CD38+ B cells affect immunotherapy for allergic rhinitis.

BACKGROUND: Allergen-specific immunotherapy (AIT) is the mainstay in the treatment of allergic diseases, but the therapeutic effects of AIT need to be improved. CD38+ B cells are an immune cell fraction involved in the pathogenesis of allergic diseases as well as in immune regulation. OBJECTIVE: We sought to elucidate the role of antigen-specific CD38+ B cells in AIT. METHODS: An analysis was carried out on AIT results of 48 patients with perennial allergic rhinitis (AR), among which peripheral blood immune cells were analyzed by flow cytometry; serum cytokine levels were determined by ELISA. An AR murine model was developed to test the role of CD38+ B cells in AIT. RESULTS: A fraction of antigen-specific CD38+ B cell was detected in AR patients. CD38+ B-cell frequency was negatively correlated with the therapeutic effects of AIT. A negative correlation was detected between the CD38+ B-cell frequency and regulatory T-cell frequency in AR patients treated with AIT. Exposure to specific antigens induced CD38+ B cells to produce IL-6, that converted Treg cells to TH17 cells. Coadministration of anti-CD38 antibody significantly promoted the therapeutic effects of AIT. CONCLUSIONS: Antigen-specific CD38+ B cells compromise AIT effects by producing IL-6 to convert regulatory T cells to TH17 cells. Inhibition of CD38+ B cells promotes the effects of AIT.

Livin in synergy with Ras induces and sustains corticosteroid resistance in the airway mucosa.

Rationale: Corticosteroid resistance (CR) seriously affects the therapeutic effects of steroids on many chronic inflammatory disorders, including airway allergy. The mechanism of CR development is unclear. Recent research indicates that livin, an apoptosis inhibitor, is associated with the regulation in cell activities. This study investigates the role of livin in the inducing and sustaining CR in the airway mucosa. Methods: Nasal epithelial cells (NECs) were isolated from surgically removed nasal mucosal tissues of patients with allergic rhinitis (AR) and nasal polyps with or without CR. Differentially expressed genes in NECs were analyzed by the RNA sequencing. A CR mouse model was developed to test the role of livin in CR development. Results: The results showed that NECs of AR patients with CR expressed high levels of livin, that was positively correlated with the thymic stromal lymphopoietin (TSLP) expression and the high Ras activation status in NECs. Livin and Ras activation mutually potentiating each other in the inducing and sustaining the TSLP expression in NECs. TSLP induced eosinophils and neutrophils to express glucocorticoid receptor-ß (GRß). Eosinophils and neutrophils with high CRß expression were resistant to corticosteroids. Depletion of livin or inhibition of TSLP markedly attenuated CR and airway allergy. Conclusions: Livin facilitates CR development in the airways by promoting TSLP expression in epithelial cells and the GRß expression in eosinophils and neutrophils. Depletion of livin or inhibiting TSLP attenuates CR development and inhibits airway allergy, this has the translational potential to be used in the treatment of airway allergy.

Interaction between Ras and Bcl2L12 in B cells suppresses IL-10 expression.

The pathogenesis of recurrent tonsillitis is to be further investigated. B cell-derived interleukin (IL)-10 plays a critical role in immune regulation. Ras activation plays an important role in cancer and many immune disorders. This study aims to investigate the role of Ras activation in down regulating IL-10 expression in tonsillar B cells. Surgically removed tonsil tissues were collected from patients with recurrent acute tonsillar inflammation; B cells were isolated from the tonsillar tissues by flow cytometry sorting to be analyzed by the Ras-specific enzyme-linked immunosorbent assay and pertinent immunological approaches. We found that, compared to peripheral B cells (pBC), B cells isolated from the tonsillar tissues with recurrent inflammation (tBC) showed higher Ras activation, lower IL-10 expression and higher Bcl2L12 expression. Bcl2L12 formed a complex with GAP (GTPase activating protein) to prevent Ras from deactivating. The Ras activation triggered the MAPK/Sp1 pathway to promote the Bcl2L12 expression in B cells. Bcl2L12 prevented the IL-10 expression in tBCs, that was counteracted by inhibition of Ras or the Ras signal transduction pathway. In conclusion, Bcl2L12 interacts with Ras activation to compromise immune tolerance in the tonsils by inhibiting the IL-10 expression in tBCs. Inhibition of Bcl2L12 can restore the IL-10 expression in tBCs.

