Secretory protein beta‐lactoglobulin in cattle stable dust may contribute to the allergy‐protective farm effect

Abstract Background Growing up on a cattle farm and consuming raw cow's milk protects against asthma and allergies. We expect a cattle‐specific protein as active component in this farm effect. Methods Dust was collected from cattle and poultry stables and from mattresses of households. Urine was obtained from cattle, and ambient aerosols were sampled. Samples were analysed for BLG by SDS PAGE/immunoblot and mass spectrometry, and for association with metals by SEC‐ICP‐MS. PBMC of healthy donors were incubated with BLG +/− zinc, and proliferation and cytokines determined. BALB/c mice were pre‐treated intranasally with stable dust extract containing BLG or depleted of BLG, and subsequent allergy response after sensitization was evaluated on antibody and symptom level. Results A major protein in dust from cattle farms and ambient air was identified as BLG. Urine from female and male cattle is a major source of BLG. In dust samples, BLG was associated with zinc. In vitro, zinc‐BLG provoked significantly lower proliferation of CD4+ and CD8+ cells while inducing significantly higher levels of IFN‐γ and IL‐6 than the apo‐BLG devoid of zinc. In vivo, pre‐treatment of mice with dust extract containing BLG resulted in lower allergy symptom scores to BLG and unrelated Bet v 1 than pre‐treatment with extract depleted of BLG. These in vitro and in vivo effects were independent of endotoxin. Conclusion The lipocalin BLG is found in large amounts in cattle urine, accumulates in bovine dust samples and is aerosolized around farms. Its association with zinc favorably shapes the human cellular immune response towards Th1‐cytokines in vitro. BLG together with zinc in stable dust protects mice from allergic sensitization. BLG with its associated ligands may in an innate manner contribute to the allergy‐protective farm effect.


| BACKGROUND
Living on a farm especially with traditional farming conditions results in a higher encounter of factors stimulating the innate immune system, for example endotoxin 1 or N-glycolylneuraminic acid (Neu5Gc). 2 These factors can also be found in indoor dust samples of farmassociated homes. The protective effect of these indoor dust samples was seen in lower prevalence numbers of children affected by asthma and allergies, 3 and was further proven in a murine animal model of OVA-induced asthma, 1 where the ubiquitin-modifying enzyme A20 was described as a responsible factor. 4 The connection between farm life and asthma-protection was also shown by differences in the indoor microbiota composition between farm and non-farm homes of Finnish birth cohorts. 5 However, not all farms were equally protective against asthma or allergies: especially cattle and pig farming provide a beneficial environment, 6,7 in addition to consumption of raw cow's milk being a protective factor for asthma. 8 The syllogism for us was that also species-specific animal-derived proteins might play a role in the protective farm effect. Our work is thus complementary to other studies focusing on farm-protective effects associated with stable microbiota.
We hypothesized that the bovine beta-lactoglobulin (BLG) might be an interesting protein candidate as (i) it is secreted via milk and could likely be contained in other bovine secretions; (ii) it is a very stable protein due to numerous disulfide bridges; (iii) it is a member of the lipocalin family for which we previously could show that its immunomodulatory functions depend on the association with binding partners: while the emptied BLG (apo-BLG) induces Th2-responses, holo-BLG loaded with compounds, for example iron-quercetin or retinoic acid, results in immune resilience and prevention of allergic reactions, a result not only shown in vitro but also in in vivo animal models. [9][10][11][12][13] To prove the relevance of these observations in real life, we specifically investigated the occurrence of BLG indoor and outdoor of cattle farms, its natural binding partners and its immunomodulatory capacity as a potential active factor in the allergy-protective farm effect.

