The transcription factor XBP1 in dendritic cells promotes the TH2 cell response in airway allergy.

Dendritic cells (DCs) that express T cell immunoglobulin domain molecule-4 (TIM4), a cell surface receptor for phosphatidylserine, induce T helper 2 (TH2) cell responses and allergic reactions. We elucidated the role of the transcription factor X-box-binding protein-1 (XBP1) in the induction of the TH2 cell response through its role in generating TIM4+ DCs. We found that XBP1 was required for TIM4 mRNA and protein expression in airway DCs in response to the cytokine interleukin-2 (IL-2) and that this pathway was required for TIM4 expression on DCs in response to the allergens PM2.5 and Derf1. The IL-2-XBP1-TIM4 axis in DCs contributed to Derf1/PM2.5-induced, aberrant TH2 cell responses in vivo. An interaction between the guanine nucleotide exchange factor Son of sevenless-1 (SOS1) and the GTPase RAS promoted XBP1 and TIM4 production in DCs. Targeting the XBP1-TIM4 pathway in DCs prevented or alleviated experimental airway allergy. Together, these data suggest that XBP1 is required for TH2 cell responses by inducing the development of TIM4+ DCs, which depends on the IL-2-XBP1-SOS1 axis. This signaling pathway provides potential therapeutic targets for the treatment of TH2 cell-dependent inflammation or allergic diseases.

An eosinophil-Sos1-RAS axis licenses corticosteroid resistance in patients with allergic rhinitis.

BACKGROUND: Corticosteroid resistance (CR) is a serious disadvantage in treating many chronic inflammatory conditions. Eosinophils are the main inflammation cells in allergic reactions. Environmental pollution, such as PM2.5, is associated with the pathogenesis of allergic disorders. The objective of this study is to elucidate the mechanism by which the exposure to PM2.5 confers eosinophil CR status. METHODS: Patients with allergic rhinitis were recruited and assigned to corticosteroid sensitive (CS) and CR groups. Eosinophils were purified from nasal lavage fluids collected from patients with allergic rhinitis. A murine AR mouse model was developed with dust mite allergens and PM2.5 as the sensitization reagents. RESULTS: CR status was detected in about 60% eosinophil collected in patients with AR. Upon exposure to eosinophil activators, CS eosinophils released a large quantity of mediators, which was suppressed by the presence of steroids in the culture. CR eosinophils demonstrated resistance to steroidal therapy. RAS activation levels in eosinophils were higher in CR eosinophils than in CS eosinophils. Higher expression of the Son of sevenless-1 (Sos1) was detected in CR eosinophils, which formed a complex with RAS and glucocorticoidreceptor-α in CR eosinophils to prevent the binding between steroids and glucocorticoidreceptor-α. The presence of an Sos1 inhibitor dissociated glucocorticoid receptor-α from RAS/Sos1 complex, that restored the sensitivity to steroids in eosinophils. Administering the Sos1 inhibitor effectively attenuated the experimental allergic rhinitis. CONCLUSIONS: CR status was detected in approximately 1/3 eosinophils sampled from patients with allergic rhinitis. Sos1 was instrumental in the development and perseverance of CR in eosinophils. Sos1 inhibition restored sensitivity to steroids in CR eosinophils, which effectively reduced experimental allergic rhinitis.

Modulating Oxidative Stress in B Cells Promotes Immunotherapy in Food Allergy.

