Inducible costimulator regulates Th2-mediated inflammation, but not Th2 differentiation, in a model of allergic airway disease

A novel costimulatory molecule expressed on activated T cells, inducible costimulator (ICOS), and its ligand, B7-related protein-1 (B7RP-1), were recently identified. ICOS costimulation leads to the induction of Th2 cytokines without augmentation of IL-2 production, suggesting a role for ICOS in Th2 cell differentiation and expansion. In the present study, a soluble form of murine ICOS, ICOS-Ig, was used to block ICOS/B7RP-1 interactions in a Th2 model of allergic airway disease. In this model, mice are sensitized with inactivated Schistosoma mansoni eggs and are subsequently challenged with soluble S. mansoni egg Ag directly in the airways. Treatment of C57BL/6 mice with ICOS-Ig during sensitization and challenge attenuated airway inflammation, as demonstrated by a decrease in cellular infiltration into the lung tissue and airways, as well as by a decrease in local IL-5 production. These inhibitory effects were not due to a lack of T cell priming nor to a defect in Th2 differentiation. In addition, blockade of ICOS/B7RP-1 interactions during ex vivo restimulation of lung Th2 effector cells prevented cytokine production. Thus, blockade of ICOS signaling can significantly reduce airway inflammation without affecting Th2 differentiation in this model of allergic airway disease.     

ICOS expression by activated human Th cells is enhanced by IL-12 and IL-23: increased ICOS expression enhances the effector function of both Th1 and Th2 cells

Previous mouse studies have shown that IL-4 increases the expression of ICOS on activated Th cells, resulting in enhanced ICOS expression on Th2 cells. In this study, we show that ICOS expression on human Th cells is not increased by IL-4, but by IL-12 and by IL-23 instead. Consequently, ICOS expression during IL-12-driven Th1 cell polarization was transiently increased compared with the levels on Th0 cells and IL-4-driven Th2 cells. Addition of IL-12 and/or IL-23 during restimulation increased ICOS expression to the same extent on pre-established Th1, Th2, and Th0 cells, indicating that ICOS levels are not stably imposed by prior polarization. In contrast to the findings in the mouse, IL-4 significantly suppressed the ICOS-enhancing effects of IL-12 and IL-23. The functional consequence of variable ICOS levels was shown in coculture experiments with cells expressing the ICOS-ligand B7-related protein 1 (either transfected Chinese hamster ovary cells or autologous dendritic cells). Ligation of ICOS on 2-day-preactivated effector cells increased their cytokine production to an extent proportional to their ICOS expression levels. As the ICOS-enhancing potentials of IL-12 and IL-23 were maintained for several days after stimulation, both on Th1 and Th2 cells, we propose the concept that local regulation of ICOS expression on activated Th cells by IL-12 and/or IL-23 may provide a powerful means to amplify effector T cell responses in peripheral tissues, independently of the polarized state of the Th cells.     

Administration of pentoxifylline during allergen sensitization dissociates pulmonary allergic inflammation from airway hyperresponsiveness

Asthma, a chronic inflammatory disease characterized by intermittent, reversible airflow obstruction and airway hyperresponsiveness (AHR), is classically characterized by an excess of Th2 cytokines (IL-13, IL-4) and depletion of Th1 cytokines (IFN-gamma, IL-12). Recent studies indicating an important role for Th1 immunity in the development of AHR with allergic inflammation suggest that Th1/Th2 balance may be important in determining the association of AHR with allergic inflammation. We hypothesized that administration of pentoxifylline (PTX), a phosphodiesterase inhibitor known to inhibit Th1 cytokine production, during allergen (OVA) sensitization and challenge would lead to attenuation of AHR in a murine model of allergic pulmonary inflammation. We found that PTX treatment led to attenuation of AHR when administered at the time of allergen sensitization without affecting other hallmarks of pulmonary allergic inflammation. Attenuation of AHR with PTX treatment was found in the presence of elevated bronchoalveolar lavage fluid levels of the Th2 cytokine IL-13 and decreased levels of the Th1 cytokine IFN-gamma. PTX treatment during allergen sensitization leads to a divergence of AHR and pulmonary inflammation following allergen challenge.     