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.

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.

Integrin αvß6 cooperates with resiquimod to restore antigen-specific immune tolerance in airway allergy.

BACKGROUND: Integrin αvß6 can convert the transforming growth factor (TGF)-ß precursor to the mature form. Resiquimod (R848) can generate TGF-ß-producing regulatory T cells (Treg). Thus, to concurrent administration of specific antigen and R848 may generate antigen-specific Tregs, that is expected to restore immune tolerance in subjects with airway allergic diseases (AAD). METHODS: A bio-nanoparticle, designated Rexo, containing an antigen/MHC II complex and R848, was naturally assembled in dendritic cells, that was released as an exosome. An AAD mouse model was developed used to test the effects of Rexo on restoring the immune tolerance in the airways. RESULTS: Exposure to R848 failed to induce Tregs in the ß6-deficient mouse airway tissues, that were successfully induced in wild type mice. The results were validated inin vitro experiments. R848 activated the TLR7/MyD88/p38 signal pathway to increase the αvß6 levels in CD4+ T cells, the αvß6 then converted the TGF-ß precursor to its mature form, and thus, induced Treg generation. Administration of Rexo restored the antigen-specific immune tolerance in the airways manifesting efficiently suppressing experimental AAD by inducing antigen-specific Tregs in the airways and inhibiting antigen-specific Th2 response. CONCLUSIONS: Rexos can inhibit experimental AAD via inducing antigen-specific Tregs to restore immune tolerance in the airway tissues, suggesting that Rexos have the translational potential to be used in the treatment of AAD.

Bcl2 like protine-12 (Bcl2L12) facilitates experimental airway allergic inflammation by inducing autocrine eotaxin in eosinophils.

BACKGROUND: The pathogenesis of airway allergic disorders (AAD) needs to be further investigated. Eosinophils (Eos) are the canonical effector cells in AAD attacks. Bcl2 like protein-12 (Bcl2L12) is an apoptosis inhibitor and an immune regulator. Eos have the defects of apoptosis. This study aims to investigate the role of Bcl2L12 in the AAD pathogenesis by regulating Eo activities. METHODS: Human nasal lavage fluids (NLF) and mouse bronchoalveolar lavage fluids (BALF) was collected. Eos in NLF and BALF were analyzed by flow cytometry. A murine AAD model was developed with ovalbumin as a specific antigen. RESULTS: We found that Eos isolated from NLF or BALF of AAD subjects expressed high levels of Bcl2L12 and showed defects of apoptosis. The Bcl2L12 expression in Eos was positively correlated with the AAD response. High lipopolysaccharide levels were detected in the AAD airways, that promoted the Bcl2L12 expression in Eos. Bcl2L12 mediated the LPS-induced autocrine eotaxin 1 expression in Eos through activating the MAPK p38/STAT6/NF-κB signal pathway. Depletion of Bcl2L12 in Eos suppressed experimental AAD in mice. CONCLUSIONS: AAD Eos express high levels of Bcl2L12, the latter is associated with AAD response by regulating the autocrine eotaxin 1 in Eos. Depletion of Bcl2L12 in Eos attenuates experimental AAD, suggesting that to suppress the Bcl2L12 Eos has the translational potential in the treatment of AAD.

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.

Identification of antigen-specific neutrophils in the tonsils with recurrent acute inflammation.