Allergen-specific immunotherapy (SIT) is the mainstay in the treatment of allergic diseases; its therapeutic efficacy is to be improved. Bacterial flagellin (FGN) has immune regulatory functions. This study investigates the role of FGN in promoting immunotherapy efficacy through modulating oxidative stress in regulatory B cells (Bregs). Blood samples were collected from patients with food allergy (FA) and healthy control (HC) subjects. CD19+ CD5+ Bregs were purified from blood samples by flow cytometry cell sorting. A murine FA model was developed with ovalbumin as the specific antigen. The results showed that peripheral Bregs from FA patients showed lower TLR5-related signals and higher apoptotic activities. The peripheral Breg frequency was negatively correlated with serum FGN levels in FA patients. Exposure to a specific antigen in culture induced antigen-specific Breg apoptosis that was counteracted by the presence of FGN. FGN diminished specific antigen-induced oxidative stress in Bregs. The STAT3/MAPKp38/NF-κB signal pathway was involved in the FGN/TLR5 signal-promoted superoxide dismutase expression in Bregs. Administration of FGN promotes the SIT efficacy in suppressing experimental FA. In summary, administration of FGN promotes SIT efficacy on FA, suggesting that the combination of FGN and SIT can be a novel therapy that has the translational potential to be employed in the treatment of allergic diseases.

Glutaminolysis is required in maintaining immune regulatory functions in B cells.

IL-10-expressing regulatory B cells (B10 cells) are dysfunctional in patients with many immune disorders. The underlying mechanism remains to be further elucidated. Glutamine is an essential nutrient for cell metabolism. This study aims to elucidate the role of glutaminolysis in maintaining the immune regulatory capacity in B10 cells. Peripheral blood samples were collected from 50 patients with allergic rhinitis and 50 healthy control subjects. B cells were isolated from blood samples by cell sorting with flow cytometry. The role of glutaminolysis in regulating B10 cell activities was assessed by immunological and biochemical approaches. The results showed that B cells from patients with allergic rhinitis expressed low levels of the transporter of glutamine and neutral amino acid. Glutaminolysis was required in the IL-10 expression in B cells. The glutamine catabolism was required in B10 cell generation. The mTOR activation mediated the glutaminolysis-associated B10 cell induction, and the suppression of the B cell glycogen synthase kinase-3 (GSK3) activation. GSK3 activation suppressed IL-10 expression in B cells. Inhibition of GSK3 enhanced IL-10 expression in B cells and alleviated experimental allergic rhinitis by generating immune competent type 1 regulatory T cells.

Intestinal Epithelial Cell-Derived CD83 Contributes to Regulatory T-Cell Generation and Inhibition of Food Allergy.

The mechanism of generation of antigen-specific regulatory T cells (Treg) is not fully understood yet. This study aimed to investigate the role of intestinal epithelial cell (IEC)-derived CD83 in the Treg generation in the intestine. In this study, the role of CD83 in the generation of Tregs was assessed in a cell-culture model and a food allergy (FA) mouse model. We found that mouse IECs expressed CD83; its levels were markedly lower in sensitized mice. Mice with CD83-deficient IECs failed to induce Tregs in the intestine. CD83 promoted the transforming growth factor-ß-inducible early gene 1 (TIEG1) expression in CD4+ T cells. Toll-like receptor 4 (TLR4)/myeloid differentiation protein-2 (MD-2) complex mediated the effects of CD83 on the expression of TIEG1. Activation of the CD83/TLR4/MD-2/TIEG1 promoted the Treg generation. Concomitant administration of CD83 and specific antigens, but not either one alone, efficiently inhibited experimental FA via inducing the Treg generation in the intestine. In Conclusion, IEC expresses CD83 that is low in sensitized mice. Concomitant administration of CD83 and specific antigens efficiently inhibits FA in a murine model via inducing Tregs in the intestine. The data suggest that CD83 has translation potential in the treatment of FA.

Chimeric antigen-guiding extracellular vesicles eliminate antigen-specific Th2 cells in subjects with food allergy.

