Activation of invariant natural killer T cells in regional lymph nodes as new antigen‐specific immunotherapy via induction of interleukin‐21 and interferon‐γ

Invariant natural killer T (iNKT) cells play important immunoregulatory functions in allergen‐induced airway hyperresponsiveness and inflammation. To clarify the role of iNKT cells in allergic rhinitis (AR), we generated bone marrow‐derived dendritic cells (BMDCs), which were pulsed by ovalbumin (OVA) and α‐galactosylceramide (OVA/α‐GalCer‐BMDCs) and administered into the oral submucosa of OVA‐sensitized mice before nasal challenge. Nasal symptoms, level of OVA‐specific immunoglobulin (IgE), and T helper type 2 (Th2) cytokine production in cervical lymph nodes (CLNs) were significantly ameliorated in wild‐type (WT) mice treated with OVA/α‐GalCer‐BMDCs, but not in WT mice treated with OVA‐BMDCs. These anti‐allergic effects were not observed in Jα18^–/– recipients that lack iNKT cells, even after similar treatment with OVA/α‐GalCer‐BMDCs in an adoptive transfer study with CD4⁺ T cells and B cells from OVA‐sensitized WT mice. In WT recipients of OVA/α‐GalCer‐BMDCs, the number of interleukin (IL)‐21‐producing iNKT cells increased significantly and the Th1/Th2 balance shifted towards the Th1 dominant state. Treatment with anti‐IL‐21 and anti‐interferon (IFN)‐γ antibodies abrogated these anti‐allergic effects in mice treated with α‐GalCer/OVA‐BMDCs. These results suggest that activation of iNKT cells in regional lymph nodes induces anti‐allergic effects through production of IL‐21 or IFN‐γ, and that these effects are enhanced by simultaneous stimulation with antigen. Thus, iNKT cells might be a useful target in development of new treatment strategies for AR.     

Topical CTLA4-Ig suppresses ongoing mucosal immune response in presensitized murine model of allergic rhinitis.

Allergic rhinitis is thought to be mediated by CD4+ T cells producing Th2-associated cytokines. Optimal Ag-specific T-cell activation requires the engagement of T-cell receptor with antigen (Ag) in the context of MHC, and the engagement of appropriate costimulatory molecules. One of the most well-characterized costimulatory pathways is the interaction of B7/CD28-CTLA4 molecules. Recent studies have suggested that the costimulatory pathway may influence the development of Th2 immune responses. The objective of this study was the examination of the role of B7/CD28-CTLA4 costimulatory pathway in the pathogenesis of ovalbumin (OVA)-induced immune response in presensitized murine model of allergic rhinitis. Systemically presensitized BALB/c mice significantly developed Ag-induced early phase nasal symptoms, nasal hyperresponsiveness to histamine, nasal eosinophilia, serum levels of OVA- specific IgE and Th2-associated cytokines following repeated topical Ag challenges. Topical administration of CTLA4-Ig during nasal challenges inhibited Ag-induced nasal symptoms and histamine hyperresponsiveness. We also found a significant reduction in nasal lavage eosinophilia and serum levels of OVA-specific IgE. Furthermore, CTLA4-Ig treatment significantly decreased interleukin (IL)-4 content in nasal tissue, while there was no significant change in IL-5 or IFN-gamma levels. These results suggest that B7/CD28-CTLA4 costimulatory pathway mediates the development of ongoing Th2 immune responses and plays a major role in regulating allergic disease, such as allergic rhinitis.     

Nanomedicine-mediated prevention of inflammatory monocytes infiltration ameliorate ovalbumin-induced allergic rhinitis in mouse model