The pathogenesis of recurrent acute tonsillitis (Rtn) is to be further investigated. Polymorphonuclear neutrophils (PMN) often associate with the pathogenesis of acute and chronic inflammation. This study aims to identify the antigen-specific PMNs (sPMNs) isolated from the tonsillar tissues with recurrent acute inflammation. In this study, CD66b+ PMNs were isolated from surgically removed tonsils (40 tonsils were from 20 Rtn patients; 24 tonsils were from 12 tonsil tumour patients) by flow cytometry cell sorting. sPMNs were identified through immunological approaches. We found that compared with the control tonsil samples (from marginal non-tumour tissues of tonsil cancer), Rtn samples showed higher PMN frequency, higher levels of myeloperoxidase (MPO) and neutrophil elastase (NE), in which positive correlation was detected between the inflammatory scores in the Rtn tissues and PMN counts (r = .7352; p = .0002), or MPO (r = .6565, p = .0017), or NE (r = .6687, p = .0013). Upon exposure to tonsillar tissue protein extracts in the culture, a portion of Rtn PMNs was activated and released inflammatory mediators. A complex of tonsillar tissue-specific IgG and FcγRI was observed on the surface of Rtn PMNs; these PMNs could specifically recognize the Rtn tissue extracts and were designated the tonsillar antigen-specific PMNs (sPMNs). A signal transduction pathway of mitogen-activated protein kinase (MAPK)-nuclear factor of T cell activation (NFAT) was activated in sPMNs after exposure to Rtn tissue extracts. In summary, a fraction of sPMN in the Rtn tonsillar tissues was identified and characterized. The sPMNs can be activated upon exposure to tonsil-specific antigens. These sPMNs may contribute to the Rtn pathogenesis.

Inhibition of livin overcomes radioresistance in nasopharyngeal carcinoma cells.

BACKGROUND AND AIMS: Radiotherapy is one of the major remedies for the treatment of cancer, including nasopharyngeal carcinoma (NPC). Radioresistance occurs very often in target cells that is a large drawback in cancer treated with radiotherapy. Livin involves the over-growth of cancer cells. This study aims to investigate the role of livin in the radioresistance formation in NPC cells. METHODS: NPC cell lines were exposed to small doses of irradiation to establish a cell model of radioresistance, in which the role of livin in the development of radioresistance was evaluated. RESULTS: The expression of livin was observed in NPC cells, which was significantly increased after exposing to small doses of irradiation. A negative correlation was detected between livin and Fas expression in NPC cells. Livin formed a complex with heat shock factor-1 (HSF1, the transcription factor of Fas) in NPC cells after irradiation, which sped up ubiquitination of HSF1. Livin was involved in suppressing Fas expression in NPC cells with radioresistance. Exposure to livin inhibitors prevented radioresistance development and overcame the established radioresistance in NPC cells. CONCLUSIONS: Livin expression in NPC cells plays a critical role in the development of radioresistance. Depletion of livin increases the sensitiveness of NPC cells to irradiation. Target therapy against livin may have the translational potential for the treatment of NPC.

A20-OVA Nanoparticles Inhibit Allergic Asthma in a Murine Model.

The skewed T helper (Th) 2 response plays a critical role in the pathogenesis of allergic asthma. Regulatory T (Treg) cells and the regulatory cytokines are required in maintaining the homeostasis in the body. This study aims to determine the effects of a poly(lactic-co-glycolic) acid (PLGA)-ovalbumin (OVA)+A20 (a ubiquitin E3 ligase) nanovaccine on inhibiting allergic asthma in a murine model. In this study, A20 and OVA (a model antigen) were encapsulated into PLGA to be a nanovaccine (PLGA-OVA+A20). An allergic asthma murine model was developed with OVA as the specific antigen to test the role of PLGA-OVA+A20 nanovaccine in maintaining the immune homeostasis in the airway tissues. The results showed that PLGA-OVA+A20 nanovaccine inhibited the asthma responses in mice by suppressing Th2 inflammatory responses, promoting the generation of Treg cells in the airway tissues. We conclude that the PLGA-OVA+A20 nanovaccine has a marked inhibitory effect on the airway allergic response in sensitized mice by significantly promoting the generation of Treg cell and IL-10. The data suggest that PLGA-OVA+A20 has translational potential in the treatment of allergic asthma.