BACKGROUND: Antigen (Ag)-specific T helper (Th)2 cells play a central role in food allergy (FA) pathogenesis. Methods can be used to eliminate Ag-specific Th2 cells that are currently lacking. This study aims to eliminate the Ag-specific Th2 cells with a novel nanoparticle, the mEV (modified extracellular vesicles, that carry a chimeric antigen peptide, MHC II and caspase 3) in a murine FA model. METHODS: mEVs were generated by exposing dendritic cells (DC) to ovalbumin (OVA, a specific Ag) and recombinant caspase 3 (Casp3) in the culture overnight. Exosomes were purified from culture supernatant by the magnetic antibody approach. A murine FA model was developed with OVA as the specific Ag. RESULTS: Purified mEVs had the molecular markers of extracellular vesicle, CD81, CD63, and CD9, cleaved Casp3 and MHC II/OVA complexes. mEVs specifically bound to the surface of Ag-specific CD4+ T cells, induced Ag-specific CD4+ T cell apoptosis both in vitro and in vivo as well as increased regulatory T cells in the intestinal tissues. Administration of mEV efficiently suppressed experimental FA. CONCLUSIONS: mEVs carry Ag/MHC II complexes and Casp3, that can induce Ag-specific Th2 cell apoptosis. Administration of mEV can efficiently suppress experimental FA. The results suggest that the mEVs have the translational potential to be used in the treatment of FA and other allergic diseases.

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.

Role of FcγRI in Antigen-Dependent Eosinophil Activation in Patients With Allergic Rhinitis.

BACKGROUND: The eosinophil (Eo) activation is a crucial factor evoking allergic rhinitis (AR) attacks; factors; the mechanism of triggering Eo activation remains to be further investigated. The interaction of antigen (Ag) and antibody plays a critical role in evoking allergy attacks. This study aims to elucidate the role of FcγRI, the high affinity receptor of IgG, in the Ag-mediated Eo activation. METHODS: Nasal lavage fluids (NLF) were collected from AR patients and healthy control (HC) subjects. Eos were isolated by flow cytometry cell sorting and analyzed by pertinent immunological approaches. RESULTS: Eos composed more than 60% of the cellular components in AR NLF. Exposure to specific Ags (sAgs) in the culture triggered Eos to release inflammatory mediators. High levels of FcγRI were detected on the surface of AR NLF Eos. Exposure to lipopolysaccharide markedly increased the FcγRI expression in naive Eos, which could be bound by Ag-specific IgG (sIgG) to form complexes on the surface of Eos; this made Eos at the sensitized status. Eos bore with the sIgG/FcγRI complexes could be activated upon exposure to sIgG in the culture; these Eos can be designated as Ag-specific Eos. Passive transfer of Ag-specific Eos resulted in profound AR response in mice upon sAg challenge. Depletion of FcγRI on Eos efficiently abolished AR response in mice. CONCLUSIONS: AR Eos express high levels FcγRI, that can be bound by sIgG to make Eos sensitized. Re-exposure to specific Ags can activate the sensitized Eos.

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.

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.

Inhibition of Bcl2L12 Attenuates Eosinophilia-Related Inflammation in the Heart.

Background: The eosinophilic inflammation plays a critical role in myocarditis (Mcd); its underlying mechanism remains to be further elucidated. This study aims to investigate the role of Bcl2-like protein 12 (Bcl2L12) in inducing the defects of apoptosis in eosinophils (Eos) of the heart tissues. Methods: Human explant heart samples were collected. Eosinophilia and myocarditis (Mcd)-like inflammation were induced in the mouse heart by immunizing with murine cardiac α-myosin heavy chain (MyHCα) peptides. Results: Markedly more Eos were observed in heart tissues from patients with Mcd than those from patients with dilated cardiomyopathy. Eos isolated from Mcd hearts showed the signs of apoptosis defects. The Eo counts in the Mcd heart tissues were positively correlated with the Bcl2L12 expression in Eos isolated from the heart tissues. Exposure to interleukin 5 in the culture induced the expression of Bcl2L12 in Eos. Bcl2L12 bound c-Myc, the transcription factor of Fas ligand (FasL), to prevent c-Myc from binding to the FasL promoter, to restrict the FasL gene transcription in Eos. Inhibition of Bcl2L12 prevented the induction of eosinophilia and Mcd-like inflammation in the mouse heart. Conclusions: The Bcl2L12 expression contributes to apoptosis defects in Eos of the Mcd heart. Blocking Bcl2L12 prevents the eosinophilia induction and alleviates Mcd-like inflammation in mice.

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.

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.

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.

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.