or TGF-β was observed in GP-SCIT groups, VitD3 supplemented GP-SCIT mice displayed a significantly increased level of IL10 compared to the control GP-SCIT group. Furthermore, only the VitD3 supplemented GP-SCIT group displayed increased levels of amphiregulin in lung tissue after GP challenges when compared to the supplemented positive controls (Fig. 3E). VitD3 supplementation had no effect on the ratios of GP-spIgG1/GP-spIgE and GP-spIgG2a/GP-spIgE after GP-SLIT, used as a measure of blocking capacity (Figs. 4G,H and S4G,H). Furthermore, we observed a striking decrease in fold induction of GP-spIgE by allergen challenges, reflecting the blunted IgE response in the SLIT treated groups, but no effect of VitD3 supplementation was observed (Figs. 4I and S4I). Finally, we measured GP-spIgA in BALF and sera taken after challenges (Post) and found that GP-SCIT injections induced increases of GP-spIgA levels in both sera and BALF (S4J,K). Moreover, addition of VitD in GP-SCIT resulted in a significant increase of GP-spIgA levels after challenges when compared to the unsupplemented GP-SCIT group (Fig. S4J,K).

VitD3 supplementation enhances specific IgG responses induced by
These data indicate that GP-SLIT treatment induced enhanced blocking, GP-specific immunoglobulin responses while providing a significant decrease of GP-spIgE after challenges. Effects of VitD3 supplementation were detected in the levels of GP-spIgG1 only.
VitD3 supplementation of GP-SLIT reduces ear swelling and airway hyperresponsiveness. Next, we assessed the effect of GP-SLIT on the early-phase response to intradermal GP injections in the ear. Ear swelling was reduced in GP-SLIT treated groups as compared to the sham treated controls. We observed a trend towards increased suppression of ear swelling in the VitD3 supplemented GP-SLIT group as compared to its unsupplemented control (Figs. 5A and S5A).
To measure the effect of GP-SLIT treatment on AHR to methacholine, we measured airway resistance (R) and compliance (C) and calculated the ED 3 values (R of 3 cmH 2 O.s/mL) in all experimental groups (Figs. 5B-D and S5B-D). The ED3 values were significantly increased only in VitD3 supplemented GP-SCIT treated mice, indicating a reduced sensitivity to methacholine (Figs. 5B and S5B). Indeed, the VitD3 supplemented GP-SLIT group displayed a significantly reduced AHR also when directly compared to GP-SLIT treatment alone (Figs. 5C and S5C). Both VitD3 GP-SLIT as well as GP-SLIT alone showed a significant improvement of lung compliance when compared to their sham-treated controls, however no differences between the groups were detected (Figs. 5D and       www.nature.com/scientificreports www.nature.com/scientificreports/ Effects of VitD3 supplemented GP-SLIT on eosinophilic inflammation and cytokine responses. To assess the effect of VitD3 supplementation on airway inflammation, we compared eosinophilic airway inflammation and levels of cytokines in lung tissue after GP-SLIT treatment (Figs. 6A-E and S6A-E). We observed a marked suppression of eosinophil numbers in BAL fluid and lung tissue after GP-SLIT (Figs. 6B,C and S6B,C). Moreover, VitD3 supplementation of GP-SLIT resulted in a significantly reduced number of eosinophils in lung tissue compared to the GP-SLIT group lacking the VitD3 supplementation (Figs. 6B,C and S6B,C). This VitD3 mediated effect on GP-SLIT was also evident when the data were presented as fold reduction in eosinophils of both GP-SLIT treated groups relative to their Sham-treated groups (Figs. 6D and S6D,E).
Finally, we also analyzed cytokine levels in lung homogenates after GP challenges and observed that levels of the type-2 cytokines IL4, IL5 and IL13 were significantly affected by GP-SLIT treatment, but no VitD3 mediated effects were observed (Fig. 6E). Similar findings were observed when other cytokines and chemokines were analyzed after VitD supplemented GP-SLIT (Fig. S6F). However, we were able to show a significant increase of IL10 in the VitD3 supplemented GP-SLIT group compared to the unsupplemented GP-SLIT group. Moreover, levels of TGF-β1 and amphiregulin in lung tissue showed a trend towards an increase in GP-SLIT treatment in the presence of VitD3 (Fig. 6E).

Discussion
In this study, we investigated whether supplementation of GP-specific immunotherapy with 10 ng VitD3 per administration could enhance the efficacy of both sublingual and subcutaneous administration of the GP allergen extract in suppressing asthmatic manifestations upon GP challenges in an experimental mouse model. We find remarkable similarity in the effects of VitD3 supplementation between GP-SCIT and GP-SLIT treatments: an enhanced GP-specific IgG2a antibody response, suppression of lung tissue eosinophils and increased IL10 levels in lung tissue after GP challenges. In GP-SLIT, we additionally observed an effect of VitD3 supplementation on GP-spIgG1 levels and GP-spIgA levels, as well as on suppression of ear swelling responses and methacholine-induced airway resistance.