Abstract

Th2 immune cells infiltration into nasal mucosa is one of the characters of allergic rhinitis (AR). We aimed to explore whether inhibition of Th2 immune cells infiltration would attenuate AR progression. AR mouse model was established by i.p. injection of ovalbumin (OVA). The infiltrated immune cells into nasal lavage fluid were detected by flow cytometry. Cytokine concentration in serum was determined by ELISA. AR mice symptoms were indicated by the number of sneezing and nasal rubbing events. In AR mice, CCL2 expression levels and CD45⁺CD11b⁺Ly6C^hi inflammatory monocytes cells significantly increased as compared with control mice. CCL2 siRNA encapsulated nanoparticles (NP_siCCL2) prevent CCL2 expression and inflammatory monocytes infiltration in AR mice. NP_siCCL2 treatment dramatically decreased the number of sneezing and nasal rubbing events in AR mice. Moreover, NP_siCCL2 treatment attenuated serum OVA-specific IgE, OVA-specific IgG1 and histamine levels. Mechanically, NP_siCCL2 treatment attenuates AR symptoms via inhibiting Th2 cytokine (IL-4, IL-5 and IL-13) production. Nanomedicine-mediated prevention of inflammatory monocytes infiltration ameliorates ovalbumin-induced allergic rhinitis in mouse model.     

Antigen‐specific airway IL‐33 production depends on FcγR‐mediated incorporation of the antigen by alveolar macrophages in sensitized mice

Although interleukin (IL)‐33 is a candidate for the aggravation of asthma, the mechanisms underlying antigen‐specific IL‐33 production in the lung are unclear. Therefore, we analysed the mechanisms in mice. Intra‐tracheal administration of ovalbumin (OVA) evoked increases in IL‐33 and IL‐33 mRNA in the lungs of both non‐sensitized and OVA‐sensitized mice, and the increases in the sensitized mice were significantly higher than in the non‐sensitized mice. However, intra‐tracheal administration of bovine serum albumin did not increase the IL‐33 level in the OVA‐sensitized mice. Depletion of neither mast cells/basophils nor CD4⁺ cells abolished the OVA‐induced IL‐33 production in sensitized mice, suggesting that the antigen recognition leading to the IL‐33 production was not related with either antigen‐specific IgE‐bearing mast cells/basophils or memory CD4⁺ Th2 cells. When a fluorogenic substrate‐labelled OVA (DQ‐OVA) was intra‐tracheally administered, the lung cells of sensitized mice incorporated more DQ‐OVA than those of non‐sensitized mice. The lung cells incorporating DQ‐OVA included B‐cells and alveolar macrophages. The allergic IL‐33 production was significantly reduced by treatment with anti‐FcγRII/III mAb. Depletion of alveolar macrophages by clodronate liposomes significantly suppressed the allergic IL‐33 production, whereas depletion of B‐cells by anti‐CD20 mAb did not. These results suggest that the administered OVA in the lung bound antigen‐specific IgG Ab, and then alveolar macrophages incorporated the immune complex through FcγRII/III on the cell surface, resulting in IL‐33 production in sensitized mice. The mechanisms underlying the antigen‐specific IL‐33 production may aid in development of new pharmacotherapies.     

SIRT1 attenuates murine allergic rhinitis by downregulated HMGB 1/TLR4 pathway

Conventional allergic rhinitis (AR) treatments have limitations due to the lack of safety and complete cure strategy. We evaluated the effects of silent information regulator 1 (SIRT1), a multifunctional molecule involved in a variety of inflammatory pathways, on murine AR model. Ovalbumin (OVA)‐induced murine model was constructed, and recombinant SIRT1 was administered into the nostril continuously. The expression of SIRT1 was measured at mRNA and protein levels, and the allergic symptoms were evaluated. Protein levels of OVA‐specific IgE, leukotriene C4 (LTC4), eosinophil cation protein (ECP), prostaglandin D2 (PGD2), as well as different inflammatory cytokine mediators in the serum and nasal lavage fluid (NLF), were assessed by ELISA. The effects of SIRT1 on human primary nasal epithelial cells challenged with tumour necrosis factor (TNF)‐α were also evaluated by investigating the HMGB1/TLR4 signalling pathway. Administration of SIRT1 significantly alleviated OVA‐induced AR symptoms with lower numbers of sneezing and nasal rubbing events, decreased levels of OVA‐specific IgE, LTC4, ECP, PGD2, less inflammatory cells and downregulated levels of Th2 type cytokines. SIRT1 also reduced the genes of HMGB1/TLR4 signalling pathway in the murine model and cultured human nasal epithelial cells. Expression of SIRT1 is impaired in OVA‐induced AR model. The administration of SIRT1 alleviates the allergic symptoms of mice, regulates the production of pro‐inflammatory mediators predominantly produced by Th2 cells in AR and attenuates expressions of proteins relevant to HMGB1/TLR4 signalling pathway. All the results showed that SIRT1 is promising as a therapeutic agent of AR.     

