Allergen specific immunotherapy regulates macrophage property in the airways.

BACKGROUND: Allergen specific immunotherapy (AIT) has been widely used in allergy clinics. The therapeutic effects of it are to be improved. Macrophages occupy the largest proportion of airway immune cells. The aim of this study is to measure the effects of nasal instillation AIT (nAIT) on airway allergy by regulating macrophage functions. METHODS: An airway allergy mouse model was established with the ovalbumin-alum protocol. nAIT was conducted for mice with airway allergy through nasal instillation. The effects of nAIT were compared with subcutaneous injection AIT (SCIT) and sublingual AIT (SLIT). RESULTS: Mice with airway allergy showed the airway allergic response, including lung inflammation, airway hyper responsiveness, serum specific IgE, increase in the amounts of eosinophil peroxidase, mouse mast cell protease-1, and Th2 cytokines in bronchoalveolar lavage fluid. nAIT had a much better therapeutic effect on the airway allergic response than SCIT and SLIT. Mechanistically, we observed better absorption of allergen in macrophages, better production of IL-10 by macrophages, and better immune suppressive functions in macrophages in mice received nAIT than SCIT and SLIT. CONCLUSIONS: The nAIT has a much better therapeutic effect on suppressing the airway allergic response, in which macrophages play a critical role.

Galectin-9 in synergy with NF-κB inhibition restores immune regulatory capability in dendritic cells of subjects with food allergy.

The pathogenesis of immune tolerance disruption is not fully understood. Galectin-9 (Gal9) has immune regulatory functions. The objective of the present study is to assess the role of Gal9 in maintaining immune tolerance. Blood and intestinal biopsies were taken from patients with food allergy (FA). The status of tolerogenic dendritic cells (tDC) and type 1 regulatory T cells (Tr1 cells) in the samples was evaluated and used as representative parameters of immune tolerance. An FA mouse model was established to assess the role of Gal9 in maintaining immune tolerance. We found that peripheral CD11c+ CD5+ CD1d+ tDC frequency was significantly lower in FA patients as compared to health control (HC) subjects. There was no significant change in CD11c+ DC frequency between the FA group and the HC group. The expression of IL-10 in peripheral tDCs was lower in the FA group than that in the HC group. A positive correlation was detected between the serum levels of IL-10 and Gal9. The expression of Gal9 was observed in intestinal biopsies, which was positively correlated with the serum levels of Gal9 as well as serum IL-10 levels. Peripheral Tr1 cells had lower frequencies in the FA group than in the non-FA (Con) group. tDCs demonstrated the ability to generate Tr1 cells, which was weaker in the FA group as compared with the Con group. Exposure of FA tDCs to Gal9 in culture restored the ability to generate Tr1 cells. In summary, the lower frequency of tDC and Tr1 cell of FA patients was associated with the levels of Gal9. The presence of Gal9 restored the capacity of tDC to generate Tr1 cells.

Quercetin inhibits the activity and function of dendritic cells through the TLR4/IRAK4/NF-κB signalling pathway.

Introduction: To investigate the inhibitory effect of quercetin (QUE) on dendritic cells (DCs) through the toll-like receptor 4/interleukin-1 receptor-associated kinase 4/nuclear factor kappa-B (TLR4/IRAK4/NF-κB) signalling pathway. Material and methods: CCK-8 and apoptosis assays were performed to determine the optimal concentration and action time of QUE to inhibit DCs. Protein extracts from treated DCs were used for Western blotting experiments to determine the relative expression levels of TLR4, IRAK4, and NF-κB p65 proteins. Changes in the ratio of CD86 and CD11c positive cells on the DCs surface were detected using flow cytometry. The molecular docking technique was used to analyse the binding site and free energy of QUE and IRAK4. Results: CCK-8 and apoptosis assays suggested that QUE inhibited the activity and function of DCs in a time-dose-dependent manner. The results of Western blotting suggested that the relative expression levels of TLR4, IRAK4, and NF-κB p65 proteins were increased in the lipopolysaccharide (LPS) group compared with the normal control group, and the relative expression of the above proteins was decreased after treatment with QUE and IRAK4-IN-4. The results of flow cytometry suggested that LPS increased the expression of CD86 and CD11c on the surface of DCs, and QUE and IRAK4-IN-4 decreased the expression of CD86 and CD11c induced by LPS. Molecular docking results showed that the binding sites of QUE and IRAK4 were stable, with the minimum binding energies comparable to that of IRAK4-IN-4. Conclusions: Quercetin may inhibit the activity and function of DCs through the TLR4/IRAK4/NF-κB signalling pathway, and IRAK4 may be its target.

