Micronutritional supplementation with a holoBLG‐based FSMP (food for special medical purposes)‐lozenge alleviates allergic symptoms in BALB/c mice: Imitating the protective farm effect

Previously, the protective farm effect was imitated using the whey protein beta‐lactoglobulin (BLG) that is spiked with iron‐flavonoid complexes. Here, we formulated for clinical translation a lozenge as food for special medical purposes (FSMP) using catechin‐iron complexes as ligands for BLG. The lozenge was tested in vitro and in a therapeutical BALB/c mice model.

milk, in particular, the whey protein content has been linked to protection against allergies. 13 Milk processing, especially heat treatment, coincides with loss of asthma-and allergy-protective effect of raw cow's milk, implicating the role of heat-sensitive milk components, such as whey proteins, in the tolerogenic feature and protective effects of raw cow's milk. [14][15][16] Notably, beta-lactoglobulin (BLG) is a major whey compound, with a great similarity to the human lipocalin-2 (LCN-2) with innate immunoregulatory potency. In allergics, LCN-2 levels are reduced and can be corrected by allergen immunotherapy. 17 Hence, interference with the regulatory lipocalin system with an exogenous lipocalin could be beneficial in allergy. We also detected BLG as a major compound in cow stable dust and aerosolized around cattle farms. 18 In previous studies, we demonstrated that BLG prevented allergic sensitization, when fulfilling its innate function shuttling ligands to immune cells when BLG carried micronutrients within its protein pocket such as iron-flavonoid complexes, 19 or retinoic acid [20][21][22] -it acted as a tolerogen, preventing antigen presentation and activating anti-inflammatory pathways. 23 Importantly, transport of these micronutrients resulted in immune resilience in an antigen-unspecific manner and prevented the onset of allergy also to other allergens, thus mimicking the farm effect. 24 Here, we went one step further seeking clinical translation for the new principle in the protective farm effect. Considering that farm dust and raw milk exposure need thorough time for mucosal absorption, we developed a dietary supplement as a lozenge from compounds compatible with EU regulations for food safety. 25 Besides zinc 18 and retinoic acid, the flavonoids catechin and epicatechin from cocoa 26,27 were chosen for complexing iron and combined with whey protein BLG derived from organic farms in EU and Switzerland.
Here, we demonstrate that the combination of the dietary components in the lozenge activated the anti-inflammatory aryl hydrogen receptor pathway (AhR) similar to the single purified components. Indeed, IgE-binding of children allergic to milk was reduced to the ligand-filled, holo-form of BLG and also antigen-specific degranulation of human mast cells was significantly diminished. The impact of the lozenge was further tested in double-sensitized mice resulting in decreased antigen presentation, promotion of regulatory T cells and reduced proliferation that was accompanied by reduced allergic symptoms to both allergens in a challenge. The results identify the new lozenge as a successful translation of the protective farm effect transporting micronutritional ligands to immune cells that initiate tolerogenic pathways in mice. The beneficial innate and allergen-non-specific effect of the lozenge has been clinically confirmed in house dust mite allergic rhinoconjunctivitis patients. 28 2 | MATERIAL S AND ME THODS

| Animals
Female BALB/c mice, 5-7 weeks of age, were obtained from Charles River Laboratories and kept under conventional housing and treated according to European Union rules of animal care, with the permission of the Austrian Ministry of Sciences (BMWF-66.009/0133-WF/V/3b/2016).

| Supplement/Placebo and raw materials
The holoBLG lozenge (ImmunoBON ® ) was produced and provided by Biomedical International R+D GmbH, Austria containing active ingredients (whey with BLG, iron, catechines, vitamin A and zinc); the placebo lozenge formulated without active ingredients, consisted of sorbitol, mountain herbs flavour and karo-coffee providing a similar appearance and taste like the verum lozenge.
All compounds used were pure, organic and 100% food grade. The source of flavonoids in the holoBLG lozenge is cocoa extract. The whey concentrate was obtained from an Austrian diary company, which complies to the stringent criteria applied for food product according to EU regulations. The delivered product conformed to the current EU food regulations as well as the Austrian food law. The inspection body AT-BIO-301 made sure that the EU regulation 834/2007 was fulfilled. In addition, each whey charge was controlled for the protein composition and comparability with BLG as the major component, before using it in the lozenge production. The lozenges are produced under GMP conditions in Gebro Pharma, Fieberbrunn, Tyrol, Austria, and each constituent undergoes regular and stringent quality checks.