| Collection and extraction of dust samples from stables, bedrooms and environmental air around stables
Dust samples from different cattle farms were collected by different methods (Table S1, set 1) to establish collection and extraction method.
A defined set of stable dust samples (Table S1,  inhabitants, as well as from urban households (n = 10) was collected by vacuum cleaner.
Ambient aerosols (total suspended particles) were collected to determine the appropriate sampling material (cellulose, teflon or quartz filters; Table S1, set 3).
For distance measurements of ambient aerosols (Table S1,

| Collection of urine samples
Urine samples (Table S2) from female and male cattle (n = 57) were collected from different stables (corresponding to stable dust samples; Table S1, set 1) noting sex, age, lactation, feeding status and season of collection time point. Additional samples were provided by the Austrian Food Safety Agency (AGES, n = 34) including information about sex, age, lactation and feeding status of the animal. As control, canine (n = 1) and human (n = 1) urine samples were used. Urine samples were frozen immediately after collection.

| Endotoxin detection in stable dust
Endotoxin was detected in dust samples of cattle and poultry farms, extracted according to Mårtensson et al., 14

| Investigation of BLG-association with trace elements
One cattle stable dust extract and 2 bedroom-dust samples were randomly selected and investigated for presence of BLG, and association of binding partners (e.g. trace elements) to BLG. The measurements were performed via size exclusion chromatography combined with inductively coupled plasma mass spectrometry (SEC-ICP-MS). The SEC method was conducted under native conditions (75 mmol/L NaCl, pH 7.4) enabling selective separation of the intact metal/protein adduct. The on-line combination with ICP-MS (dynamic reaction cell with oxygen as reaction gas) allowed the sensitive detection of the proteins via sulfur (as SO + ) and the elements aluminum, manganese, iron, nickel, copper and zinc.

| Mapping of zinc cations on BLG epitopes
Crystal structures 4LZU and 4LZV of zinc-BLG complexes in the presence of 2 and 20 mM zinc chloride, 15 respectively, were retrieved from the Protein Data Bank (PDB). 16  with SDE depleted of BLG via magnetic beads (Protein G Magnetic Beads, Pierce™, ThermoFischer) coupled with human IgG1 and IgG4 antibodies specific for bovine BLG 19 acc. to manufacturer's instructions (SDE-; BLG content below detection limit in ELISA; n = 5), or (C) with MQ water (n = 4; Figure S1A). The overall composition of dust extract (original and depleted of BLG) was controlled in SDS-PAGE via silver staining, showing that after antigen-specific removal of BLG via magnetic beads coated with BLG-specific antibody, other major components remained included (data not shown).
One day before sacrifice, animals were challenged i.p. with BLG   Figure 1E).

| BLG is transferred into households and detected in bed dust samples
BLG levels were significantly higher in bed dust of cattle farms than in bed dust of poultry farms and urban apartments (Table S3), tested by BLG-specific ELISA ( Figure 1I).

| BLG in stable and bed dust is associated with zinc
SEC-ICP-MS showed that stable dust and bed dust extracts from cattle farms contained BLG, comparing with BLG standard, which eluted as 2 peaks according to monomer at 9 min and dimer at 7.8 min ( Figure 3A). In both, cattle stable dust extract ( Figure 3B) as well as in bed dust extract ( Figure 3C), the zinc-peak was associated to the dimer of BLG, eluted at 7.8 min.
The association of zinc to BLG was confirmed via ANS-assay by fluorescence measurement, where addition of zinc to apo-BLG increased the fluorescence signal in a concentration-dependent manner, as due to slight unfolding of BLG by zinc, more ANS molecules might bind ( Figure 3D).

| Zinc is closely associated to a B-cell epitope on BLG
In epitope mapping, 20-23 one zinc ion near residue E74 was  cation was found to be associated to a T-cell epitope on BLG ( Figure 4B).