5-HT is associated with the dysfunction of regulating T cells in patients with allergic rhinitis.

The dysfunction of regulating T lymphocytes (Treg) is associated with the pathogenesis of many diseases. 5-hydroxytryptamine (5-HT) is capable of interacting with immune cells. The objective of the present study is to shed light on the role of 5-HT in regulating Treg activities. Blood samples were collected from patients with perennial allergic rhinitis (AR). Tregs were isolated from blood samples by magnetic cell sorting. The levels of 5-HT and other cytokines were determined by enzyme-linked immunosorbent assay. The results showed that serum 5-HT levels in patients with AR were higher than in healthy control (HC) subjects. A positive correlation was identified in the data between 5-HT concentrations and AR-related cytokine concentrations in the serum. A negative correlation was found between serum levels of 5-HT and the peripheral frequency of Treg. Exposure to 5-HT enhanced the expression of IL-6 and IL-21 in dendritic cells (DC). Co-culture of 5-HT-primed DCs with Tregs led to the conversion of Th17 cells. STAT3 blockade efficiently abolished the 5-HT-associated conversion of Th17 cells from Tregs. In summary, patients with AR exhibited higher serum concentrations of 5-HT. 5-HT-primed DCs could convert Tregs to Th17 cells.

FcγRI plays a critical role in patients with ulcerative colitis relapse.

Ulcerative colitis (UC) is a disease that frequently relapses and affects more than 0.1% general population; the underlying mechanism is poorly understood. Published data show that polymorphonuclear neutrophils (PMN) contribute to the pathogenesis of UC. This study aims to identify antigen (Ag)-specific PMNs and investigate their role in UC relapse. In this study, the correlation between PMN activities and UC relapse was assessed in a group of UC patients. A UC mouse model was developed to expand the findings of UC patient study. The results showed that a positive correlation was detected between the high PMN activities and the food Ag-specific IgG amounts in colon biopsies of UC patients. UC patient-derived Ag-specific PMNs could be activated upon exposure to food specific Ag. The Ag/FcγRI complexes were detected on the surface of PMNs in UC patients. Re-exposure of sensitized PMNs to specific Ag triggered PMN activation and induced UC-like inflammation in the mouse colon. We conclude that FcγRI plays a critical role in UC relapse. Inhibition of FcγRI can efficiently inhibits experimental UC.

Epithelial cell-derived CD83 restores immune tolerance in the airway mucosa by inducing regulatory T-cell differentiation.

The mechanism of generation of regulatory T cells (Treg) remains incompletely understood. Recent studies show that CD83 has immune regulatory functions. This study aims to investigate the role of epithelial cell-derived CD83 in the restoration of immune tolerance in the airway mucosa by inducing the Treg differentiation. In this study, CD83 and ovalbumin (OVA)-carrying exosomes were generated from airway epithelial cells. An airway allergy mouse model was developed to test the role of CD83/OVA-carrying exosomes in the suppression of airway allergy by inducing Treg generation. We observed that mouse airway epithelial cells expressed CD83 that could be up-regulated by CD40 ligand. The CD83 deficiency in epithelial cells retarded the Treg generation in the airway mucosa. CD83 up-regulated transforming growth factor-ß-inducible early gene 1 expression in CD4+ T cells to promote Foxp3 expression. Exposure of primed CD4+ T cells to CD83/OVA-carrying exosomes promoted antigen-specific Treg generation. Administration of CD83/OVA-carrying exosomes inhibited experimental airway allergic response. In summary, airway epithelial cells express CD83 that is required in the Treg differentiation in the airway mucosa. Administration of CD83/OVA-carrying exosomes can inhibit airway allergy that has the translation potential in the treatment of airway allergic disorders.

Twist1 sustains the apoptosis resistance in eosinophils in nasal mucosa of allergic rhinitis.

Eosinophils (Eos) are the canonical effector cells in allergic rhinitis (AR) and many inflammatory diseases. The mechanism of eosinophilia occurring in the lesion sites is not fully understood yet. Twist1 protein (Twist, in short) is an apoptosis inhibitor that also has immune regulatory functions. This study aims to investigate the role of Twist in the pathogenesis of eosinophilia in AR. In this study, surgically removed human nasal mucosal samples were obtained from patients with chronic sinusitis and nasal polyps with AR (the AR group) or without AR (the nAR group). Eos were isolated from the samples by flow cytometry. We found that abundant Eos were obtained from the surgically removed nasal mucosa tissues of both nAR and AR groups. Significantly higher Ras activation was detected in AR Eos than that in nAR Eos. Ras activation was associated with the apoptosis resistance in AR Eos. The Twist (an apoptosis inhibitor) expression was higher in AR Eos, which was positively correlated with the Ras activation status. The sensitization to IgG induced Twist expression in Eos, in which Ras activated the MAPK-HIF-1α pathway, the latter promoted the Twist gene transcription. Twist bound Rac GTPase activating protein-1 to sustain the Ras activation in Eos. Ras activation sustained the apoptosis resistance in Eos. In conclusion, high Ras activation was detected in the AR nasal mucosal tissue-isolated Eos. IgG-sensitization induced Ras activation and Twist expression in Eos, that conferred Eos the apoptosis resistance.