IFN-gamma-inducing factor (IL-18) increases allergic sensitization, serum IgE, Th2 cytokines, and airway eosinophilia in a mouse model of allergic asthma

We investigated the effects of IFN-gamma-inducing factor (IL-18) in a ragweed (RW) mouse model of allergic asthma. Administration of IL-18 in conjunction with allergic sensitization and challenge in wild-type, but not IFN-gamma -/- mice, inhibited the bronchoalveolar lavage (BAL) eosinophilia induced by RW challenge, and increased serum levels of RW-specific IgG2a and production of IFN-gamma from splenocytes cultured with RW, indicating a critical role for IFN-gamma in mediating these effects. Paradoxically, the same treatment schedule in WT mice increased serum levels of RW-specific IgE and IgG1, and production of IL-4 and IL-5 from splenocytes cultured with RW. When the effects of the same IL-18 treatment schedule were allowed to mature for 3 wk, the inhibition of lung eosinophil recruitment was replaced by augmentation of lung eosinophil recruitment. In another experiment, IL-18 administered only with allergic sensitization increased BAL eosinophilia and lung expression of IL-5 and IFN-gamma, while IL-18 administered only with RW challenge decreased BAL eosinophilia and increased lung IFN-gamma expression, while lung expression of IL-5 remained unchanged. IL-18 administered without RW or adjuvant to naive mice increased total serum IgE levels. Finally, intrapulmonary administrations of IL-18 plus RW in naive mice dramatically increased Th2 cytokine production, IgE levels, eosinophil recruitment, and airway mucus, demonstrating induction of allergic sensitization. This is the first report demonstrating that IL-18 promotes a Th2 phenotype in vivo, and potently induces allergic sensitization. These results suggest that IL-18 may contribute to the pathogenesis of allergic asthma.     

Notch ligand delta-like 4 regulates development and pathogenesis of allergic airway responses by modulating IL-2 production and Th2 immunity

Activation of the canonical Notch pathways has been implicated in Th cell differentiation, but the role of specific Notch ligands in Th2-mediated allergic airway responses has not been completely elucidated. In this study, we show that delta-like ligand 4 (Dll4) was upregulated on dendritic cells in response to cockroach allergen. Blocking Dll4 in vivo during either the primary or secondary response enhanced allergen-induced pathogenic consequences including airway hyperresponsiveness and mucus production via increased Th2 cytokines. In vitro assays demonstrated that Dll4 regulates IL-2 in T cells from established Th2 responses as well as during primary stimulation. Notably, Dll4 blockade during the primary, but not the secondary, response increased IL-2 levels in lung and lymph node of allergic mice. The in vivo neutralization of Dll4 was associated with increased expansion and decreased apoptosis during the primary allergen sensitization. Moreover, Dll4-mediated Notch activation of T cells during primary stimulation in vitro increased apoptosis during the contraction/resting phase of the response, which could be rescued by exogenous IL-2. Consistent with the role for Dll4-mediated IL-2 regulation in overall T cell function, the frequency of IL-4-producing cells was also significantly altered by Dll4 both in vivo and in vitro. These data demonstrate a regulatory role of Dll4 both in initial Th2 differentiation and in Th2 cytokine production in established allergic responses.     

CpG oligodeoxynucleotides can reverse Th2-associated allergic airway responses and alter the B7.1/B7.2 expression in a murine model of asthma

CpG oligodeoxynucleotides (CpG-ODN) administered during Ag sensitization or before Ag challenge can inhibit allergic pulmonary inflammation and airway hyperreactivity in murine models of asthma. In this study, we investigated whether CpG-ODN can reverse an ongoing allergic pulmonary reaction in a mouse model of asthma. AKR mice were sensitized with conalbumin followed by two intratracheal challenges at weekly intervals. CpG-ODN was administered 24 h after the first Ag challenge. CpG-ODN administration reduced Ag-specific IgE levels, bronchoalveolar lavage fluid eosinophils, mucus production, and airway hyperreactivity. We found that postchallenge CpG-ODN treatment significantly increased IFN-gamma concentrations and decreased IL-13, IL-4, and IL-5 concentrations in bronchoalveolar lavage fluids and spleen cell culture supernatants. Postchallenge CpG-ODN treatment also increased B7.1 mRNA expression and decreased B7.2 mRNA expression in lung tissues. These results suggest that CpG-ODN may have potential for treatment of allergic asthma by suppressing Th2 responses during IgE-dependent allergic airway reactions. The down-regulation of Th2 responses by CPG-ODN may be associated with regulation of the costimulatory factors B7.1 and B7.2.     

