Probiotic bacteria and their metabolites, particularly the short chain fatty acid butyrate, possess beneficial immunomodulatory and anti-inflammatory properties and have been shown to protect against gastrointestinal and respiratory diseases including IgE-mediated allergic inflammation.1-3 However, underlying mechanisms such as modulation of dendritic cell maturation and induction of regulatory T (Treg) cells are complex and not fully understood.2, 4 Moreover, to achieve a strong immune-modulatory response, it is crucial to provide multiple bacteria in optimal concentration and simultaneously. Here, we investigated for the first time a well-defined commercially available probiotic formulation (BactoFlor® 10/20, BF), consisting of 10 large and small intestine-specific strains of Lacto- and Bifidobacteria and the prebiotic carbohydrate inulin, in comparison to butyrate in a well-established and valid humanized mouse model of allergen-induced gut and lung inflammation.5 Nonobese diabetic severe-combined-immunodeficient γc−/− (NSG) mice were intraperitoneally injected with PBMC from highly-sensitized birch or grass pollen allergic human donors together with the respective allergen or PBS as control. During the whole experiment, mice were orally treated with BF or butyrate as described in the Appendix S1. In this model, sufficient numbers of human cells are detectable 3 weeks after PBMC transfer, and inflammation of the gut and lungs can be measured by high-resolution video mini-endoscopy, evaluating the parameters translucency, granularity, fibrin production, vascularity, and stool, or by invasive body plethysmography after rectal or intranasal allergen challenge, respectively.5 Figure 1A,B shows that the strong inflammation of the gut in PBMC plus allergen-treated mice was completely prevented by oral administration of BF or butyrate. In addition, disturbed intestinal barrier integrity, as measured by FITC-dextran assay, was significantly restored (Figure 1C). Analysis of lung allergic inflammation further revealed reduced lung airway resistance, reduced mucosal hypertrophy, and reduced mucus-producing goblet cells in BF- or butyrate-treated mice (Figure 1D,E). Interestingly, despite equal numbers of human CD45+ cells in all organs, an increased migration of FoxP3+ Treg cells into the lung and gut tissue was found in BF- as well as butyrate-treated mice after respective allergen challenge (Figure 1F). In the spleen, a slight enhancement of CD4+FoxP3 expression was also detected without reaching significance (Figure 1G). Engraftment of BF- or butyrate-pretreated PBMC also significantly reduced gut and lung inflammation, but less pronounced as compared to oral treatment (Figure S1A–D). This superior protective effect of orally applied probiotics/butyrate suggests that besides their immunomodulatory effect on PBMC, they also directly affect the mucosal epithelium and barrier. Moreover, a slight but not significant increase in butyrate concentration was observed in stool of BF-treated mice (Figure S2). This implicated that other microbiota-derived metabolites than butyrate, possibly tryptophan metabolites or retinoid acid, might also be involved in the induction of the regulatory immune response.2, 6 To analyze whether Treg cells are responsible for the protective effect of BF and butyrate, CD25high-expressing cells were depleted from the PBMC prior to injection (Figure S3). In these CD25negPBMC engrafted NSG mice, gut and lung inflammation was only marginally reduced by BF or butyrate (Figure 2A–D). This is in accordance with former studies demonstrating the strong Treg cell dependency in prevention of allergic diseases.5 Notably, neutralization of allergy promoting group 2 innate lymphoid cells (ILC2), which are known to be controlled by Treg,1 by anti-ST2 mAbs restored the protective effect of BF and butyrate in Treg-depleted mice (Figure S4). Other cell types might also be involved which is currently under investigation. Taken together, our study support the relevance of Treg cells regarding the combined effect of defined beneficial intestinal microbial species and their products on allergic intestinal and airway inflammation. Hence, it advances our current understanding of the previously suspected adjuvant effect of certain probiotics in allergic desensitization. Our results may also be applicable to other inflammatory diseases of the gastrointestinal and respiratory tract. DS, JS, and IB generated the idea and supervised the work; RK, BW, FS, SS, and IB performed laboratory assays and contributed to data analysis; RK, DS, and IB wrote the manuscript; all authors have read and approved the manuscript. The authors would like to thank Mrs. Claudia Braun and Mrs. Anke Heinz for their excellent technical assistance. Open Access funding enabled and organized by Projekt DEAL. This work was supported by Deutsche Forschungsgemeinschaft (DFG), grants BE 4504/3-3 (I.B.), SFB1181-B02 (B.W.), SCHU 646/20-3 and CRC TR355/1 project B08 (D.S.). The authors have no conflicting financial interests to disclose. The data that support the findings of this study are available from the corresponding author upon reasonable request. Appendix S1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Background Approximately 70 % of individuals allergic to birch pollen (Bet v 1.01 [Bet]) develop a secondary food allergy (e.g. hazelnut: Cor a 1.04 [Cor]), due to allergen cross-reactivity. However, standard immunotherapy for type I allergies often does not improve the food allergy sufficiently. We analyzed the allergen-specific and cross-reactive suppressive capacity of primary human regulatory T cells (Treg) induced by autologous IL-10-modulated dendritic cells (IL-10 DC) in vitro and in vivo . Methods CD4 T cells of patients with birch pollen and associated hazelnut allergies were differentiated into Bet-specific or non-specific induced Treg (iTreg). After Bet- or Cor- specific restimulation the phenotype, proliferation and suppressive capacity of iTreg subsets were analyzed. iTreg function was further investigated in humanized mouse models of airway and intestinal allergy, generated by engraftment of peripheral blood mononuclear cells from allergic donors into immunodeficient animals. Results After IL-10 DC priming and allergen-specific restimulation (Bet or Cor) non-specific control iTreg remained anergic, whereas Bet-specific iTreg proliferated extensively and exhibited a regulatory phenotype (enhanced expression of CTLA-4, PD-1, TNFR2, IL-10). Accordingly, activated Bet-specific iTreg displayed a high capacity to suppress Bet- and Cor-induced responder T 2 cell responses in vitro , indicating induction of both allergen-specific (birch) and cross-reactive tolerance (hazelnut). In vivo , the beneficial effect of Bet-specific iTreg was verified in humanized mouse models of allergic airway and intestinal inflammation, resulting in reduced allergen-induced clinical symptoms and immune responses. Conclusion Human IL-10 DC-induced iTreg facilitate allergen-specific and cross-reactive tolerance. Therefore, they are potential candidates for regulatory cell therapy in allergic and autoimmune diseases.