Specific antigen-guiding exosomes inhibit food allergies by inducing regulatory T cells.

The therapies for food allergy (FA) need to be improved. The generation of inducible regulatory T cells (Tregs) can support immune tolerance in the body. This study aims to suppress experimental FA by inducing Tregs through the employment of modified exosomes (mExosomes). In this study, mExosomes were prepared by incubating dendritic cells with interleukin (IL)-2 and ovalbumin (OVA, used as a specific antigen) in the culture. Exosomes were purified from culture supernatant and used as the mExosomes. A murine FA model was developed to test the effects of mExosomes on the generation of Tregs in the mouse intestinal tissues and inhibiting FA. The results showed that mExosomes, which carried IL-2 and a complex of OVA peptide-major histocompatibility complex class II on the surface of exosomes, bound to OVA-specific CD4+ T cells and induced CD4+ T cells to differentiate into Tregs. In the FA mouse intestinal tissues, we found low IL-2 levels that were positively correlated with the number of Tregs. Depletion of IL-2 in mice prevented the generation of Tregs. The levels of peroxisome proliferator-activated receptor-γ were increased in the FA intestinal tissues with inhibited IL-2 production. Administration of mExosomes induced Tregs in the intestinal tissues and efficiently suppressed FA in mice. We conclude that the mExosomes can suppress FA in mice through inducing Tregs. The data suggest that the mExosomes have translational potential in the treatment of FA and other allergic disorders.

Chimeric specific antigen epitope-carrying dendritic cells induce interleukin-17(+) regulatory T cells to suppress food allergy.

BACKGROUND: The on-purpose-modulated dendritic cells (DCs) have shown charming effects on restoring immune regulatory functions in subjects with immune diseases. OBJECTIVE: This study aims to construct DCs carrying chimerical antigen (Ag) peptides (CAP-DCs) to induce interleukin (IL)-17+ inducible Tregs (iTregs) to alleviate food allergy (FA) in a murine model. METHODS: In this study, we constructed CAP-DCs. The CAP is a fusion protein, consisting of a segment of recombinant scFv of anti-DEC205 antibody and an ovalbumin (OVA) epitope (IC). A murine OVA-FA model was developed to test the effects of CAP-DCs on suppressing the allergic response in the intestine. RESULTS: The CAP-DCs are characterized as that a complex of scFv-IC is presented on the surface of the cells, moderately express CD80 and CD86 as well as IL-6, IL-23, transforming growth factor (TGF)-ß and CCR9. After being passively transferred with CAP-DCs or injection of scFv-IC, Ag-specific IL-17+ Foxp3+ iTregs were induced in the intestinal lamina propria of FA mice. The iTregs showed immune suppressive effects on Ag-specific Th2 response. FA mice were adoptively transferred with the CAP-DCs or scFv-IC injection, which resulted in a significant decrease in the number of Ag-specific Th2 cells and suppression of FA response in an Ag-specific manner. CONCLUSIONS AND CLINICAL RELEVANCE: CAP-DCs can ameliorate FA response by inducing Ag-specific IL-17+ Foxp3+ iTregs and suppressing Ag-specific Th2 response. To generate CAP-DCs has the translational potential in the treatment of FA.

Cold stress promotes IL-33 expression in intestinal epithelial cells to facilitate food allergy development.

BACKGROUND: The causative factors and pathogenesis of food allergy (FA) is not fully understood yet. Cold stress (CS) occurs frequently in human life that influences physiological activities in the body. In this study, we aimed to investigate the chronic CS (CS) effects on promoting the expression of IL-33 in intestinal epithelial cells. METHODS: CS was carried out by placing mice at 4 °C for 1 h daily for 7 consecutive days. We developed a mouse model used to test the effects of CS on the FA development. RESULTS: We found that, similar to conventional FA mouse model, CS induced the core body temperature to drop markedly in mice, increased intestinal epithelial barrier permeability and facilitated FA development. CS promoted interleukin (IL)-33 expression in intestinal epithelial cells through the adrenocorticotropic hormone (ACTH)/cortisol axis and via inducing the Il33 promoter methylation. CS facilitated the FA development in mice, that could be blocked by depletion of IL-33 expression in intestinal epithelial cells. CONCLUSIONS: CS induces IL-33 expression in intestinal epithelial cells to promote Th2 polarization in the intestinal tissues and facilitates FA development.