Vitamin D insufficiency is widespread, and is thought to contribute to asthma 18 . In some cases, supplementation of VitD3 in clinical studies has resulted in a clear benefit. For instance, VitD3 supplementation during pregnancy reduces the risk of recurrent wheeze and acute respiratory tract infections in early life 18,19 . Moreover, VitD3 supplementation in asthma patients has been shown to reduce the rate of asthma exacerbations requiring treatment with systemic corticosteroids 20 . The mechanism of action is thought to include both steering of the immune system towards a more tolerogenic response, as well as reinforcing the barrier and antiviral properties of the bronchial epithelium 18 . Based on these tolerogenic properties of VitD3, we previously used an experimental SCIT mouse model to show that injection of VitD3 enhanced the therapeutic effects of SCIT in this OVA-driven mouse model for allergic airway inflammation 8 . However, conflicting data have since been obtained in clinical studies using allergen-based SCIT and SLIT treatment protocols 12,13 . These recent studies indicate that VitD3 supplementation had limited positive effects on HDM-SCIT treatment, with asthma symptom score as the only improvement compared to control HDM-SCIT treatment 12 . In contrast, VitD3 supplementation of GP-SLIT was reported to suppress nasal and asthmatic symptoms to the control GP-SLIT treated group 13 . The discrepancy between these studies might be due to differences in allergen used (HDM versus GP), duration of treatment (12 versus 5 months) or the route of application of the allergen vaccine. To resolve whether VitD3 supplementation has the potential to enhance efficacy of both SCIT and SLIT, we here aimed to perform a side-by-side comparison of VitD3 supplementation in SCIT versus SLIT using the same allergen extract in a mouse model of GP-driven allergic airway inflammation.
To our knowledge, this is the first study comparing the adjuvant effects of VitD3 supplementation in GP-SCIT and GP-SLIT treatments in an experimental model for allergic airway disease. Strikingly, and in contrast to previous results using unsupplemented AIT 14 , we here report a prominent Treg cytokine profile in lung tissue after VitD3 supplemented GP-SCIT and GP-SLIT, as demonstrated by the increased levels of IL10 and in SLIT also of TGF-β1. In contrast, clear suppression of Th2 cytokine responses by VitD3 supplementation was not observed. The selective reduction of eosinophils by VitD3 supplementation in GP-SCIT treated mice in absence of a clear suppression of Th2 cell cytokines (Fig. 3D,E), might indicate increased Treg activity, as we have previously shown that Treg depletion prior to allergen challenges mainly affects eosinophilic airway inflammation in SIT-treated mice 21 . However, other sources of IL10 might include regulatory B cells, dendritic cells or innate lymphoid cells [22][23][24][25] . Although IL10 and TGF-β1 are not necessarily exclusively produced by regulatory T cells, several studies confirmed the need of IL10 for a successful induction of allergen tolerance 24 . These results are in line with the previously reported biological effects of VitD3 on DCs, which was shown to result in enhanced generation of adaptive Treg cells and IL10 and TGF-β1 production 26,27 . These data support further clinical studies on VitD3 supplementation in allergen-specific immunotherapy treatment.
In literature, supplementation of standard VitD3 levels from 2,000 IU/kg in standard chow to 10,000 IU/kg in supplemented chow or in drinking water resulted in decreased AHR and airway inflammation in mouse model of asthma 28,29 . These studies indicate that systemic levels of VitD do affect airway inflammation and hyperresponsiveness in experimental mouse models. In our study, all mice were fed a standard hypo-allergen diet containing 2,900 IU/kg throughout the experiment, and VitD was applied together with the GP extract, securing high local concentrations at the site of injection, whilst not making a strong contribution to systemic VitD levels (all below 25 ng/mL in serum; data not shown). Therefore, it seems likely that an effect on the phenotype of the local antigen-presenting cell is sufficient to mediate the enhanced effects of VitD3 supplementation on SLIT and SCIT in our experimental mouse models.
In addition, we observe enhanced induction of spIgG1 (SCIT) and spIgG2a (SCIT and SLIT) after VitD3 supplementation. However, we have previously reported that blocking antibodies are not required for suppression of Th2 cell activity and eosinophilic airway inflammation in an experimental OVA-SCIT mouse model 30   300kSQ SLIT with 10 ng VitD3. *P < 0.05, **P < 0.01, ***P < 0.001 compared to PC or PCD respectively (300 vs PC and 300D vs PCD), unless otherwise specified.