Interleukin-4-independent production of Th2 cytokines by nasal lymphocytes and nasal eosinophilia in murine allergic rhinitis

BACKGROUND: Interleukin (IL)-4 is believed to play an important role in the atopic pathogenesis. However, the precise role of IL-4 in the in vivo initiation of allergic rhinitis is not fully understood. We have recently found that BALB/c mice sensitized intranasally with Schistosoma mansoni egg antigen (SEA) mount a Th2 response that initiates allergic rhinitis. Thus, we sought to determine the role of IL-4 in the initiation of allergic rhinitis in vivo with this model. METHODS: IL-4 gene-deficient (IL-4 -/-) BALB/c and wild-type (IL-4 +/+) control mice were sensitized by intranasal SEA administration, and their immunologic responses were examined both in vivo and in vitro. RESULTS: IL-4 +/+ mice sensitized with SEA displayed significantly higher titers of SEA-specific IgG1 and IgE antibodies than IL-4-/- mice, while the latter produced significantly more SEA-specific IgG2a. Antigen-stimulated nasal lymphocytes from SEA-sensitized IL-4 -/- and IL-4 +/+ mice produced similar amounts of IL-5 and IL-10, but neither produced IFN-gamma. Furthermore, the severity of nasal eosinophilia was similar in both groups. CONCLUSIONS: These results indicate that although IL-4 is necessary for the production of Th2-associated antibodies--in particular, IgE--it is not required for either the production of the Th2-associated cytokines IL-5 and IL-10, or the induction of nasal eosinophilia.     

Pathogenesis of rhinitis

Rhinitis is a heterogeneous condition that has been associated with inflammatory responses as in allergic rhinitis but can also occur in the absence of inflammation such as in so‐called idiopathic (previously ‘vasomotor’) rhinitis. Allergic rhinitis affects approximately one in four of the population of westernized countries and is characterized by typical symptoms of nasal itching, sneezing, watery discharge and congestion. The intention of this review is to illustrate key concepts of the pathogenesis of rhinitis. Imbalance in innate and adaptive immunity together with environmental factors is likely to play major roles. In allergic rhinitis, initial allergen exposure and sensitization involves antigen‐presenting cells, T and B lymphocytes and results in the generation of allergen‐specific T cells and allergen‐specific IgE antibodies. On re‐exposure to relevant allergens, cross‐linking of IgE on mast cells results in the release of mediators of hypersensitivity such as histamine and immediate nasal symptoms. Within hours, there is an infiltration by inflammatory cells, particularly Th2 T lymphocytes, eosinophils and basophils into nasal mucosal tissue that results in the late‐phase allergic response. Evidence for nasal priming and whether or not remodelling may be a feature of allergic rhinitis will be reviewed. The occurrence of so‐called local allergic rhinitis in the absence of systemic IgE will be discussed. Non‐allergic (non‐IgE‐mediated) rhinitis will be considered in the context of inflammatory and non‐inflammatory disorders.     

Fcγ receptor‐mediated antigen uptake by lung DC contributes to allergic airway hyper‐responsiveness and inflammation

During asthma, lung DC capture and process antigens to initiate and maintain allergic Th2 cell responses to inhaled allergens. The aim of the study was to investigate whether allergen‐specific IgG, generated during sensitization, can potentiate the acute airway inflammation through Fcγ receptor (FcγR)‐mediated antigen uptake and enhance antigen presentation resulting in augmented T‐cell proliferation. We examined the impact of antigen presentation and T‐cell stimulation on allergic airway hyperresponsiveness and inflammation using transgenic and gene‐deficient mice. Both airway inflammation and eosinophilia in bronchoalveolar lavage fluid were markedly reduced in sensitized and challenged FcγR‐deficient mice. Lung DC of WT, but not FcγR‐deficient mice, induced increased antigen‐specific CD4⁺ T‐cell proliferation when pulsed with anti‐OVA IgG immune complexes. Intranasal application of anti‐OVA IgG immune complexes resulted in enhanced airway inflammation, eosinophilia and Th2 cytokine release, mediated through enhanced antigen‐specific T‐cell proliferation in vivo. Finally, antigen‐specific IgG in the serum of sensitized mice led to a significant increase of antigen‐specific CD4⁺ T‐cell proliferation induced by WT, but not FcγR‐deficient, lung DC. We conclude that FcγR‐mediated enhanced antigen presentation and T‐cell stimulation by lung DC has a significant impact on inflammatory responses following allergen challenge in asthma.     