| Association of BLG with zinc induces a predominant Th1-response in vitro
Apo-and zinc-BLG or zinc alone were used to stimulate PBMC of healthy human donors. Endotoxin levels were comparable in both samples (apo-BLG: 132.0 EU/ml, zinc-BLG: 157.5 EU/ml). The efficacy of the zinc-unloading method to generate apo-BLG, and of zincloading to generate zinc-BLG was verified by flame atomic absorption spectroscopy ( Figure S1).  Figure 5C) and CD8 + cells ( Figure 5D).
In cells without PHA-stimulation, zinc on BLG (but not zinc alone) reduced the number of CD14 + cells among PBMC in a concentrationdependent manner ( Figure S2A); within the CD14 + cell population, CD4-expression was lower when increasing zinc-concentrations were offered together with BLG ( Figure S2B).
Incubation of PHA-stimulated PBMC with zinc-BLG compared to apo-BLG resulted in significantly higher levels of IL-6 and especially IFN-γ in supernatants of PBMC. No significantly higher induction of IL-2, IL-4, IL-5, or IL-13 was observed with zinc-BLG compared to apo-BLG ( Figure 6). When zinc alone was applied to PHA-stimulated PBMC, in addition to IFN-γ and IL-6, also high levels of cytokines IL-2, IL-4, IL-5 and IL-13 were released, diminishing the Th1-dominance in the cytokine pattern ( Figure 6). Levels of TNF-α and IL-10 did not significantly differ between the different stimulation conditions (data not shown). We aimed here to test whether the pre-exposure to SDE+ has an impact on sensitization (i) in an antigen-unspecific way by using the unrelated allergen Bet v 1 from birch pollen, or (ii) in an antigenspecific manner using BLG itself for sensitization. Challenge with Bet v 1 in the double sensitized mice resulted in significantly lower symptom scores in the group pre-treated with SDE+ than groups treated with SDE-or water ( Figure S3B). Hence, SDE+ in a nonspecific manner protected against sensitization to an independent allergen. A similar trend could be observed after BLG-challenge (n.s.; Figure S3B). For Bet v 1-and BLG-specific IgE, lower levels were found in the SDE+ pre-treated animals, paralleled by higher amounts of BLG-specific IgG2a, resulting in the highest IgG2a/IgE-ratios for