| Allergic therapeutic model
Sample size for the mouse experiments were based on the literature and described in the approved protocol of the animal ethic (BMWF-66.009/0133-WF/V/3b/2016). No randomization was performed. Female BALB/c mice, 5-7 weeks of age were split in 3 groups: (n = 8 for lozenge, n = 8 for placebo, n = 6 for naïve).
Animals were immunized three times with 5 µg BLG and 5 µg Bet v 1 in conjunction with Al(OH)3 in biweekly intervals, thereafter mice received oral gavages of aliquots of crushed lozenges containing active ingredients (5 mg holoBLG using iron-catechin 3 complexes as ligand; molar ratio BLG: iron: catechin = 1:1:3, 10 µg retinol and 176 µg zinc) or not (placebo-group). Mice were orally gavaged on 3 consecutive days per week over the course of 6 weeks to ensure ingestion of the crushed aliquots. Subsequently, all mice were challenged intraperitoneally with 50 µg BLG, followed 1 week later by 50 µg Bet v 1.
Body temperature and movements were monitored for 20 minutes after i.p. challenge using an Imaging system. 30 The allergic symptoms of challenged animals were scored as previously described 31 ; 0 points for no symptoms; 1 for scratching and rubbing around the nose and head, 2 for puffiness around the eyes and mouth, diarrhoea, pilar erection, reduced activity and/or decreased activity with increased respiratory rate; 3 for wheezing, laboured respiration, cyanosis around the mouth and the tail and 4 for no activity after prodding or tremor and convulsion. After euthanasia with CO 2 , blood was collected by cardiac puncture, and sera were stored at −80°C until further processing. Spleens were collected. Results of two separate, independent experiments were compared.
Tetramethylbenzidine (eBioscience) was used as substrate, and 1.8 M sulfuric acid was used as stop solution followed by optical density measurement at 450 nm.

| Spectral analysis
For spectral analysis, deionized water was used as buffer to minimize iron contamination from the air. The pH was kept constant at pH 7 by addition of NaOH. Optical density was measured at a constant concentration of 100 µM quercetin or 300 µM catechin with incremental increase of iron. All measurements were repeated at least three times with similar results.

| In vitro stimulation of splenocytes
Isolated splenocytes of individual mice were plated at a density of 5 × 10 6 cells/ml and cultured with 25 μg/ml BLG or Bet v 1 (Sigma) or medium alone for 72 h at 37°C/5% CO 2 .
Secreted mouse cytokines were measured with the corresponding commercial ELISAs (Invitrogen/eBioscience, for mouse IL-5, IL-10, IL-13 and IFNγ) according to the manufacturer's instructions.

| AZ-AhR reporter assay
AZ-AhR cells were incubated at 37°C, 5% CO 2 on 96-well plates at a density of 2 × 10 4 cells/well for 18 h. Subsequently cells were stimulated for 18 h in triplets with 90 µM catechin/epicatechin alone or in complex with iron and increasing concentrations of BLG (10 µM). Compounds were initially incubated together for 15 minutes and the pH was adjusted to pH 7 prior addition of BLG.
The positive control cells were treated with 20 nM indirubin. Cells were washed once with 0.89% NaCl, and lysis buffer was added.
After a single freeze-thaw cycle, 20 μl/well of lysates were transferred into a black 96-well flat-bottom plate (Thermo Scientific) and bioluminescent reaction were started with addition of 100 μl/ well of luciferase assay reagent (Promega). Chemiluminescence was measured (10 sec/well) using a spectrophotometer (Tecan InfiniteM200 PRO).