The central role of CD30L/CD30 interactions in allergic rhinitis pathogenesis in mice

CD30 ligand (CD30L) plays an important role in the amplification and/or activation of effector CD4(+) T cells, irrespective of Th cell subset. To examine the role of CD30L in allergic rhinitis, we evaluated an OVA model of allergic rhinitis in CD30L knock out (KO) mice on a BALB/c background sensitized with OVA. Symptoms of allergic rhinitis such as eosinophil infiltration into the nasal mucosa were drastically diminished in OVA-sensitized CD30L KO mice following intranasal challenge with OVA. The levels of OVA-specific IgE in the sera and the Th2 response in nasopharynx-associated lymphoid tissues and cervical LNs of CD30L KO mice were significantly lower than those of WT mice following intranasal challenge with OVA. Intranasal administration of CD30-Ig during the effector phase with OVA significantly prevented the development of allergic rhinitis in WT mice. These results suggest that CD30L plays an important role in allergic rhinitis and that the inhibition of CD30L/CD30 signaling might be useful as a novel biological therapy for allergic rhinitis.     

Obesity promotes prolonged ovalbumin‐induced airway inflammation modulating T helper type 1 (Th1), Th2 and Th17 immune responses in BALB/c mice

Clinical and epidemiological studies indicate that obesity affects the development and phenotype of asthma by inducing inflammatory mechanisms in addition to eosinophilic inflammation. The aim of this study was to assess the effect of obesity on allergic airway inflammation and T helper type 2 (Th2) immune responses using an experimental model of asthma in BALB/c mice. Mice fed a high‐fat diet (HFD) for 10 weeks were sensitized and challenged with ovalbumin (OVA), and analyses were performed at 24 and 48 h after the last OVA challenge. Obesity induced an increase of inducible nitric oxide synthase (iNOS)‐expressing macrophages and neutrophils which peaked at 48 h after the last OVA challenge, and was associated with higher levels of interleukin (IL)‐4, IL‐9, IL‐17A, leptin and interferon (IFN)‐γ in the lungs. Higher goblet cell hyperplasia was associated with elevated mast cell influx into the lungs and trachea in the obese allergic mice. In contrast, early eosinophil influx and lower levels of IL‐25, thymic stromal lymphopoietin (TSLP), CCL11 and OVA‐specific immunoglobulin (IgE) were observed in the obese allergic mice in comparison to non‐obese allergic mice. Moreover, obese mice showed higher numbers of mast cells regardless of OVA challenge. These results indicate that obesity affects allergic airway inflammation through mechanisms involving mast cell influx and the release of TSLP and IL‐25, which favoured a delayed immune response with an exacerbated Th1, Th2 and Th17 profile. In this scenario, an intense mixed inflammatory granulocyte influx, classically activated macrophage accumulation and intense mucus production may contribute to a refractory therapeutic response and exacerbate asthma severity.     

Allergen-specific regulation of allergic rhinitis in mice by intranasal exposure to IgG1 monoclonal antibody Fab fragments against pathogenic allergen

Fab fragments (Fabs) have the ability to bind to specific antigens but lack the Fc portion for binding to receptors on immune and inflammatory cells that play a critical role in allergic diseases. In the present study, we investigated whether Fabs of an allergen-specific IgG1 monoclonal antibody (mAb) inhibited allergic rhinitis in mice. BALB/c mice sensitized by intraperitoneal injections of ovalbumin (OVA) plus alum on days 0 and 14 were intranasally challenged with OVA on days 28–30, and 35. Fabs prepared by the digestion of an anti-OVA IgG1 mAb (O1–10) with papain were also intranasally administered 15 min before each OVA challenge. The results showed that treatment with O1–10 Fabs significantly suppressed the sneezing frequency, associated with decrease of OVA-specific IgE in the serum and infiltration by mast cells in the nasal mucosa seen following the fourth antigenic challenge; additionally, the level of mouse mast cell protease-1, a marker of mast cell activation, in serum was decreased. Furthermore, infiltration of eosinophils and goblet cell hyperplasia in the nasal mucosa at the fourth challenge were inhibited by treatment with O1–10 Fabs. In conclusion, these results suggest that intranasal exposure to Fabs of a pathogenic antigen-specific IgG1 mAb may be effective in regulating allergic rhinitis through allergen capture by Fabs in the nasal mucosa before the interaction of the intact antibody and allergen.     