IL-10- and IL-12-independent down-regulation of allergic sensitization by stimulation of CD40 signaling

Interaction between CD154 (CD40 ligand) on activated T lymphocytes and its receptor CD40 has been shown to be critically involved in the generation of cell-mediated as well as humoral immunity. CD40 triggering activates dendritic cells (DC), enhances their cytokine production, up-regulates the expression of costimulatory molecules, and induces their maturation. It is unknown how stimulation of CD40 during sensitization to an airborne allergen may affect the outcome of allergic airway inflammation. We took advantage of a mouse model of allergic asthma and a stimulatory mAb to CD40 (FGK45) to study the effects of CD40-mediated DC activation on sensitization to OVA and subsequent development of OVA-induced airway inflammation. Agonistic anti-CD40 mAb (FGK45) injected during sensitization with OVA abrogated the development of allergic airway inflammation upon repeated airway challenges with OVA. Inhibition of bronchial eosinophilia corresponded with reduced Th2 cytokine production and was independent of IL-12, as evidenced by a similar down-regulatory effect of anti-CD40 mAb in IL-12 p40-deficient mice. In addition, FGK45 equally down-regulated allergic airway inflammation in IL-10-deficient mice, indicating an IL-10-independent mechanism of action of FGK45. In conclusion, our results show that CD40 signaling during sensitization shifts the immune response away from Th2 cytokine production and suppresses allergic airway inflammation in an IL-12- and IL-10-independent way, presumably resulting from enhanced DC activation during sensitization.     

Anti-inflammatory non-steroidal drug able to modulate IL-10 in allergic asthma

Our studies target alternative/adjuvant therapies in allergic diseases, able to qualitatively/quantitatively modify cytokine profiles produced by both CD4+ T-cell subsets (mainly Th1 and Th2) and B-cells, macrophages, etc. Current investigations aim to identify compounds capable to down-regulate IL-10 as an exponent of Th2 cell function and, consequently, to up-regulate Th1 cytokine levels. Experiments on ten allergic asthmatic patients and ten healthy subjects as control were performed. Cytokine production, triggered in PBMCs culture systems by PHA, was modulated with Indomethacin, a non-steroidal anti-inflammatory drug and IL-10 was measured in 24 hours culture supernatants. According to our experimental data, IL-10 level of asthmatic patients' PBMCs in the resting state is not significantly different from control. PHA-activated PBMCs from asthmatic patients do not display significantly higher IL-10 levels than the normal subjects. The results obtained up-to-date reveal the fact that Indomethacin strongly down-regulates IL-10 levels in PBMCs cultures, in both asthmatic allergic patients and healthy subjects. It is obvious that the inhibitory effect of Indomethacin on IL-10 released by PBMCs is higher in the case of allergic asthmatic patients. The results obtained in this study demonstrate that Indomethacin is a possible therapeutic candidate in allergic asthma.     

Cyclooxygenase inhibition during allergic sensitization increases STAT6-independent primary and memory Th2 responses

Immune sensitization and memory generation are required for the development of allergic inflammation. Our previous studies demonstrate that the cyclooxygenase (COX) metabolic pathway is actively involved in allergic responses and COX inhibition increases allergic airway inflammation in a STAT6-independent fashion. To test the hypothesis that COX inhibition augments allergic inflammation by enhancing immune sensitization and memory, we sensitized STAT6 knockout mice with an i.p. injection of OVA with aluminum hydroxide as an adjuvant and treated the mice with the COX inhibitor indomethacin or vehicle for analyses of the primary and memory immune responses. We found that COX inhibition during immune sensitization, but not the allergic challenge phase, was necessary and sufficient to increase allergic inflammation. COX inhibition during sensitization increased the numbers of mature dendritic cells and activated CD4 T cells in the spleen and augmented OVA-specific IL-5 and IL-13 responses of the splenic CD4 T cells at day 5 after sensitization. COX inhibition during sensitization also augmented allergic Th2 response to OVA challenge 90 days after the sensitization. Therefore, COX inhibition during allergic sensitization augments allergic responses by enhancing Th2 cell activation and memory generation and the proallergic effect is STAT6-independent. These findings provide a mechanistic explanation for the increased allergic inflammation previously shown in the mice treated with COX inhibitors and in COX-deficient mice and suggest that use of COX-inhibiting drugs during initial allergen exposure may increase the risk of developing allergic responses.     