| In vitro stimulation of peripheral blood mononuclear cells
Peripheral blood mononuclear cells (PBMCs) from sixteen birch and/ or grass pollen allergic subjects were isolated via Ficoll-Paque (GE Healthcare) and washed with 0.9% NaCl, before cells were incubated with apoBLG (5 μM) or holoBLG (5 μM BLG plus 15 μM catechine and 5 μM iron) in media neither containing phenol red nor fetal calf serum for 18 hours. Only a single time-point was measured due to the technical limitations that were encountered when working with iron, as media and buffers had to be iron-free, although iron is essential for cell survival.

| Flow cytometric analyses of human cells
To determine the intracellular iron content and the expression of co-

| Human mast cell generation
Human peripheral blood mononuclear cell-derived mast cells were

| Statistical analyses
Mouse groups and murine cellular studies were compared by ordinary one-way ANOVA following Tukey's multiple comparisons test with a single pooled variance, for the symptom score non-parametric Kruskal-Wallis test with uncorrected Dunn's test was employed.
Concentration-dependent activation of AhR was compared with RM 2-way ANOVA, for the cellular studies with AZ-AHR cells and mast cells one-way ANOVA following Tukey's multiple comparisons test was employed.
Flow cytometric analysis of PBMCs were compared by repeated measures one-way ANOVA following the Tukey multiple comparisons test. For the binding of patient sera to different BLG forms Wilcoxon matched-pairs signed test was applied. All tests were twosided and considered significant when p < .05.

| BLG binds to iron-catechin complexes
Catechin with a complex stability constant log ß of 47.4 at physiological pH 34,35 binds strongly to ferric iron. Indeed, binding to ferric iron leads to colour formation and can be monitored by UV-VIS spectroscopy ( Figure 1B). Importantly, BLG binds strongly to these iron(catechin) 3 and iron(epicatechin) 3 complexes with calculated affinity constants reaching the lower nM-range with 16.7 and 46.1nM respectively ( Figure 1A, C). This leads to a discolouration, which is visible macroscopically and spectroscopically. The data confirm that the flavonoid catechin at physiological aerobic conditions is usually present in complex with iron and that BLG is able to bind to these compounds.

| Ligand binding masks B and T cell epitopes of BLG affecting IgE binding and antigen-specific mast cell degranulation
As previously published with quercetin as ligand, 23 also catechin ( Figure 2) and epicatechin ( Figure S1) clearly affected the experimentally deduced epitope regions of BLG. When we compared the ligandbinding site with the two described dominant IgE epitope regions at residues K75-D85 located in the loop and E127-P144 at the site of the alpha helix 26 and the "weaker" IgE epitopes described at segments L31-P48, K47-K60, and L57-I78, it became apparent that also the iron(catechin)3 complex was in near proximity to the major B cell epitope 1 (Figure 2A), but not of epitope 2, which is located at the opposite site of epitope 1 on the alpha helix. Importantly, we determined the clinical relevance of epitope masking, by comparing IgE binding to BLG with or without catechin-iron ligands, in milk-sensitized Ligand binding also affected the immune-dominant T cell epitope at residues 97-117 of BLG as also described for retinoic acid 20 and for quercetin 23 with the most important core residues spanning from Y102-E112 (YLLFCMENSAE). 36,37 As depicted in Figure 2A

| BLG facilitates AhR activation by (epi) catechin-iron complexes
As our dietary supplement lozenge contained cocoa extract as a source for BLG ligands, which is rich of the flavonoids epicatechin and catechin, we assessed whether anti-inflammatory pathways such as the cytoplasmic arylhydrocarbon receptor (AhR) were activated by these flavonoids. AhR is described to interact with many exogenous ligands 40 and to mediate primarily anti-inflammatory feedback mechanisms 41 that promote regulatory T cells, 39,42,43 while preventing Th2-skewing 44,45 and impairing antigen presentation. 46 Using reporter cells, we could demonstrate that catechin and epicatechin could concentration-dependently activate AhR and interestingly activation was significantly enhanced when they were present as a complex with iron ( Figure 3A) implying active transport of the complex. Similarly, to our previous report, addition of BLG significantly enhanced (epi)catechin-dependent activation of the AhR pathway suggesting synergistic pathways which directed targeting to the putative BLG receptors ( Figure 3B). We also tested whether the single constituents of the lozenge and combinations thereof were able to activate the AhR pathway ( Figure 3C)

