IL-10-modulated dendritic cells from birch pollen- and hazelnut-allergic patients facilitate Treg-mediated allergen-specific and cross-reactive tolerance.

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 Th2 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.

Depletion of CD56+CD3+ invariant natural killer T cells prevents allergen-induced inflammation in humanized mice.

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.

Cinnamon extract inhibits allergen-specific immune responses in human and murine allergy models.

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.

Review of biologics in allergic contact dermatitis.

The resistant and recalcitrant nature of severe allergic contact dermatitis (ACD) makes its management challenging. With advances in the understanding of the cellular and molecular pathogenesis of ACD, newer therapeutic targets are becoming apparent. In particular, the use of biologics has gained momentum, given the specificity of their action. This article aims to review the presently available data on the use of biologics in ACD. English-language-based literature available on the use of biological therapy was thoroughly probed in the following databases as on October 14, 2019: PubMed, Google Scholar, The Cochrane library, Embase, Scopus, and EBSCO. The following keywords were used: "contact dermatitis", "allergens", "delayed-type hypersensitivity reaction", "biologics", "biological therapy", "monoclonal antibodies", "patch testing", "TNF-α inhibitors", "infliximab", "adalimumab", "etanercept", "dupilumab", "omalizumab", "secukinumab", "ustekinumab", "rituximab".

Wheat amylase-trypsin inhibitors exacerbate intestinal and airway allergic immune responses in humanized mice.

BACKGROUND: Amylase-trypsin inhibitors (ATIs) in wheat and related cereals are potent activators of myeloid innate immune cells via engagement of TLR4. Furthermore, ATIs have been shown to serve as adjuvants in experimental intestinal inflammatory diseases. OBJECTIVE: The aim of this study was to analyze whether ATIs are also modifiers of allergic inflammation. METHODS: Therefore, CD4+ T cells from donors sensitized to grass or birch pollen were stimulated with autologous allergen-pulsed dendritic cells in the presence or absence of ATIs or the control storage protein zein from corn. To analyze allergen-induced gut and lung inflammation, immunodeficient mice were engrafted with PBMCs from these allergic donors plus the respective allergen, and fed with selected diets. Three weeks later, inflammation was induced by rectal or intranasal allergen challenge and monitored by mini endoscopy or airway hyperreactivity, respectively. RESULTS: Allergen-specific T-cell proliferation and cytokine production was significantly exacerbated by ATIs and not by zein. In vivo, allergen-specific human IgE level was strongly elevated in sera of mice receiving an ATI-containing diet compared with mice that were fed gluten-free and thus ATI-free diet. Importantly, allergen-induced IgE-dependent colitis and airway hyperreactivity were also enhanced in ATI-fed mice. Gut inflammation was further increased in mice receiving an additional ATI injection and even detectable in the absence of the aeroallergen, whereas zein had no such effect. Injection of anti-human TLR4 mAbs or the anti-human IgE mAb omalizumab completely abolished ATI-induced allergic inflammation. CONCLUSIONS: These results underline that wheat ATIs are important nutritional activators and adjuvants of allergy, which might be exploited for nutritional therapeutic strategies.

Air Pollution and Climate Change Effects on Allergies in the Anthropocene: Abundance, Interaction, and Modification of Allergens and Adjuvants.

Air pollution and climate change are potential drivers for the increasing burden of allergic diseases. The molecular mechanisms by which air pollutants and climate parameters may influence allergic diseases, however, are complex and elusive. This article provides an overview of physical, chemical and biological interactions between air pollution, climate change, allergens, adjuvants and the immune system, addressing how these interactions may promote the development of allergies. We reviewed and synthesized key findings from atmospheric, climate, and biomedical research. The current state of knowledge, open questions, and future research perspectives are outlined and discussed. The Anthropocene, as the present era of globally pervasive anthropogenic influence on planet Earth and, thus, on the human environment, is characterized by a strong increase of carbon dioxide, ozone, nitrogen oxides, and combustion- or traffic-related particulate matter in the atmosphere. These environmental factors can enhance the abundance and induce chemical modifications of allergens, increase oxidative stress in the human body, and skew the immune system toward allergic reactions. In particular, air pollutants can act as adjuvants and alter the immunogenicity of allergenic proteins, while climate change affects the atmospheric abundance and human exposure to bioaerosols and aeroallergens. To fully understand and effectively mitigate the adverse effects of air pollution and climate change on allergic diseases, several challenges remain to be resolved. Among these are the identification and quantification of immunochemical reaction pathways involving allergens and adjuvants under relevant environmental and physiological conditions.