| Stable dust extract with BLG prevents allergy symptoms in mice
Bet v 1 as well as for BLG in the SDE+ group ( Figure S3C). This was accompanied by highest Th1-cytokine IFN-γ parallel to low IL-6 levels in supernatants of BLG-stimulated splenocytes of the SDE + pretreated group ( Figure S3D). However, differences were not statistically significant due to our small group size in this pilot experiment. No significant differences were seen in total IgE, specific Bet v 1-or BLG-specific IgG1, or BLG-specific IgA; Bet v 1-specific IgG2a or IgA did not increase upon i.p. sensitization (data not shown). In addition to microbes and their products, specific proteins may also play a role in the protective effect of farms against asthma, atopic eczema and allergies. 24 Additionally, the fact that raw milk consumption per se is an independent protective factor, 25 and especially the whey protein fraction seems to be important 26 with 50% of whey proteins constituted by BLG, led us to the assumption that this protein could play an additional role here.
Although BLG's cognate matrix is milk, we demonstrate here that it is also secreted in large amounts via cattle urine, explaining its delivery into stable dust. Using poultry stable dust as control, we could show that BLG indeed was specifically present in cattle farm samples, associated to zinc. In addition to BLG, endotoxin was found at higher levels in stable dust of cattle than poultry farms. 27 This might be due to a specific microbiota composition in the different stables. In a recent paper, the microbiome of farm home dust was shown to contain cattle-associated microbes that were typically absent from non-farm households. 5 However, even though BLG is a well-known carrier for endotoxin, we found no statistically significant correlation between BLG and endotoxin levels. In previous studies the limits for the protective farm effect were set within a distance below 390 m from the nearest cattle stable. 7 We show here that also the concentration of aerosolized BLG declines steadily in a distance-dependent gradient from the cattle stable, again implying a potential contribution of BLG to the farm effect.
As expected, endotoxin levels were highest close to the stable. While BLG levels gradually declined with increasing distance to the stable, endotoxin declined more rapidly outside the stable. As BLG is a known endotoxin-carrier, it was important to dissect the impact of endotoxin and zinc-BLG. In vitro, PBMC were stimulated with apo-BLG or zinc-BLG, with both samples containing similar endotoxin levels. Also in our preliminary in vivo mouse model the applied SDE+ and SDE-both contained high endotoxin levels. Interestingly, SDEcontained even higher levels of endotoxin, probably due to more handling steps during depletion. These results show that differences in vitro and in vivo were independent of endotoxin levels, and therefore favour BLG as likely contributor to the protective farm effect.
The amounts of BLG were put in relation to the mean pulmonary capacity. Certainly, the composition of ambient air with different sizes of dust particles also impacts the pulmonary distribution of the inhaled particles, meaning that smaller carrier particles of BLG in the inhaled air could enter deeper lung compartments. We calculated the mean lung capacity without considering a potential difference in the uptake site for particles and/or BLG as such.
Urine was revealed as the major source of BLG in cattle stable dust. The concentration of BLG was higher in young animals and in dairy cattle, however, it was independent of sex, lactation status, season as well as breed. Interestingly, the human equivalent to BLG, human lipocalin-2 (huLCN2), is used as a diagnostic urinary marker in chronic kidney disease 28 ; but LCN2 also has a functional role: in cancer, where immune tolerance is detrimental, elevated huLCN2 levels are prognostic 29,30 ; in contrast, its levels are reduced in allergics and can be corrected by allergen immunotherapy, pointing towards a role in immune tolerance. 31 Our novel collected evidence suggests that the lipocalin BLG might act tolerogenic also via aerosols.
As not all family members of a farm may be working or staying routinely and regularly in the stable, it was important to clarify the presence of BLG in the household. Even non-farm homes can be protective when a farm-like microbial environment is present in the home. 5 We thus collected dust from cattle and poultry stables and corresponding mattress and pillow dust from farms and, for comparison, bed dust samples from urban apartments. There were significantly higher levels of BLG in dust of cattle farm households, compared to poultry farm or urban samples. Higher BLG levels in bed dust compared to respective stable dust are presumably the result of relatively higher protein concentration per weight of dust due to increased accumulation by vacuum-cleaning. Moreover, bed dust contains less contaminants than the stable environment, where higher levels of non-proteinic material are present, for example straw, hay, fodder, fine stone-dust etc. In correlation with higher BLG concentrations at cattle farms, we recorded that relatively lower numbers of family members living on cattle farms reported to be affected by allergy (14.3%) compared to poultry farms (25%) or urban flats (60%). There was no clear correlation for BLG content in households of cattle farmers and their life-style and habits (hair washing, mattress cleaning, pets in house), or number of animals on farms, again probably due to the low sample number. We also compared the farms with highest (n = 2) and lowest (n = 1) indoor-BLG levels, but could not find obvious differences in behavior of farmers or other conditions, except for the fact that the household with lowest BLG in bed dust (but not in the stable) also had the lowest number of cattle (n = 1). Also for BLG in city apartments, no characteristic could explain high levels in bed dust, for example eating in bedroom, pets in household, type of mattress, or frequency of washing mattress or hair. Still, another group observed that newer mattresses contain more food allergens, and that a longer distance from kitchen to bedroom, the size of the dwelling, and cleaning equipment influenced the occurrence of food allergens in bed dust, incl. milk allergens. 