Bovine milk fat enriched in conjugated linoleic and vaccenic acids attenuates allergic airway disease in mice

Background It has been argued that a reduction in the Western diet of anti‐inflammatory unsaturated lipids, such as n‐3 polyunsaturated fatty acids, has contributed to the increase in the frequency and severity of allergic diseases. Objective We investigated whether feeding milk fat enriched in conjugated linoleic acid and vaccenic acids (VAs) (‘enriched’ milk fat), produced by supplementing the diet of pasture‐fed cows with fish and sunflower oil, will prevent development of allergic airway responses. Methods C57BL/6 mice were fed a control diet containing soybean oil and diets supplemented with milk lipids. They were sensitized by intraperitoneal injection of ovalbumin (OVA) on days 14 and 28, and challenged intranasally with OVA on day 42. Bronchoalveolar lavage fluid, lung tissues and serum samples were collected 6 days after the intranasal challenge. Results Feeding of enriched milk fat led to marked suppression of airway inflammation as evidenced by reductions in eosinophilia and lymphocytosis in the airways, compared with feeding of normal milk fat and control diet. Enriched milk fat significantly reduced circulating allergen‐specific IgE and IgG1 levels, together with reductions in bronchoalveolar lavage fluid of IL‐5 and CCL11. Treatment significantly inhibited changes in the airway including airway epithelial cell hypertrophy, goblet cell metaplasia and mucus hypersecretion. The two major components of enriched milk fat, cis‐9, trans‐11 conjugated linoleic acid and VA, inhibited airway inflammation when fed together to mice, whereas alone they were not effective. Conclusion Milk fat enriched in conjugated linoleic and VAs suppresses inflammation and changes to the airways in an animal model of allergic airway disease.     

Intravenous immunoglobulin attenuates airway hyperresponsiveness in a murine model of allergic asthma

Background Intravenous immunoglobulin (IVIG) has potent anti‐inflammatory and immune‐modulating properties. IVIG has been utilized as a steroid‐sparing agent in severe asthma, but the results of clinical trials have been conflicting. Objective To determine whether IVIG is able to attenuate bronchial reactivity, pulmonary inflammation and T cell function using a murine model of allergic airways disease. Methods BALB/c or C57BL/6 mice were sensitized to ovalbumin (OVA) or a phosphate‐buffered saline control using local nasal sensitization, and then received five intranasal challenges on days 28–32 before sacrifice. Mice were treated intraperitoneally with either IVIG (1–2 g/kg) or equivalent human serum albumin 24 h before the first OVA challenge. Bronchial reactivity to methacholine was examined using the FlexiVent small animal ventilator. We evaluated pulmonary histology, mRNA from lung digests for T‐helper type 2 (Th2)‐related genes and bronchoalveolar lavage for cell counts and cytokines. Splenocytes were utilized to study OVA‐induced cell proliferation, cytokine production and dendritic cell maturation. Results IVIG markedly attenuated the perivascular and peribronchial pulmonary inflammation, and decreased bronchial hyperresponsiveness to methacholine. IVIG treatment of splenocytes from sensitized animals diminished cellular proliferation to OVA, whereas IVIG treatment in vivo markedly attenuated OVA‐driven splenocyte proliferation. This is accompanied by diminished IL‐13 and TNF‐α levels in splenocyte culture, decreased expression of Jagged‐1, increased Delta‐4 and decreased GATA‐3 mRNA levels, signs that IVIG has suppressed the expected Th2 response that accompanies repeated allergen exposure. Increased regulatory T cells were found in draining pulmonary lymph nodes in IVIG‐treated mice but not in controls. Conclusions and Clinical Relevance IVIG was effective in ameliorating allergic airway disease in our model. IVIG may be a promising adjunct therapy requiring further study for patients with severe asthma. Cite this as: G. N. Kaufman, A. H. Massoud, S. Audusseau, A.‐A. Banville‐Langelier, Y. Wang, J. Guay, J. A. Garellek, W. Mourad, C. A. Piccirillo, C. McCusker and B. D. Mazer, Clinical & Experimental Allergy, 2011 (41) 718–728.     

