Worldwide 300 million children and adults are affected by asthma. The development of asthma is influenced by environmental and other exogenous factors synergizing with genetic predisposition, and shaping the lung microbiome especially during birth and in very early life. The healthy lung microbial composition is characterized by a prevalence of bacteria belonging to the phyla Bacteroidetes, Actinobacteria, and Firmicutes. However, viral respiratory infections are associated with an abundance of Proteobacteria with genera Haemophilus and Moraxella in young children and adult asthmatics. This dysbiosis supports the activation of inflammatory pathways and contributes to bronchoconstriction and bronchial hyperresponsiveness. Exogenous factors can affect the natural lung microbiota composition positively (farming environment) or negatively (allergens, air pollutants). It is evident that also gut microbiota dysbiosis has a high influence on asthma pathogenesis. Antibiotics, antiulcer medications, and other drugs severely impair gut as well as lung microbiota. Resulting dysbiosis and reduced microbial diversity dysregulate the bidirectional crosstalk across the gut-lung axis, resulting in hypersensitivity and hyperreactivity to respiratory and food allergens. Efforts are undertaken to reconstitute the microbiota and immune balance by probiotics and engineered bacteria, but results from human studies do not yet support their efficacy in asthma prevention or treatment. Overall, dysbiosis of gut and lung seem to be critical causes of the increased emergence of asthma.

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The major cow’s milk allergen Bos d 5 belongs to the lipocalin protein family, with an intramolecular pocket for hydrophobic ligands. We investigated whether Bos d 5 when loaded with the active vitamin A metabolite retinoic acid (RA), would elicit differential immune responses compared to the unloaded state. By in silico docking an affinity energy of −7.8 kcal/mol was calculated for RA into Bos d 5. Loading of RA to Bos d 5 could be achieved in vitro, as demonstrated by ANS displacement assay, but had no effect on serum IgE binding in tolerant or challenge-positive milk allergic children. Bioinformatic analysis revealed that RA binds to the immunodominant T-cell epitope region of Bos d 5. In accordance, Bos d 5 significantly suppressed the CD3+ CD4+ cell numbers, proliferative response and IL-10, IL-13 and IFN-γ secretion from stimulated human PBMCs only when complexed with RA. This phenomenon was neither associated with apoptosis of T-cells nor with the activation of Foxp3+ T-cells, but correlated likely with enhanced stability to lysosomal digestion due to a predicted overlap of Cathepsin S cleavage sites with the RA binding site. Taken together, proper loading of Bos d 5 with RA may suppress its immunogenicity and prevent its allergenicity.

Background Macrophages can be converted in vitro into immunoregulatory M2b macrophages in the presence of immune complexes ( IC s), but the role of the specific subclasses IgG1 or IgG4 in this phenotypic and functional change is not known. Objective We aimed to refine the original method by applying precisely defined IC s of the subclasses IgG4 or IgG1 constructed by two independent methods. Methods Monocyte‐derived macrophages ( MDM s) were treated with M‐ CSF , followed by IL ‐4/ IL ‐13 to induce the M2a allergic phenotype. To mimic unspecific or allergen‐specific IC s, plates were coated with myeloma IgG1 or IgG4, or with grass pollen allergen Phl p 5 followed by recombinant human Phl p 5‐specific IgG1 or IgG4. M2a polarized macrophages were then added, cultured, and examined for cellular markers and cytokines by flow cytometry, ELISA , and rt PCR . Alternatively, immune complexes with IgG1 or IgG4 were formed using protein L. Results IgG4 IC s down regulated CD 163 and CD 206 on M2a cells, and significantly increased IL ‐10, IL ‐6, TNF α, and CCL 1 secretion, indicating a shift to an M2b‐like phenotype. Treatment with IgG4 IC s resulted in expression of Fcγ RII and down modulation of Fcγ RII compared with IgG1 treated cells ( P = 0.0335) or untreated cells ( P < 0.00001). Conclusion Immune complexes with subclasses IgG1 and IgG4 can in vitro be generated by plate absorption, and in fluid form by protein L. Cross‐linking of Fcγ RII b by the IgG4 subclass redirects pro‐allergic M2a macrophages to an M2b‐like immunosuppressive phenotype. This suggests an interplay of macrophages with IgG4 in immune tolerance, likely relevant in allergen immunotherapy.