The chemical modification of aeroallergens by reactive oxygen and nitrogen species (ROS/RNS) may contribute to the growing prevalence of respiratory allergies in industrialized countries. Post-translational modifications can alter the immunological properties of proteins, but the underlying mechanisms and effects are not well understood. In this study, we investigate the Toll-like receptor 4 (TLR4) activation of the major birch and grass pollen allergens Bet v 1 and Phl p 5, and how the physiological oxidant peroxynitrite (ONOO-) changes the TLR4 activation through protein nitration and the formation of protein dimers and higher oligomers. Of the two allergens, Bet v 1 exhibited no TLR4 activation, but we found TLR4 activation of Phl p 5, which increased after modification with ONOO- and may play a role in the sensitization against this grass pollen allergen. We attribute the TLR4 activation mainly to the two-domain structure of Phl p 5 which may promote TLR4 dimerization and activation. The enhanced TLR4 signaling of the modified allergen indicates that the ONOO--induced modifications affect relevant protein-receptor interactions. This may lead to increased sensitization to the grass pollen allergen and thus contribute to the increasing prevalence of allergies in the Anthropocene, the present era of globally pervasive anthropogenic influence on the environment.

Over the past decades, atopic diseases, including allergic rhinitis, asthma, atopic dermatitis, and food allergy, increased strongly worldwide, reaching up to 50% in industrialized countries. These diseases are characterized by a dominating type 2 immune response and reduced numbers of allergen-specific regulatory T (Treg) cells. Conventional allergen-specific immunotherapy is able to tip the balance towards immunoregulation. However, in mouse models of allergy adaptive transfer of Treg cells did not always lead to convincing beneficial results, partially because of limited stability of their regulatory phenotype activity. Besides genetic predisposition, it has become evident that environmental factors like a westernized lifestyle linked to modern sanitized living, the early use of antibiotics, and the consumption of unhealthy foods leads to epithelial barrier defects and dysbiotic microbiota, thereby preventing immune tolerance and favoring the development of allergic diseases. Epigenetic modification of Treg cells has been described as one important mechanism in this context. In this review, we summarize how environmental factors affect the number and function of Treg cells in allergic inflammation and how this knowledge can be exploited in future allergy prevention strategies as well as novel therapeutic approaches.