Association studies of CTLA-4, CD28, and ICOS gene polymorphisms with type 1 diabetes in the Japanese population

Co-stimulatory molecules of CD28, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), and the newly identified inducible co-stimulator (ICOS) are expressed on cell surfaces and provide regulatory signals for T-cell activation. Their genes are candidate susceptibility genes for type 1 diabetes because they co-localize to Chromosome 2q33 with the IDDM12 locus. After determining the genomic structure and screening for polymorphisms of the ICOS gene, we performed association studies between newly identified polymorphisms of the ICOS gene, together with known polymorphisms of CD28 and CTLA-4 genes, and type 1 diabetes. The 49A/G dimorphism in exon 1 and the (AT)n in the 3' untranslated region of the CTLA-4 gene were significantly associated with type 1 diabetes. Evaluation of the CTLA-4 49A-3'(AT)n 86-bp haplotype frequency in patients and controls confirmed the results from the analysis of each polymorphic site. Dimorphism in intron 3 of the CD28 gene was associated with type 1 diabetes only in the early-onset group. In contrast, there was no association with the microsatellite polymorphisms in the ICOS gene or dimorphisms in the promotor region of CTLA-4. Of the three genes encoding co-stimulatory molecules, the CTLA-4 gene appears to confer risks for the development of type 1 diabetes.     

Vaccination with heat-killed Listeria as adjuvant reverses established allergen-induced airway hyperreactivity and inflammation: role of CD8+ T cells and IL-18

Asthma is a respiratory disorder characterized by airway hyperreactivity (AHR) and inflammation and is associated with high serum IgE and overproduction of IL-4, IL-5, and IL-13 by allergen-specific Th2 cells. Our previous studies demonstrated that heat-killed Listeria monocytogenes (HKL) as an adjuvant in immunotherapy successfully reversed ongoing Ag-specific Th2-dominated responses toward Th1-dominated responses, but it was unclear if such immune modulation could reverse ongoing, established disease in target organs such as the lung. In this paper we show that a single dose of Ag plus HKL as adjuvant significantly reduced AHR in a murine model for asthma and reversed established AHR when given late after allergen sensitization. HKL as adjuvant also dramatically inhibited airway inflammation, eosinophilia, and mucus production, significantly reduced Ag-specific IgE and IL-4 production, and dramatically increased Ag-specific IFN-gamma synthesis. The inhibitory effect of HKL on AHR depended on the presence of IL-12 and CD8+ T cells and was associated with an increase of IL-18 mRNA expression. Thus, our results demonstrate that HKL as an adjuvant for immunotherapy mediates immune deviation from a pathological Th2-dominated response toward a protective immune response in peripheral lymphoid tissues and in the lungs and may be clinically effective in the treatment of patients with established asthma and allergic disease.     

Engineered recombinant ovomucoid third domain can modulate allergenic response in Balb/c mice model