Exosomes carry IL-10 and antigen/MHC II complexes to induce antigen-specific oral tolerance.

BACKGROUND: It is known that the immune tolerance can be naturally established in the intestine, while the mechanism by which the immune tolerance development in the intestine is not fully understood yet. Vasoactive intestinal peptides (VIP) has the immune regulatory functions. This study aims to investigate the role of VIP in the immune tolerance development in the intestine. METHODS: Intestinal epithelial cell (IEC)-derived exosomes were prepared. The exosomes carried IL-10 and antigen/MHC II complexes. VIP-deficient (VIPd) mice and wild type mice were employed to test the role of VIP in the development of immune tolerance in the intestine. RESULTS: VIPd mice failed to induce type 1 regulatory T cells (Tr1 cells) in the intestine and retarded the establishment of antigen (Ag)-specific immune tolerance. Exposure to VIP in the culture induced IL-10 expression in intestinal epithelial cells (IECs). Exosomes derived from ovalbumin (OVA, used as a specific Ag)/VIP-primed IECs carried IL-10 and OVA/MHC II complexes; these exosomes were designated IL10CARs (IL-10/chimeric antigen receptor-carrying exosomes). IL10CARs could recognize OVA-specific CD4+ T cells and converted OVA-specific CD4± T cells to OVA-specific Tr1 cells. Administration of IL10CARs suppressed experimental food allergy. CONCLUSIONS: The data show that IL10CARs are capable of suppressing experimental FA by inducing antigen-specific Tr1 cells, which has the translation potential for FA treatment.

CARsomes inhibit airway allergic inflammation in mice by inducing antigen-specific Th2 cell apoptosis.

BACKGROUND: Skewed T helper (Th)2 response plays a crucial role in the pathogenesis of allergic diseases. The therapeutic efficacy for allergic diseases is unsatisfactory currently. This study aims to regulate the skewed Th2 response with CARsomes. METHODS: The CARsome consisted of an epitope of Dermatophagoides farina-1 (Derf1), a segment of the anti-DEC205 antibody, the scFv, and an open reading frame of perforin. This fusion protein binds to DEC205 molecule on the surface of exosomes derived from dendritic cells (DC). The effects of CARsome on inducing antigen (Ag)-specific Th2 cell apoptosis were assessed both in vivo and in vitro. RESULTS: Exposure to CARsomes in the culture induced Ag-specific Th2 cell apoptosis. Injection of CARsomes through the vein puncture also induced Ag-specific Th2 cell apoptosis in the lungs of sensitized mice. CARsomes could induce Ag-specific regulatory T cells. Administration of CARsomes efficiently inhibited experimental allergic airway inflammation. CONCLUSIONS: The CARsomes can inhibit allergic airway inflammation by inducing Ag-specific Th2 cell apoptosis and induce Ag-specific regulatory T cells. The data suggest that CARsomes have the translational potential to be used to treat allergic airway inflammation.

Bcl2L12 Contributes to Th2-Biased Inflammation in the Intestinal Mucosa by Regulating CD4+ T Cell Activities.

The Th2-biased inflammation and immune deregulation play a critical role in the pathogenesis of ulcerative colitis (UC). Recent studies indicate that the Bcl2-like protein 12 (Bcl2L12) is associated with immune deregulation of UC. This study aims to investigate the role of Bcl2L12 in the induction of aberrant Th2-biased inflammation. In this study, peripheral blood samples were collected from patients with inflammatory bowel disease. The Th2 cell activities were analyzed by flow cytometry, real-time quantitative RT-PCR, and Western blotting. Mice with Bcl2L12-knockout CD4+ T cells were used in the experiments. The results showed that the expression of Bcl2L12 was detected in peripheral CD4+ T cells, which was significantly higher in UC patients than in healthy subjects. A positive correlation between the expression of Bcl2L12 and Th2 cytokines was detected in CD4+ T cells from UC patients. Naive CD4+ T cells with Bcl2L12 overexpression were prone to differentiate into Th2 cells. Mice with Bcl2L12 deficiency failed to induce the Th2-biased inflammation in the intestine. Bcl2L12 bound GATA3 to form a complex to enhance the binding between GATA3 and the Il4 promoter to enhance the expression of IL-4 in CD4+ T cells. CD4+ T cells with Bcl2L12 overexpression were resistant to apoptosis. In conclusion, the Bcl2L12 is a critical factor in the induction of aberrant Th2 polarization by upregulating Th2 responses and downregulating Th2 cell apoptosis. Bcl2L12 may be a novel therapeutic target in the management of the disorders with Th2-biased inflammation.