| Dietary supplementation with verum lozenge containing holoBLG reduced clinical reactivity in vivo
We analysed whether holoBLG with catechin-iron complexes as ligands in a dietary supplement could be used therapeutically. A lozenge containing BLG, iron-catechins as well as zinc and vitamin A were formulated to be suitable also for human use.
Mice were simultaneously sensitized with alum against BLG and Bet v 1, before treating them orally with crushed aliquots of verum lozenges containing 5mg BLG with iron-catechin complexes, 10 µg retinol and 175 µg zinc, or placebo lozenges devoid of active ingredients. Allergic reactivity was analysed by systemic challenges first with BLG and in a second step also with Bet v 1 ( Figure 4A).
Treatment in the verum lozenge group resulted in a significant decrease of BLG-specific IgG1 only, and Bet v 1-specific IgG1 and IgE antibodies ( Figure 4B,C). Though BLG-specific IgE did not differ after verum supplementation, allergic mice treated with holoBLG, but not with the placebo substances, were protected against anaphylaxis to both antigens BLG and Bet v 1 ( Figure 4D).

| Lozenge treatment decreased immune reactivity by promoting tolerance: less lymphocytic and B cell proliferation, more Tregs and decreased antigen presentation in vivo
In line with a suppressed immune response, treatment with the verum lozenge rendered comparable splenocyte numbers as the naïve group ( Figure 5A). Also, the expression of the proliferation marker CD71 on lymphocytes and B cells did not significantly differ from the naïve group ( Figure 5B,C), in contrast to the group treated with the placebo lozenge. However, splenocytes of double-sensitized mice still had the capacity to respond similarly to in vitro stimulation with BLG and Bet v 1, irrespective whether the groups were treated with the verum or placebo lozenges ( Figure S2). Regulatory T cells were significantly elevated after verum lozenge treatment compared to the placebo lozenge-treated group ( Figure 5D) presumably in an antigen-unspecific manner. However, the data on the verum lozenge treatment were accompanied by impaired antigen presentation, in line with a decreased expression of co-stimulatory markers ( Figure 5E). Hence, supplementation with the verum lozenge established immune resilience in an ongoing Th2-response.

| Iron transport by holoBLG into CD14+ monocytic cells is accompanied by decreased antigen presentation
In a next step, we assess whether similar results could be obtained Moreover, in line with previous reports, 19 the relative number of

CD3+CD4+ T cells was significantly up-regulated in cells stimulated
with apoBLG ( Figure S3a). Relative CD3+CD4+ numbers did not differ upon holoBLG treatment from medium control, whereas the ironcatechine complex alone was sufficient to reduce CD3+CD4+numbers which may indicate that particularly T helper cells were sensitive to the catechine-iron complex. Also, overnight incubation with holoBLG significantly promoted regulatory T cells, (Figure S3b) though also here the data suggest a role of the catechine-iron complex in this process.
Cytokine analysis ( Figure S4) revealed that, in particular the release of pro-inflammatory cytokine TNFα was significantly reduced upon overnight stimulation with holoBLG but not with apoBLG. Additionally, released IL-4 and IL-6 levels tended to be lower when cells were incubated with holoBLG, whereas IFNγ, IL-10 and IL-5 levels did not differ in cells treated either with apo-or holoBLG.
Summing up, the increased iron levels in antigen presenting cells went along with decreased expression of co-stimulatory markers and an increase of T cells with a supposed regulatory phenotype upon treatment with holoBLG. In this study, iron-catechine complexes were used as ligands for BLG to provide antigen presenting cells with micronutrients and to promote an immune resilience state.