Soluble CD83 alleviates experimental allergic rhinitis through modulating antigen-specific Th2 cell property.

Background and aims: Dysfunction of the immune regulatory system plays a role in the pathogenesis of allergic rhinitis (AR). The underlying mechanism needs to be further investigated. Published data indicate that soluble CD83 (sCD83) has immune regulatory activities. This study aims to investigate the role of sCD83 in the alleviation of experimental AR. Methods: Peripheral blood samples were obtained from AR patients. Serum levels of sCD83 were determined by enzyme-linked immunosorbent assay. A murine AR model was developed to test the effects of sCD83 on suppressing experimental AR. Results: We found that serum levels of sCD83 in the AR group were lower than that in the healthy control group. A negative correlation was identified between the serum sCD83 levels and the frequency of T helper-2 (Th2) cells. The low serum sCD83 levels were also associated with the Bcl2L12 expression in antigen-specific Th2 cells. Exposure to sCD83 enhanced the responsiveness of antigen-specific Th2 cells to apoptosis inducers via suppressing the Bcl2L12 expression. Administration of sCD83 efficiently suppressed experimental AR. Conclusions: sCD83 contributes to immune homeostasis by regulating CD4+ T cell activities. Administration of sCD83 may have translational potential for the treatment of AR or other allergic diseases.

Regulating Bcl2L12 expression in mast cells inhibits food allergy.

Rationale: Mast cells play a crucial role in allergic diseases. Yet, the regulation of mast cell bioactivities is not fully understood. This study aims to elucidate the role of B cell lymphoma 2 like protein 12 (Bcl2L12), one of the anti-apoptosis proteins, in regulating mast cell apoptosis. Methods: A food allergy (FA) mouse model was developed to establish mast cell over population in the intestinal tissue. Either compound 48/80 (C48/80) or specific antigens were used to activate mast cells in the intestinal mucosa. Results: After treating with C48/80, apoptosis was induced in mast cells of the intestine of naive control mice, but not in FA mice. The expression of Fas ligand (FasL) was lower in the mast cells of FA mice. Interleukin (IL)-5 was responsible for the suppression of FasL by upregulating the expression of Bcl2L12 in mast cells. Bcl2L12 prevented c-Myc, the major transcription factor of FasL, from binding the FasL promoter to inhibit the expression of FasL in mast cells. Inhibition of Bcl2L12 restored the apoptosis machinery of mast cells in the FA mouse intestine. Conclusions: The apoptosis machinery in mast cells is impaired in an allergic environment. Inhibition of Bcl2L12 restores the apoptosis machinery in mast cells in the FA mouse intestine.

Survivin induces defects in apoptosis in eosinophils in intestine with food allergy.

Survivin is an anti-apoptosis protein that may be associated with the development of eosinophilia; the latter is associated with the pathogenesis of many immune disorders. Here we report that less apoptotic eosinophils (Eos) were induced in those isolated from mice suffering from food allergy (FA) than those from naive mice after treating with cisplatin in vitro. Exposure to cisplatin induced more Fas ligand (FasL) expression in Eos isolated from naive mice than in those of FA mouse. Survivin was detected in the intestinal tissue extracts in much higher amounts in the FA group than in the naive group. Immunohistochemistry showed that epithelial cells were the major source of survivin in the intestine. Exposure to IL-4 or IL-13 up-regulated the expression of survivin in intestinal epithelial cells. Survivin interfered with the expression of FasL in Eos. Inhibition of survivin attenuated the eosinophilia-related inflammation in the intestine. In conclusion, intestinal epithelial cell-produced survivin induced defects in apoptosis in Eos to contribute to eosinophilia in the intestine. Inhibition of survivin can suppress the eosinophilia-related intestinal inflammation. The data suggest that survivin may be a novel target for the treatment of FA.