IgE-mediated allergic reactions to egg white are a serious health problem and ovomucoid being the dominant egg white allergen has been on focus in the past decade. Engineered hypoallergens with reduced reactivity for IgE antibodies are being examined to modulate the allergic response and develop prophylactic allergen vaccines. In this study, we evaluated the immunomodulatory effect of a genetic variant of the third domain of ovomucoid (GMFA) which showed reduced IgE binding with egg allergic patient's sera in comparison to the native form of the third domain of ovomucoid (DIII) in a murine model system. Balb/c mice were injected intraperitoneally with DIII and GMFA antigens. Allergen-specific serum IgG, IgG1, IgG2a, and IgE responses were evaluated using enzyme-linked immunosorbent assay. Splenocyte cytokine levels in the medium of the cultured cells were examined by ELISA and levels of IL-4, INF-gamma, and IL-12 (p70) cytokines were quantified. Neutralization with anti-IL-12 monoclonal antibody was assayed and cytokine levels with respect to GMFA mutant antigen stimulation were measured. GMFA mutant form was found to have significantly reduced levels of specific IgE when compared to the DIII suggesting a mutation-induced abrogation of the IgE binding epitope in mice. The increase in IgG2a levels in GMFA together with the decline of IgE and IgG1 points to a shift from a Th2 response to a Th1 dominated response. The cytokine profile showed a modulation of anti-allergic Th1 phenotype in GMFA from a proallergic Th2 response observed with DIII. Low levels of IL-4 and increased levels of INF-gamma and IL-12 were observed and anti-IL-12 monoclonal antibody restored the levels of IL-4 and suppressed levels of INF-gamma and IL-12 in the GMFA sensitized group. These results indicate that GMFA has a marked suppressive effect on the allergic response of ovomucoid and caused a shift towards a Th1 pathway, thereby modulating the Th1/Th2 cytokine balance and could be used as a potential hypoallergenic candidate for allergen-immunotherapy in the treatment of egg white allergy.     

Blocking IL-15 prevents the induction of allergen-specific T cells and allergic inflammation in vivo

IL-15 has been shown to accelerate and boost allergic sensitization in mice. Using a murine model of allergic sensitization to OVA, we present evidence that blocking endogenous IL-15 during the sensitization phase using a soluble IL-15Ralpha (sIL-15Ralpha) suppresses the induction of Ag-specific, Th2-differentiated T cells. This significantly reduces the production of OVA-specific IgE and IgG and prevents the induction of a pulmonary inflammation. Release of proinflammatory TNF-alpha, IL-1beta, IL-6, and IL-12 as well as that of Th2 cytokines IL-4, IL-5, and IL-13 into the bronchi are significantly reduced, resulting in suppressed recruitment of eosinophils and lymphocytes after allergen challenge. It is of clinical relevance that the airway hyper-responsiveness, a major symptom of human asthma bronchiale, is significantly reduced by sIL-15Ralpha treatment. Ex vivo analysis of the draining lymph nodes revealed reduced numbers of CD8, but not CD4, memory cells and the inability of T cells of sIL-15Ralpha-treated mice to proliferate and to produce Th2 cytokines after in vitro OVA restimulation. This phenomenon is not mediated by enhanced numbers of CD4(+)/CD25(+) T cells. These results show that IL-15 is important for the induction of allergen-specific, Th2-differentiated T cells and induction of allergic inflammation in vivo.     

ICOS costimulation expands Th2 immunity by augmenting migration of lymphocytes to draining lymph nodes

The T cell costimulatory molecule ICOS regulates Th2 effector function in allergic airway disease. Recently, several studies with ICOS(-/-) mice have also demonstrated a role for ICOS in Th2 differentiation. To determine the effects of ICOS on the early immune response, we investigated augmenting ICOS costimulation in a Th2-mediated immune response to Schistosoma mansoni Ags. We found that augmenting ICOS costimulation with B7RP-1-Fc increased the accumulation of T and B cells in the draining lymph nodes postimmunization. Interestingly, the increased numbers were due in part to increased migration of undivided Ag-specific TCR transgenic T cells and surprisingly B cells, as well as non-TCR transgenic T cells. B7RP-1-Fc also increased the levels of the chemokines CCL21 and CXCL13 in the draining lymph node, suggesting ICOS costimulation contributes to migration by direct or indirect effects on dendritic cells, stromal cells and high endothelial venules. Further, the effects of B7RP-1-Fc were not dependent on immunization. Our data support a model in which ICOS costimulation augments the pool of lymphocytes in the draining lymph nodes, leading to an increase in the frequency of potentially reactive T and B cells.     