BACKGROUND:CD56-expressing natural killer (NK) cells as well as invariant NK T (iNKT) cells have been shown to either promote or inhibit allergic immune responses. OBJECTIVE:The aim of the present study was to investigate the impact of these cells in a recently developed humanized mouse model of allergen-induced IgE-dependent gut and lung inflammation. METHODS:Nonobese diabetic-severe combined immunodeficiency γ-chain knockout mice were injected intraperitoneally with human PBMCs or CD56-depleted (CD56neg) PBMCs from highly sensitized donors with birch or grass pollen allergy together with the respective allergen or with NaCl as a control. Three weeks later, the mice were challenged with the allergen rectally and gut inflammation was monitored by video miniendoscopy and by histology. Furthermore, airway inflammation was measured after an additional intranasal allergen challenge. RESULTS:Allergen-specific human IgE in mouse sera, detectable only after coinjection of the respective allergen, was reduced in mice being injected with CD56neg PBMCs compared with in mice receiving nondepleted PBMCs. Consequently, allergen-induced IgE-dependent colitis, airway hyperreactivity, and mucus-producing goblet cells were significantly inhibited in these mice. Interestingly, reconstitution of CD56neg PBMCs with nondepleted CD56+ cells and with CD56+CD3+ iNKT cells restored gut as well as lung inflammation, whereas addition of CD3-depleted CD56+ cells did not. CONCLUSION:These results demonstrate that allergen-specific gut and lung inflammation in PBMC-engrafted humanized mice is promoted by CD56+CD3+ iNKT cells, which opens new possibilities of therapeutic intervention in allergic diseases.

Background Ceylon cinnamon has been shown to possess anti-inflammatory properties in many diseases including allergic inflammation. Objective The aim of this study was to analyse in more detail the effects of cinnamon extract (CE) and its major compounds p-cymene and trans-cinnamaldehyde (CA) on allergen-specific immune responses in vitro and in vivo. Methods Therefore, monocyte-derived mature dendritic cells (DC) from grass or birch pollen allergic donors were pulsed with the respective allergen in the presence or absence of CE, p-cymene, CA or the solvent ethanol and co-cultured with autologous CD4(+) T cells. Furthermore, basophil activation test was performed with or without CE or ethanol treatment. For the in vivo experiments, BALB/c mice were immunized with ovalbumin (OVA) and orally treated with CE or ethanol. Results Addition of CE, p-cymene or CA, but not ethanol significantly inhibited DC maturation and subsequent allergen-specific T cell proliferation as well as Th1 and Th2 cytokine production. Sulphidoleukotriene release and CD63 expression by basophils were also significantly diminished after addition of CE. In vivo, treatment of OVA-sensitized mice with CE led to a significant shift from OVA-specific IgE towards IgG2a production and to a strong inhibition of OVA-specific proliferation. Moreover, airway inflammation as well as anaphylaxis after intranasal or systemic allergen challenge was significantly reduced in CE-treated mice. Furthermore, topical application of CE prevented calcipotriol-induced atopic dermatitis-like inflammation in these mice. Conclusions and Clinical Relevance Taken together, our data indicate that the anti-inflammatory effect of cinnamon might be exploited for treatment of allergic inflammation, which needs to be further investigated.

Toll-like receptor 4 (TLR4) plays a crucial role in the recognition of invading pathogens. Upon activation by lipopolysaccharides (LPS), TLR4 is recruited into specific membrane domains and dimerizes. In addition to LPS, TLR4 can be stimulated by wheat amylase-trypsin inhibitors (ATI). ATI are proteins associated with gluten containing grains, whose ingestion promotes intestinal and extraintestinal inflammation. However, the effect of ATI vs. LPS on the membrane distribution of TLR4 at the nanoscale has not been analyzed. In this study, we investigated the effect of LPS and ATI stimulation on the membrane distribution of TLR4 in primary human macrophages using single molecule localization microscopy (SMLM). We found that in unstimulated macrophages the majority of TLR4 molecules are located in clusters, but with donor-dependent variations from ∼51% to ∼75%. Depending on pre-clustering, we found pronounced variations in the fraction of clustered molecules and density of clusters on the membrane upon LPS and ATI stimulation. Although clustering differed greatly among the human donors, we found an almost constant cluster diameter of ∼44 nm for all donors, independent of treatment. Together, our results show donor-dependent but comparable effects between ATI and LPS stimulation on the membrane distribution of TLR4. This may indicate a general mechanism of TLR4 activation in primary human macrophages. Furthermore, our methodology visualizes TLR4 receptor clustering and underlines its functional role as a signaling platform.