Toll‐like receptor 4 agonists adsorbed to aluminium hydroxide adjuvant attenuate ovalbumin‐specific allergic airway disease: role of MyD88 adaptor molecule and interleukin‐12/interferon‐γ axis

Background Epidemiological and experimental data suggest that bacterial lipopolysaccharides (LPS) can either protect from or exacerbate allergic asthma. Lipopolysaccharides trigger immune responses through toll‐like receptor 4 (TLR4) that in turn activates two major signalling pathways via either MyD88 or TRIF adaptor proteins. The LPS is a pro‐Type 1 T helper cells (Th1) adjuvant while aluminium hydroxide (alum) is a strong Type 2 T helper cells (Th2) adjuvant, but the effect of the mixing of both adjuvants on the development of lung allergy has not been investigated. Objective We determined whether natural (LPS) or synthetic (ER‐803022) TLR4 agonists adsorbed onto alum adjuvant affect allergen sensitization and development of airway allergic disease. To dissect LPS‐induced molecular pathways, we used TLR4‐, MyD88‐, TRIF‐, or IL‐12/IFN‐γ‐deficient mice. Methods Mice were sensitized with subcutaneous injections of ovalbumin (OVA) with or without TLR4 agonists co‐adsorbed onto alum and challenged with intranasally with OVA. The development of allergic lung disease was evaluated 24 h after last OVA challenge. Results Sensitization with OVA plus LPS co‐adsorbed onto alum impaired in dose‐dependent manner OVA‐induced Th2‐mediated allergic responses such as airway eosinophilia, type‐2 cytokines secretion, airway hyper‐reactivity, mucus hyper production and serum levels of IgE or IgG1 anaphylactic antibodies. Although the levels of IgG2a, Th1‐affiliated isotype increased, investigation into the lung‐specific effects revealed that LPS did not induce a Th1 pattern of inflammation. Lipopolysaccharides impaired the development of Th2 immunity, signaling via TLR4 and MyD88 molecules and via the IL‐12/IFN‐γ axis, but not through TRIF pathway. Moreover, the synthetic TLR4 agonists that proved to have a less systemic inflammatory response than LPS also protected against allergic asthma development. Conclusion Toll‐like receptor 4 agonists co‐adsorbed with allergen onto alum down‐modulate allergic lung disease and prevent the development of polarized T cell‐mediated airway inflammation.     

IgE/antigen‐mediated enhancement of IgE production is a mechanism underlying the exacerbation of airway inflammation and remodelling in mice

IgE is known to enhance some antibody responses to specific antigens, but whether this contributes to allergic asthma remains unclear. We have previously found that repeated antigen challenges in mice sensitized with antigen‐specific IgE monoclonal antibody (mAb) exacerbated airway inflammation and remodelling accompanied by increased levels of endogenous antigen‐specific IgE and IgG1. Here, we investigated whether IgE/antigen‐mediated enhancement of endogenous IgE production contributes to the exacerbation of airway inflammation and remodelling. BALB/c mice passively sensitized with ovalbumin (OVA) ‐specific IgE mAb were challenged with OVA intratracheally seven times; anti‐IgE mAb was intraperitoneally administered 1 day before the fourth challenge. Treatment with anti‐IgE mAb inhibited the increased level of endogenous OVA‐specific IgE in serum, but not OVA‐specific IgG1, and a biphasic increase in airway resistance at the fourth challenge. Furthermore, a biphasic increase in airway resistance, airway hyper‐responsiveness to methacholine, OVA‐specific IgE and IgG1 production, and infiltrations by neutrophils and eosinophils in the lungs at the seventh challenge were suppressed by treatment; airway remodelling, such as goblet cell hyperplasia and sub‐epithelial fibrosis, was also reduced. In addition, the production of interleukin‐17A, interleukin‐33 and CXCL1 in the lungs related to these IgE‐mediated responses was decreased by treatment. Collectively, we found that the mechanism leading to the exacerbation of allergic asthma is closely related to IgE/antigen‐mediated enhancement of IgE production, suggesting that this may create a vicious circle leading to the chronic status in asthmatic patients having levels of antigen‐specific IgE ready to form complexes with antigen.     