F I G U R E 4
Oral supplementation with a lozenge containing holoBLG reduces clinical reactivity in mice. (A) As depicted in the treatment scheme, mice were sensitized 3x with BLG and Bet v 1 in combination with Alum, before receiving in 6 cycles aliquots of crushed lozenges containing active ingredients (5 mg holoBLG in verum lozenge) or not (placebo lozenge). Subsequently, mice were challenged with BLG and Bet v 1 one week apart; (B) BLG-and Bet v 1-specific antibodies before and (C) after oral treatment; (D) clinical response after BLG and Bet v 1 challenge of double-sensitized mice after treatment with immune or placebo lozenges. Data from one experiment are shown (n = 8 for lozenge groups, n = 6 for naïve group). Groups were compared by one-way ANOVA followed by Tukey's multiple comparison, for symptom score analyses ANOVA followed by Kruskal-Wallis test was used. *p < .05, **p < .01, ***p < .001

| DISCUSS ION
We have previously shown that sensitization to BLG can be prevented when BLG is filled with ligands, as they initiate anti-inflammatory pathways leading to tolerance induction and immune resilience. 23 Here, we went a step further and addressed whether the concept of holoBLG, which is a novel player within the protective farm effect, may be applied and exploited for allergic subjects by using BLG as a carrier for micronutrients, nourishing antigen presenting cells in a non-antigen-specific manner. We formulated a lozenge containing food and dietary supplements approved for human use: whey concentrate as a source of BLG, cocoa as a source rich of epicatechin and catechin, furthermore ferric iron, retinoic acid and zinc.
We confirm here that catechins are strong iron chelators, activate the AhR pathway and most importantly that BLG can bind to these complexes and facilitate activation of AhR pathway, irrespective of whether it is co-applied with whey concentrate or applied in pure form. Similarly, as with other ligands, we show for the holo-form  Oral supplementation of allergic mice with the lozenge containing holoBLG led to reduced allergic reactivity in an antigen-unspecific manner as clinical symptoms improved not only against BLG but also towards Bet v 1. This effect was accompanied by reduced proliferation, antigen presentation and promotion of regulatory T cells.
Importantly, this is in agreement with our previous results, when the single component BLG complexed with iron-quercetin applied via the nasal route prevented allergy development in an antigen-unspecific manner in vivo. 24 The data are also in line with our in vitro generated data with human immune cells 23 showing that holoBLG shuttled catechin-iron complexes specifically into monocytic cells and thereby hindered their activation and maturation. It provides also a rational for the antigen-unspecific effect against allergies as the monocytic cells provided with micronutrients are the antigen presenting cells that link the innate with the adaptive immune branch.
Particularly, the provision of iron seems pivotal in modulating an immune response. The iron status deeply affects immunity, with iron deficiency known to cause immune hyperactivity 49-50 prone initially towards Th2 51-53 and being associated with atopic dermatitis, 54 allergic rhinitis 55,56 and asthma. [57][58][59][60][61] In contrast, in iron sated conditions, immune cells seem to shift towards a more resilient state with macrophages polarizing towards an anti-inflammatory M2 subtype in vivo and in vitro, 62 antigen presentation being decreased, 23,63 CD4+ counts 64 and to a lesser extent CD8+ cells being negatively affected. 65 The lozenge was also spiked with vitamin A and zinc, essential contributors to immunity: Vitamin A supports mucosal regulation and predominantly anti-inflammatory pathways, 22 whereas zinc deficiencies potentially cause lymphopenia, as zinc functions as a second messenger for innate immunity. Zinc efflux is required for dendritic cell maturation and zinc influx for macrophage activation. 66 Importantly, we have described vitamin A and zinc have been described as natural ligands for BLG before. 18,20 Similarly, numerous studies have shown that initiation of the aryl hydrocarbon receptor-highly expressed in immune cells capable of antigen presentation such as monocytes, 67  The AhR pathway may also be an underappreciated contributor to inhibit class switch and may participate in the protective farm effect, where specifically the class switch along the IgG1/IgG4/ IgE-Th2 axis seems to be suppressed. 75 Based on the preclinical data, several clinical studies are underway with a recent published clinical trial showing that supplementation with holoBLG lozenges for 3 months resulted in a significant (antigen-non-specific) reduction of the symptom burden in house dust mite allergic patients, whose symptoms were tested before and after supplementation in an allergen exposure chamber. 28

| CON CLUS ION
Based on our previous findings, we generated a lozenge as FSMP (food for special medical purpose) of allergies, exploiting the molecular mechanism of targeted micronutrition via holoBLG. The

ACK N OWLED G M ENT
We thank Manuela Czernohaus for help in the experimental setup and technical assistance.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.