Amylase trypsin inhibitors (ATI) can be found in all gluten containing cereals and are, therefore, ingredient of basic foods like bread or pasta. In the gut ATI can mediate innate immunity via activation of the Toll-like receptor 4 (TLR4) on immune cells residing in the lamina propria, promoting intestinal, as well as extra-intestinal, inflammation. Inflammatory conditions can induce formation of peroxynitrite (ONOO-) and, thereby, endogenous protein nitration in the body. Moreover, air pollutants like ozone (O3) and nitrogen dioxide (NO2) can cause exogenous protein nitration in the environment. Both reaction pathways may lead to the nitration of ATI. To investigate if and how nitration modulates the immunostimulatory properties of ATI, they were chemically modified by three different methods simulating endogenous and exogenous protein nitration and tested in vitro. Here we show that ATI nitration was achieved by all three methods and lead to increased immune reactions. We found that ATI nitrated by tetranitromethane (TNM) or ONOO- lead to a significantly enhanced TLR4 activation. Furthermore, in human primary immune cells, TNM nitrated ATI induced a significantly higher T cell proliferation and release of Th1 and Th2 cytokines compared to unmodified ATI. Our findings implicate a causative chain between nitration, enhanced TLR4 stimulation, and adaptive immune responses, providing major implications for public health, as nitrated ATI may strongly promote inhalative wheat allergies (baker's asthma), non-celiac wheat sensitivity (NCWS), other allergies, and autoimmune diseases. This underlines the importance of future work analyzing the relationship between endo- and exogenous protein nitration, and the rise in incidence of ATI-related and other food hypersensitivities.

Recently, we have developed a humanized mouse model of allergen-induced IgE-dependent gut and lung inflammation in PBMC-engrafted immunodeficient mice. As natural killer (NK) cells have been shown to promote allergen sensitization, type-2 immune responses and airway hyperreactivity, the aim of the present study was to investigate the impact of NK cells in this model. Therefore, NOD-scid-γc-/- mice were injected intraperitoneally with human PBMC or NK cell-depleted PBMC from highly sensitized birch or grass pollen allergic donors together with the respective allergen or with NaCl as control. After an additional allergen boost one week later, mice were challenged with the allergen rectally on day 21 and gut inflammation was monitored by video mini-endoscopy evaluating translucency, granularity, fibrin production, vascularity, and stool. Then, mice were further challenged intranasally on two subsequent days and airway inflammation was measured by invasive body plethysmography and by histology. Allergen-specific human IgE in mouse sera, if detectable after co-injection of the respective allergen, was reduced in mice being injected with NK cell-depleted PBMC compared to mice which received non-depleted PBMC. Additionally, allergen-induced IgE-dependent colitis, airway hyperreactivity and mucus-producing goblet cells were significantly inhibited in these mice. Importantly, infiltration of the colon and lung with human CD45+ cells was similar in all groups. These results demonstrate that allergen-specific gut and lung inflammation in PBMC-engrafted humanized mice can be diminished by depletion of NK cells prior to PBMC transfer, which may be of great interest for therapeutic intervention of allergic diseases.

SummaryBackgroundTo date, only limited information on structure, expression levels and IgE binding of Bet v 1 variants, which are simultaneously expressed in birch pollen, is available.ObjectiveTo analyse and compare structure and serum IgE/IgG binding of rBet v 1 variants to Bet v 1.0101.MethodsRecombinant Bet v 1 variants were studied with sera of 20 subjects allergic to birch pollen. Folding, aggregation and solubility of the rBet v 1 variants were analysed to attribute diverging IgE binding to either allergen structure or methodological features. IgE/IgG binding was studied with rBet v 1 in solution or adsorbed to solid phases. Allergen‐mediated cross‐linking of FcεRI receptors was determined by mediator release of sensitized humanized rat basophil leukaemia cells.ResultsAll variants, except for rBet v 1.0113, were monomeric and had Bet v 1‐type conformation. Serum IgE binding to variants adsorbed to solid phase was reduced to 6.6%‐36.5% compared with Bet v 1.0101. In contrast, inhibition of IgE binding to Bet v 1.0101 by rBet v 1 variants ranged from 62% to 83%. Similarly, mediator release ranged from 30.7% to 55.2% for all variants and was only clearly reduced for rBet v 1.0301 (10.4%). The IgE‐binding potency of rBet v 1 variants representing their native quantities in birch pollen was only slightly lower compared to extract. IgG binding to variants was between 50.9% and 134.5% compared with rBet v 1.0101 (100%).Conclusion and Clinical RelevanceBet v 1 variants previously classified as hypoallergenic can exhibit similar functional IgE binding as Bet v 1.0101. Eight rBet v 1 variants largely reproduce total Bet v 1‐specific IgE binding of birch pollen extracts. Assay format‐dependent variation in IgE‐binding properties needs to be considered in the development of diagnostic or therapeutic products.