32 BLG, belonging to the lipocalin protein family, harbors an intramolecular pocket for small molecules or elements. This is relevant as in our previous studies we have shown that proteins from the lipocalin family modulate the immune response depending on their loading state: when the cavity is filled with compounds like siderophore-iron 13,33,34 or vitamin A, 11,35 lipocalins like BLG act tolerogenic, and an allergic immune response is avoided. Our SEC-ICP-MS analysis showed that the trace element zinc is associated to BLG in dust of stables and households. In contrast to the forementioned binding partners, zinc is not found within the calyx of BLG, but rather attached to the protein surface, which was also confirmed in the ANS-assay, in analogy to crystallization studies. 11,15,35 Zinc can induce a slight unfolding of BLG, 36 giving access to more binding sites for ANS, seen as increasing fluorescence signal in the ANS-assay. well as CD8+ T-cells in a concentration-dependent manner. These results indicate that addition of zinc, converting apo-BLG into zinc-BLG, can prevent the activation/proliferation of immune cells, probably also down-regulating or preventing an inflammatory response, which we recently termed immune resilience. 13 Antigen presentation by B-cells could be affected as zinc ions might hinder binding and thus presentation to T-cells in general. BLG complexed with zinc also induced a favorable Th1-dominated pattern with high IFN-γ levels versus low IL-4, IL-5 or IL-13 levels, while zinc alone induced Th2cytokines IL-4 and IL-13 in pre-stimulated PBMC of healthy donors, as also previously reported by others. 38 Despite lowered proliferation of CD4+ and CD8+ cells by zinc-BLG, a Th1-response was promoted by this stimulant, which may be attributed to innate cells rather than T-cells. In particular IL-6 and IFN-γ were associated with zinc-BLG stimulation. IL-6 is predominantly secreted by macrophages, whereas IFN-γ is predominantly secreted by NK cells, making them the likely source for the Th1-bias. Hence, zinc associated to BLG may add to the protective farm effect in an innate manner via Th1-cytokine induction. Such Th1-skewing was also described in human and animal studies. [39][40][41] Regarding these Th1-associated cytokines, we are also aware of the potential effect of LPS in our system. Simultaneously, we acknowledge that (i) complete LPS removal is hard to achieve without removing BLG from the sample itself, and (ii) BLG is an important natural carrier of LPS. Therefore, we measured endotoxin in our stimulation samples and could prove that they are similar in LPScontent. We propose that this confirms that differences in proliferation and/or cytokine release in apo-BLG versus zinc-BLG were not due to LPS.
The proof-of-concept experiments in mice here suggest that the protective capacity of stable dust is dependent on BLG as major carrier component, since stable dust extract, in which BLG was depleted, showed a reduced allergy-protective capacity.
Importantly, the protective effect was independent of the endotoxin-levels present in the samples. Rather, in line with our recently published animal studies, this immunomodulatory effect was dependent on BLG fulfilling a shuttle function for micronutrients like iron-siderophore complexes, vitamin A and D, 12,13 and zinc, targeting them to innate immune cells. Importantly, a dietary lozenge containing whey protein with iron-quercetin, vitamin A, and zinc could reduce symptoms in human birch-pollen and house dust mite allergic patients, 42,43 as well as in the mouse model. 10 Our study has some limitations: even though BLG represents a major fraction among the whey proteins, we can only speculate whether BLG with its binding partners also represents the allergyprotective factor associated with raw milk. However, the facts that (i) milk processing destroys the tertiary structure of heat-labile BLG, 44,45 (ii) this results in a loss of associated molecules or elements, and (iii) processed milk loses its allergy-protective effect, makes this concept very plausible. Finally, the factors which determine natural BLG secretion and loading with zinc, such as cattle feed, health status and animal welfare, need to be determined.

| CONCLUSION
Our data show that the secretory protein BLG is not only a major whey protein but that it is secreted by bovine urine, aerosolized around cattle farms and finally precipitated as a major compound in cattle farm dust. We have also shown that BLG complexed with zinc is able to induce a favorable Th1-milieu in PBMC of healthy donors.
Stable dust containing BLG resulted in better protection against allergic sensitization in an allergen-nonspecific manner than dust depleted of BLG in vivo in the mouse model. We thus propose that-in addition to endotoxin and microbial load-BLG with its binding partners has innate immunomodulatory potency and may represent an active component in the well-known allergy-protective effect of cattle farms.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.