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The lipocalin beta-lactoglobulin (BLG) is a major protein compound in cow’s milk, and we detected it in cattle stable dust. BLG may be a novel player in the farm protective effect against atopic sensitization and hayfever. In previous studies, we demonstrated that only the ligand-filled holo-form of BLG prevented sensitization to itself. Here, we investigated whether holo-BLG could, in an innate manner, also protect against allergic sensitization to unrelated birch pollen allergens using a murine model. BALB/c mice were nasally pretreated four times in biweekly intervals with holo-BLG containing quercetin–iron complexes as ligands, with empty apo-BLG, or were sham-treated. Subsequently, mice were intraperitoneally sensitized two times with apo-BLG or with the unrelated birch pollen allergen apo-Bet v 1, adjuvanted with aluminum hydroxide. After subsequent systemic challenge with BLG or Bet v 1, body temperature drop was monitored by anaphylaxis imaging. Specific antibodies in serum and cytokines of BLG- and Bet v 1-stimulated splenocytes were analyzed by ELISA. Additionally, human peripheral blood mononuclear cells of pollen allergic subjects were stimulated with apo- versus holo-BLG before assessment by FACS. Prophylactic treatment with the holo-BLG resulted in protection against allergic sensitization and clinical reactivity also to Bet v 1 in an unspecific manner. Pretreatment with holo-BLG resulted in significantly lower BLG-as well as Bet v 1-specific antibodies and impaired antigen-presentation with significantly lower numbers of CD11c+MHCII+ cells expressing CD86. Pretreatment with holo-BLG also reduced the release of Th2-associated cytokines from Splenocytes in BLG-sensitized mice. Similarly, in vitro stimulation of PBMCs from birch pollen allergic subjects with holo-BLG resulted in a relative decrease of CD3+CD4+ and CD4+CRTh2 cells, but not of CD4+CD25+CD127− Treg cells, compared to apo-BLG stimulation. In conclusion, prophylactic treatment with holo-BLG protected against allergy in an antigen-specific and -unspecific manner by decreasing antigen presentation, specific antibody production and abrogating a Th2-response. Holo-BLG therefore promotes immune resilience against pollen allergens in an innate manner and may thereby contribute to the farm protective effect against atopic sensitization.

Background The hygiene hypothesis implies that microbial agents including probiotic bacteria may modulate foetal/neonatal immune programming and hence offer effective strategies for primary allergy prevention; however their mechanisms of action are poorly understood. We investigated whether oral administration of Lactobacillus paracasei NCC 2461 to mothers during gestation/lactation can protect against airway inflammation in offspring in a mouse model of birch pollen allergy, and examined the immune mechanisms involved. Methods BALB/c mice were treated daily with L. paracasei in drinking water or drinking water alone in the last week of gestation and during lactation. Their offspring were sensitized with recombinant Bet v 1, followed by aerosol challenge with birch pollen extract. Results Maternal exposure to L. paracasei prevented the development of airway inflammation in offspring, as demonstrated by attenuation of eosinophil influx in the lungs; reduction of IL-5 levels in bronchoalveolar lavage, and in lung and mediastinal lymph node cell cultures; and reduced peribronchial inflammatory infiltrate and mucus hypersecretion. While allergen-specific IgE and IgG antibody levels remained unchanged by the treatment, IL-4 and IL-5 production in spleen cell cultures were significantly reduced upon allergen stimulation in offspring of L. paracasei treated mice. Offspring of L. paracasei supplemented mothers had significantly reduced Bet v 1-specific as well as Concanavalin A-induced responses in spleen and mesenteric lymph node cell cultures, suggesting the modulation of both antigen-specific and mitogen-induced immune responses in offspring. These effects were associated with increased Foxp3 mRNA expression in the lungs and increased TGF-beta in serum. Conclusion Our data show that in a mouse model of birch pollen allergy, perinatal administration of L. paracasei NCC 2461 to pregnant/lactating mothers protects against the development of airway inflammation in offspring by activating regulatory pathways, likely through TLR2/4 signalling.