Conditioned polarized Caco-2 cell monolayers allow to discriminate for the ability of gut-derived microorganisms to modulate permeability and antigen-induced basophil degranulation

Background Food allergy is a common allergic disorder – especially in early childhood. The avoidance of the allergenic food is the only available method to prevent further reactions in sensitized patients. A better understanding of the immunologic mechanisms involved in this reaction would help to develop therapeutic approaches applicable to the prevention of food allergy. Objective To establish a multi‐cell in vitro model of sensitized intestinal epithelium that mimics the intestinal epithelial barrier to study the capacity of probiotic microorganisms to modulate permeability, translocation and immunoreactivity of ovalbumin (OVA) used as a model antigen. Methods Polarized Caco‐2 cell monolayers were conditioned by basolateral basophils and used to examine apical to basolateral transport of OVA by ELISA. Activation of basophils with translocated OVA was measured by β‐hexosaminidase release assay. This experimental setting was used to assess how microorganisms added apically affected these parameters. Basolateral secretion of cytokine/chemokines by polarized Caco‐2 cell monolayers was analysed by ELISA. Results Basophils loaded with OVA‐specific IgE responded to OVA in a dose‐dependent manner. OVA transported across polarized Caco‐2 cell monolayers was found to trigger basolateral basophil activation. Microorganisms including lactobacilli and Escherichia coli increased transepithelial electrical resistance while promoting OVA passage capable to trigger basophil activation. Non‐inflammatory levels of IL‐8 and thymic stromal lymphopoietin were produced basolaterally by Caco‐2 cells exposed to microorganisms. Conclusion The complex model designed inhere is adequate to learn about the consequence of the interaction between microorganisms and epithelial cells vis‐a‐vis the barrier function and antigen translocation, two parameters essential to mucosal homeostasis. It can further serve as a direct tool to search for microorganisms with anti‐allergic and anti‐inflammatory properties.     

Systemic Administration of Olygodeoxynucleotides with CpG Motifs at Priming Phase Reduces Local Th2 Response and Late Allergic Rhinitis in BALB/c Mice

Oligodeoxynucleotides (ODN) with CpG motifs (CpG ODN) induce T helper (Th)1-type reaction. We aimed to evaluate the therapeutic effect of CpG ODN in the development of late allergic rhinitis induced by ovalbumin (OVA), which is one of Th2 diseaes, in BALB/c mice. Effects of a single dose of synthetic CpG-ODN (50 μg) intraperitoneally (i.p.) at the priming phase (on day 0) by OVA on the development of late eosinophilic rhinitis at respiratory areas were compared to the control mice treated with its vehicle (ODN without CpG motifs; 50 μg). Animals were again sensitized by OVA (on day 10) i.p., and 4 days after second sensitization animals were challenged by OVA intranasally (on day 14). Four days after challenge, eosinophilic reactions, nasal lesions and local cytokine values were examined. Compared to the control group, the CpG ODN-administration increased production of OVA-specific Th1 cytokine (interferon-γ) and decreased productions of ovalubmin-specific Th2 cytokines [interleukin (IL)-5 and IL-13] in nasal cavity fluids, supernatants of splenocytes and/or sera. Also, eosinophilia and increased total IgE values were decreased in mice treated with the CpG ODN compared to the control group. Moreover, nasal lesions with infiltration of eosinophils were prominently reduced by the CpG ODN-treatment compared to the control mice. The present study suggests that the systemic administration of CpG ODN at the priming phase may reduce local OVA-specific Th2 responses, resulting in decreased nasal pathology in the late allergic eosinophilic rhinitis. The authors wish it to be known, in their opinion, Toshiharu Hayashi and Keiko Hasegawa contributed equally to this work.