A20 Restores Impaired Intestinal Permeability and Inhibits Th2 Response in Mice with Colitis.

BACKGROUND/AIMS: The etiology of inflammatory bowel disease is multifactorial and still obscure. The protective role of ubiquitin E3 ligase A20 (A20) in colitis needs to be further elucidated. This study aimed to investigate whether A20 exogenous administration restored impaired intestinal permeability and inhibited T helper (Th)2 response in mice with colitis. METHODS: The effect of A20 overexpression in colonic mucosa on epithelial barrier function and T cell differentiation was evaluated in mice with dextran sulfate sodium (DSS)-induced chronic colitis. RESULTS: A20 rectal treatment alleviated DSS-induced chronic colitis and restored impaired intestinal permeability. Oral challenge with 2% DSS elicited a Th2-type response in mice with colitis, and A20 rectal treatment inhibited CD4+ interleukin (IL)-4+ T cell differentiation and proliferation. In addition, the RNA expressions of Th2-related costimulatory molecular T-cell immunoglobulin and mucin domain (TIM)-1 and IL-4 were suppressed, while thrombospondin (TSP)-1 and interferon (IFN)-γ expressions were upregulated, after A20 rectal administration. CONCLUSION: A20 rectal treatment restores impaired intestinal permeability and inhibits activated Th2 cell response in mice with colitis.

Thrombospondin-1 (TSP1)-producing B cells restore antigen (Ag)-specific immune tolerance in an allergic environment.

Restoration of the antigen (Ag)-specific immune tolerance in an allergic environment is refractory. B cells are involved in immune regulation. Whether B cells facilitate the generation of Ag-specific immune tolerance in an allergic environment requires further investigation. This paper aims to elucidate the mechanism by which B cells restore the Ag-specific immune tolerance in an allergic environment. In this study, a B cell-deficient mouse model was created by injecting an anti-CD20 antibody. The frequency of tolerogenic dendritic cell (TolDC) was assessed by flow cytometry. The levels of cytokines were determined by enzyme-linked immunosorbent assay. The expression of thrombospondin-1 (TSP1) was assessed by quantitative real-time RT-PCR, Western blotting, and methylation-specific PCR. The results showed that B cells were required in the generation of the TGF-ß-producing TolDCs in mice. B cell-derived TSP1 converted the latent TGF-ß to the active TGF-ß in DCs, which generated TGF-ß-producing TolDCs. Exposure to IL-13 inhibited the expression of TSP1 in B cells by enhancing the TSP1 gene DNA methylation. Treating food allergy mice with Ag-specific immunotherapy and IL-13 antagonists restored the generation of TolDCs and enhanced the effect of specific immunotherapy. In conclusion, B cells play a critical role in the restoration of specific immune tolerance in an allergic environment. Blocking IL-13 in an allergic environment facilitated the generation of TolDCs and enhanced the therapeutic effect of immunotherapy.

Allergen-specific immune response suppresses interleukin 10 expression in B cells via increasing micro-RNA-17-92 cluster.

Interleukin (IL)-10-expressing B cells play a critical role in the immune homeostasis in the body; its regulation has not been fully understood. Micro-RNA (miR)-17-92 cluster has strong regulation in the immunity. This study tests a hypothesis that miR-17-92 cluster suppresses IL-10 expression in B cells. In this study, peripheral B cells were collected from patients with allergic rhinitis (AR). The B cells were treated with specific allergens, dust mite extracts, in the culture. The expressions of miR-17-92 cluster and IL-10 in the culture were assessed by real-time quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. The results showed that the levels of miR-19a, but not the rest of the 5 members (miR-17, miR-18a, miR-19b, miR-20a, and miR-92a), were significantly higher in peripheral B cells from AR patients as than in B cells from healthy participants. Exposure of B cells from AR patients to specific allergen, dust mite extracts, significantly increased the levels if miR-19a and suppressed the expression of IL-10 in B cells. The levels of histone deacetylase 11 and acetylated H3K9 were higher, and the RNA polymerase II and c-Maf (the IL-10 transcription factor) were lower, at the IL-10 promoter locus. In conclusion, miR-19a mediates the allergen-specific immune response-decreased IL-10 expression in B cells.