Preventative Effect of an Herbal Preparation (HemoHIM) on Development of Airway Inflammation in Mice via Modulation of Th1/2 Cells Differentiation

HemoHIM, an herbal preparation of three edible herbs (Angelica gigas Nakai, Cnidium officinale Makino, Paeonia japonica Miyabe) is known to increase the Th1 immune response as well as reduce the allergic response in human mast cells. Here, our goal was to determine whether or not HemoHIM could induce Th1 cell differentiation as well as inhibit the development of airway inflammation. To study Th1/Th2 cell differentiation, naive CD4⁺ T cells isolated from C57BL/6 mouse spleens were cultured with or without HemoHIM. To examine airway inflammation, C57BL/6 mice were fed HemoHIM for 4 weeks before sensitization and provocation with ovalbumin (OVA). In an in vitro experiment, naive CD4⁺ T cells displayed increased Th1 (IFN-γ⁺ cell) as well as decreased Th2 (IL-4⁺ cell) differentiation in a HemoHIM concentration-dependent manner. Furthermore, in an airway inflammation mice model, eosinophil numbers in BALF, serum levels of OVA-specific IgE and IgG1, and cytokine (IL-4, IL-5, and IL-13) levels in BALF and the supernatant of splenocytes all decreased upon HemoHIM (100 mg/kg body weight) pretreatment (4 weeks). These results show that HemoHIM attenuated allergic airway inflammation in the mouse model through regulation of the Th1/Th2 balance.     

A critical role for C5L2 in the pathogenesis of experimental allergic asthma

The complement fragment C5a plays dual roles in the development of experimental allergic asthma. It protects from pulmonary allergy by a regulatory effect on dendritic cells during allergen sensitization, but is proallergic during the effector phase. C5a can bind to two distinct receptors (i.e., C5a receptor and C5a receptor-like 2 [C5L2]). The functional role of C5L2 in vivo remains enigmatic. In this study, we show in two models of OVA- and house dust mite (HDM)-induced experimental allergic asthma that C5L2-deficient mice are protected from the development of airway hyperresponsiveness, Th2 cytokine production, eosinophilic airway inflammation, serum IgE, or mucus production. Surprisingly, HDM-induced experimental asthma in C5L2-deficient mice was associated with increased pulmonary IL-17A production and increased airway neutrophil numbers. To directly assess the role of C5L2 on myeloid dendritic cells (mDCs) during allergen sensitization, we performed single or repeated adoptive transfers of C5L2-deficient mDCs into wild-type mice. HDM-pulsed C5L2-deficient mDCs induced strong Th2 cytokine production, which was associated with marked IFN-γ and IL-17A production, decreased eosinophil numbers, and reduced IgE production as compared with HDM-pulsed mDCs from wild-type mice. HDM stimulation of C5L2(-/-) mDCs in vitro resulted in production of Th17-promoting cytokine IL-23, which was absent in wild-type mDCs. Our findings suggest that C5L2 acts at the mDC/T cell interface to control the development of Th1 and Th17 cells in response to airway HDM exposure. Furthermore, it drives Th2 immune responses independent of mDCs, suggesting a complex role for C5L2 in the development of experimental allergic asthma.     

ICOS Mediates the Generation and Function of CD4+CD25+Foxp3+ Regulatory T Cells Conveying Respiratory Tolerance

Costimulatory molecules like ICOS are crucial in mediating T cell differentiation and function after allergen contact and thereby strongly affect the immunologic decision between tolerance or allergy development. In this study, we show in two independent approaches that interruption of the ICOS signaling pathway by application of a blocking anti-ICOSL mAb in wild-type (WT) mice and in ICOS(-/-) mice inhibited respiratory tolerance development leading to eosinophilic airway inflammation, mucus hypersecretion, and Th2 cytokine production in response to OVA sensitization. Respiratory Ag application almost doubled the number of CD4(+)Foxp3(+) regulatory T cells (Tregs) in the lung of WT mice with 77% of lung-derived Tregs expressing ICOS. In contrast, in ICOS(-/-) mice the number of CD4(+)CD25(+)Foxp3(+) Tregs did not increase after respiratory Ag application, and ICOS(-/-) Tregs produced significantly lower amounts of IL-10 than those of WT Tregs. Most importantly, in contrast to WT Tregs, ICOS(-/-) Tregs did not convey protection when transferred to "asthmatic" recipients demonstrating a strongly impaired Treg function in the absence of ICOS signaling. Our findings demonstrate a crucial role of ICOS for the generation and suppressive function of Tregs conveying respiratory tolerance and support the importance of ICOS as a target for primary prevention strategies.