Anti-inflammation action of xanthones from Swertia chirayita by regulating COX-2/NF-κB/MAPKs/Akt signaling pathways in RAW 264.7 macrophage cells.

BACKGROUND: Swertia chirayita, has been commonly used under the name "Zang-yin-chen" for the treatment of liver infections, inflammation, abdominal pain, and bacterial infection in traditional Tibetan medicine. However, the bioactive components with anti-inflammatory activities and underlying mechanisms remain poorly evaluated. STUDY DESIGN/METHODS: Repeated column chromatography yielded two main xanthones from petroleum ether (PE) and ethyl acetate fractions of whole plants of S. chirayita, and their structures were determined as bellidifolin (1) and swerchirin (2) on the basis of spectroscopic data and literature analysis. The anti-inflammatory activities and mechanisms of anti-inflammation of these two isolated xanthones were determined via enzyme-linked immunosorbent assay (ELISA) and western blot in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophages in vitro. RESULTS: Anti-inflammation assay demonstrated that 1 and 2 inhibit the production of the pro-inflammatory cytokines interleukin-6 (IL-6) and TNF-α in LPS-stimulated RAW 264.7 macrophages. Xanthone 1 also potently inhibited the production of prostaglandin E2 (PGE2) by suppressing the protein expression of cyclooxygenase-2 (COX-2) in LPS-stimulated RAW 264.7 macrophages. Western blot showed that the phosphorylation of c-Jun N-terminal kinases (JNK), extracellular signal-regulated kinase (ERK), and p38 MAPKs were remarkably attenuated by 1 in a concentration-dependent manner. Particularly, Compound 1 suppressed the phosphorylation of the inhibitor κB kinase-ß (IKK-ß), Akt, and p65 subunit of nuclear factor-kappaB (NF-κB). CONCLUSION: The potent suppressive effects of 1 from S. chirayita on inflammatory mediators by blocking the expression of COX-2 and phosphorylation of Akt, IKK-ß, MAPK and NF-κB, activation in LPS-stimulated macrophages suggest that 1 can be a preventive therapeutic candidate for the management of inflammatory-mediated immune disorders.

Interleukin-5 induces apoptotic defects in CD4+ T cells of patients with allergic rhinitis.

T helper (Th)2 polarization plays an important role in the pathogenesis of allergic diseases; the underlying mechanism remains to be further investigated. B cell lymphoma protein-2 like protein-12 (Bcl2L12) has the anti-apoptotic function. This study aims to elucidate the contribution of Bcl2L12 to Th2 polarization in patients with allergic rhinitis (AR). In this study, human CD4+ T cells were isolated from blood samples collected from AR patients and healthy control (HC) subjects. The immune response profiles of CD4+ T cells were analyzed by immunologic approaches. The results showed that AR CD4+ T cells (CD4+ T cells collected from AR patients) showed defects of apoptosis. The expression of FasL in AR CD4+ T cells was lower than that of HC CD4+ T cells. Serum IL-5 levels were negatively correlated with the expression of FasL in AR CD4+ T cells. Exposure of CD4+ T cells to IL-5 in the culture suppressed the expression of FasL and increased the expression of Bcl2L12. IL-5 increased the levels of Bcl2L12 in CD4+ T cells, the latter bound to the FasL promoter to prevent FasL gene transcription. Inhibition of Bcl2L12 restored the apoptosis machinery in AR CD4+ T cells. In conclusion, overexpression of Bcl2L12 in CD4+ T cells compromises the apoptosis machinery; the latter can be restored by inhibition of Bcl2L12. BcL2L12 in CD4+ T cells may be a novel target for the treatment of AR and other allergic disorders.