Analysis of allergic immune responses in humanized mice.

Nowadays, more than 25% of the population in industrial countries are affected by IgE-mediated (atopic) allergic diseases such as allergic rhinitis, asthma and atopic eczema. Due to intensive research on basis of in vitro studies with human immune cells and different murine in vivo models of allergy fundamental mechanisms of allergic immune responses have been elucidated during the last years. However, human studies are restricted and the immune system of mice differs from the human immune system in several aspects so that the transferability of experimental results from mice to men is limited. Humanized mice represent a new tool to analyze the interaction of human immune cells under physiological conditions as far as possible, particularly to test novel therapeutic strategies. This review summarizes the impact of humanized mouse models for the investigation and treatment of allergic diseases.

Activated glycoprotein A repetitions predominant (GARP)-expressing regulatory T cells inhibit allergen-induced intestinal inflammation in humanized mice.

BACKGROUND: Recently, we developed a humanized mouse model of allergen-induced IgE-dependent gut inflammation in PBMC-engrafted immunodeficient mice. OBJECTIVE: In the present study, we wanted to investigate the role of regulatory T (Treg) cells and their activation status in this model. METHODS: Nonobese diabetic-severe combined immunodeficiency-γc(-/-) mice were injected intraperitoneally with human PBMCs from allergic donors together with the respective allergen or NaCl as control in the presence or absence of different concentrations of CD4(+)CD25(+) Treg cells of the same donor. After an additional allergen boost 1 week later, mice were challenged with the allergen rectally on day 21 and gut inflammation was monitored by a high-resolution video mini-endoscopic system evaluating translucency, granularity, fibrin production, vascularity, and stool. RESULTS: Allergen-specific human IgE in mouse sera, which was detectable only in PBMC plus allergen-treated mice, was strongly inhibited by coinjection of Treg cells at a ratio of at least 1:10. Consequently, the presence of Treg cells significantly decreased IgE-dependent allergen-induced gut inflammation after rectal allergen challenge. In addition, Treg cells reduced allergen-specific proliferation and cytokine production of recovered human CD4(+) T cells in vitro. Activation of Treg cells before injection further increased all inhibitory effects. Prevention of gut inflammation also occurred by the administration of glycoprotein A repetitions predominant, a molecule expressed by activated Treg cells, whereas its blockade completely abrogated inhibition by Treg cells. CONCLUSIONS: These results demonstrate that allergen-specific gut inflammation in human PBMC-engrafted mice can be avoided by enhancing the numbers or activity of autologous Treg cells, which is of great interest for therapeutic intervention of allergic diseases of the intestine.

Biodegradable pH-Sensitive Poly(ethylene glycol) Nanocarriers for Allergen Encapsulation and Controlled Release.

In the last decades, the number of allergic patients has increased dramatically. Allergen-specific immunotherapy (SIT) is the only available cause-oriented therapy so far. SIT reduces the allergic symptoms, but also exhibits some disadvantages; that is, it is a long-lasting procedure and severe side effects like anaphylactic shock can occur. In this work, we introduce a method to encapsulate allergens into nanoparticles to avoid severe side effects during SIT. Degradable nanocarriers combine the advantage of providing a physical barrier between the encapsulated cargo and the biological environment as well as responding to certain local stimuli (like pH) to release their cargo. This work introduces a facile strategy for the synthesis of acid-labile poly(ethylene glycol) (PEG)-macromonomers that degrade at pH 5 (physiological pH inside the endolysosome) and can be used for nanocarrier synthesis. The difunctional, water-soluble PEG dimethacrylate (PEG-acetal-DMA) macromonomers with cleavable acetal units were analyzed with 1H NMR, SEC, and MALDI-ToF-MS. Both the allergen and the macromonomers were entrapped inside liposomes as templates, which were produced by dual centrifugation (DAC). Radical polymerization of the methacrylate units inside the liposomes generated allergen-loaded PEG nanocarriers. In vitro studies demonstrated that dendritic cells (DCs) internalize the protein-loaded, nontoxic PEG-nanocarriers. Furthermore, we demonstrate by cellular antigen stimulation tests that the nanocarriers effectively shield the allergen cargo from detection by immunoglobulins on the surface of basophilic leucocytes. Uptake of nanocarriers into DCs does not lead to cell maturation; however, the internalized allergen was capable to induce T cell immune responses.

Cor a 1-reactive T cells and IgE are predominantly cross-reactive to Bet v 1 in patients with birch pollen-associated food allergy to hazelnut.

BACKGROUND: IgE- and T-cell cross-reactivity contribute to the birch pollen-food syndrome. OBJECTIVES: We performed a comprehensive analysis of T-cell cross-reactivity in primary cell cultures, facilitating the identification of allergen-specific T-cell subpopulations from individual patients. METHODS: Patients with birch pollen allergy and associated food allergy to hazelnuts, carrots, or both were analyzed for IgE cross-reactivity, T-cell responses, and T-cell cross-reactivity to recombinant Bet v 1.0101 (Bet v 1; birch), Cor a 1.0401 (Cor a 1; hazelnut), and Dau c 1.0104 (Dau c 1; carrot). A novel flow cytometry-based method using a 2-step staining process with fluorescent dyes was established to identify subpopulations of cross-reactive T cells. RESULTS: IgE-binding inhibition tests of individual sera revealed that the vast majority of Cor a 1-reactive IgE was cross-reactive to Bet v 1, whereas Bet v 1-reactive IgE was only partially inhibited by preincubation with Cor a 1. Primary stimulation of T cells with Bet v 1 or Cor a 1 resulted in a significant increase in specific responses to Cor a 1 or Bet v 1 after secondary stimulation, respectively, indicating T-cell cross-reactivity between birch and hazelnut allergens in all patients of the study cohort. Preactivation with Dau c 1 induced less pronounced effects. A novel flow cytometry-based proliferation assay identified a predominant Cor a 1/Bet v 1-cross-reactive T-cell subpopulation within highly Bet v 1/Cor a 1-responsive T cells. CONCLUSION: Analysis of primary allergen-specific T cells combined with flow cytometry-based proliferation assays facilitates investigation of allergen-specific T-cell subpopulations in subjects and might be helpful to evaluate the effect of birch-specific immunotherapy on pollen-associated food allergies.

CD4-mediated regulatory T-cell activation inhibits the development of disease in a humanized mouse model of allergic airway disease.

BACKGROUND: Based on their potency to control allergic diseases, regulatory T (Treg) cells represent a promising target for novel strategies to interfere with allergic airway inflammation. We have previously demonstrated that stimulation of the CD4 molecule on human Treg cells activates their suppressive activity in vitro and in vivo. OBJECTIVE: We sought to determine the effect of CD4-mediated Treg-cell activation on pulmonary inflammation in a humanized mouse model of allergic airway inflammation. METHODS: PBMCs obtained from donors allergic to birch pollen or from healthy donors were injected into NOD-severe combined immunodeficiency γc(-/-) mice, followed by allergen airway challenges and analysis of airway responsiveness and inflammation. For Treg-cell activation, mice were treated with the CD4-binding, lck-activating recombinant HIV-1 surface protein gp120 after sensitization prior to allergen challenge. Control experiments with CD25-depleted PBMCs were performed to evaluate the role of Treg cells. RESULTS: PBMCs from allergic donors but not from healthy donors induced airway inflammation and airway hyperresponsiveness. Treatment with gp120 prior to allergen challenge abrogated airway hyperresponsiveness and reduced the inflammatory immune response. In contrast, treatment had no effect on inflammation and airway hyperresponsiveness in mice that received CD25-depleted PBMCs, demonstrating Treg-cell dependency of disease prevention. CONCLUSION: Allergic airway inflammation can be prevented by stimulation of human Treg cells by CD4. These results suggest a clinical potential of Treg-cell activation by high-affinity CD4 ligands in allergic diseases.

Enhanced production of CCL18 by tolerogenic dendritic cells is associated with inhibition of allergic airway reactivity.

BACKGROUND: IL-10-treated dendritic cells (DCs) have been shown to inhibit T-cell responses through induction of anergy and regulatory T cells in various model systems, including allergic inflammation, but the factors being involved in this inhibition are still unclear. OBJECTIVE: This study set out to analyze such factors produced or induced by IL-10-treated DCs by using gene expression profiling and to explore their function. METHODS: CD4(+) T cells from allergic donors were stimulated with autologous monocyte-derived allergen-pulsed mature DCs or IL-10-treated DCs. After 24 hours, the transcriptional profile was analyzed by using Affymetrix technology. Results were validated by using quantitative real-time PCR, protein expression, and functional in vitro and in vivo studies. RESULTS: In CD4(+) T-cell/IL-10-treated DC cocultures the expression of several known genes, such as IL13, IL5 and OX40, was suppressed. Interestingly, there was only one factor that was strongly upregulated: the DC-derived chemokine CCL18. In vitro addition of CCL18 to cocultures of CD4(+) T cells and allergen-pulsed DCs resulted in a similar inhibition of T(H)2 cytokine production as induced by allergen-pulsed IL-10-treated DCs without exogenous CCL18, whereas T(H)1 cytokine production, IL-10 production, and proliferation were not affected. Furthermore, in a humanized mouse model of allergy using PBMC-engrafted NOD-scid-γc(-/-) mice, CCL18, but not another T(H)2-associated chemokine, CCL17, inhibited airway reactivity and lung inflammation. Chemotaxis assays revealed that CCL18 preferentially attracted regulatory T cells and, less efficiently, T(H)2 cells. CONCLUSION: These data demonstrate that CCL18 might represent a molecule of significant importance in immunoregulation and might be a therapeutic target in patients with allergic airway diseases.

Allergen-induced IgE-dependent gut inflammation in a human PBMC-engrafted murine model of allergy.

BACKGROUND: Humanized murine models comprise a new tool to analyze novel therapeutic strategies for allergic diseases of the intestine. OBJECTIVE: In this study we developed a human PBMC-engrafted murine model of allergen-driven gut inflammation and analyzed the underlying immunologic mechanisms. METHODS: Nonobese diabetic (NOD)-scid-γc(-/-) mice were injected intraperitoneally with human PBMCs from allergic donors together with the respective allergen or not. Three weeks later, mice were challenged with the allergen orally or rectally, and gut inflammation was monitored with a high-resolution video miniendoscopic system, as well as histologically. RESULTS: Using the aeroallergens birch or grass pollen as model allergens and, for some donors, also hazelnut allergen, we show that allergen-specific human IgE in murine sera and allergen-specific proliferation and cytokine production of human CD4(+) T cells recovered from spleens after 3 weeks could only be measured in mice treated with PBMCs plus allergen. Importantly, these mice had the highest endoscopic scores evaluating translucent structure, granularity, fibrin, vascularity, and stool after oral or rectal allergen challenge and a strong histologic inflammation of the colon. Analyzing the underlying mechanisms, we demonstrate that allergen-associated colitis was dependent on IgE, human IgE receptor-expressing effector cells, and the mediators histamine and platelet-activating factor. CONCLUSION: These results demonstrate that allergic gut inflammation can be induced in human PBMC-engrafted mice, allowing the investigation of pathophysiologic mechanisms of allergic diseases of the intestine and evaluation of therapeutic interventions.

Chemical modification by peroxynitrite enhances TLR4 activation of the grass pollen allergen Phl p 5.

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.