Interferon-regulatory factor 4 is essential for the developmental program of T helper 9 cells.

Interferon-regulatory factor 4 (IRF4) is essential for the development of T helper 2 (Th2) and Th17 cells. Herein, we report that IRF4 is also crucial for the development and function of an interleukin-9 (IL-9)-producing CD4(+) T cell subset designated Th9. IRF4-deficient CD4(+) T cells failed to develop into IL-9-producing Th9 cells, and IRF4-specific siRNA inhibited IL-9 production in wild-type CD4(+) T cells. Chromatin-immunoprecipitation (ChIP) analyses revealed direct IRF4 binding to the Il9 promoter in Th9 cells. In a Th9-dependent asthma model, neutralization of IL-9 substantially ameliorated asthma symptoms. The relevance of these findings is emphasized by the fact that the induction of IL-9 production also occurs in human CD4(+) T cells accompanied by the upregulation of IRF4. Our data clearly demonstrate the central function of IRF4 in the development of Th9 cells and underline the contribution of this T helper cell subset to the pathogenesis of asthma.

TLR3 but not TLR7/8 ligand induces allergic sensitization to inhaled allergen.

Epidemiological studies suggest that viral infections during childhood are a risk factor for the development of asthma. However, the role of virus-specific pattern recognition receptors in this process is not well defined. In the current study, we compare the effects of the inhaled viral TLR ligands polyinosinic-polycytidylic acid (TLR3) and resiquimod (TLR7/8) on sensitization to a model allergen (OVA) in a murine model. Both compounds enhance the migration, activation, and Ag-processing of myeloid dendritic cells from the lung to the draining lymph nodes comparable to the effects of LPS. Application of polyinosinic-polycytidylic acid [poly(I:C)] or LPS induces production of allergen-specific IgE and IgG1, whereas resiquimod (R848) had no effect. In addition, rechallenge of mice with OVA resulted in airway inflammation and mucus production in animals that received either poly(I:C) or LPS but not after application of R848. In summary, these results show that activation of TLR3 in combination with inhaled allergen results in induction of dendritic cell activation and migration similar to the effects of LPS. This leads to the development of allergic airway disease after allergen rechallenge, whereas mice treated with R848 did not develop allergic airway disease. These findings give further insight into the effects of stimulation of different TLRs on the development of asthma.

The tick salivary protein sialostatin L inhibits the Th9-derived production of the asthma-promoting cytokine IL-9 and is effective in the prevention of experimental asthma.

Ticks developed a multitude of different immune evasion strategies to obtain a blood meal. Sialostatin L is an immunosuppressive cysteine protease inhibitor present in the saliva of the hard tick Ixodes scapularis. In this study, we demonstrate that sialostatin L strongly inhibits the production of IL-9 by Th9 cells. Because we could show recently that Th9-derived IL-9 is essentially involved in the induction of asthma symptoms, sialostatin L was used for the treatment of experimental asthma. Application of sialostatin L in a model of experimental asthma almost completely abrogated airway hyperresponsiveness and eosinophilia. Our data suggest that sialostatin L can prevent experimental asthma, most likely by inhibiting the IL-9 production of Th9 cells. Thus, alternative to IL-9 neutralization sialostatin L provides the basis for the development of innovative therapeutic strategies to treat asthma.

Regulatory T cells more effectively suppress Th1-induced airway inflammation compared with Th2.

Asthma is a syndrome with different inflammatory phenotypes. Animal models have shown that, after sensitization and allergen challenge, Th2 and Th1 cells contribute to the development of allergic airway disease. We have previously demonstrated that naturally occurring regulatory T cells (nTregs) can only marginally suppress Th2-induced airway inflammation and airway hyperresponsiveness. In this study, we investigated nTreg-mediated suppression of Th2-induced and Th1-induced acute allergic airway disease. We demonstrate in vivo that nTregs exert their suppressive potency via cAMP transfer on Th2- and Th1-induced airway disease. A comparison of both phenotypes revealed that, despite similar cAMP transfers, Th1-driven airway hyperresponsiveness and inflammation are more susceptible to nTreg-dependent suppression, suggesting that potential nTreg-based therapeutic strategies might be more effective in patients with predominantly neutrophilic airway inflammation based on deregulated Th1 response.

CD4-mediated regulatory T-cell activation inhibits the development of disease in a humanized mouse model of allergic airway disease.

BACKGROUND: Based on their potency to control allergic diseases, regulatory T (Treg) cells represent a promising target for novel strategies to interfere with allergic airway inflammation. We have previously demonstrated that stimulation of the CD4 molecule on human Treg cells activates their suppressive activity in vitro and in vivo. OBJECTIVE: We sought to determine the effect of CD4-mediated Treg-cell activation on pulmonary inflammation in a humanized mouse model of allergic airway inflammation. METHODS: PBMCs obtained from donors allergic to birch pollen or from healthy donors were injected into NOD-severe combined immunodeficiency γc(-/-) mice, followed by allergen airway challenges and analysis of airway responsiveness and inflammation. For Treg-cell activation, mice were treated with the CD4-binding, lck-activating recombinant HIV-1 surface protein gp120 after sensitization prior to allergen challenge. Control experiments with CD25-depleted PBMCs were performed to evaluate the role of Treg cells. RESULTS: PBMCs from allergic donors but not from healthy donors induced airway inflammation and airway hyperresponsiveness. Treatment with gp120 prior to allergen challenge abrogated airway hyperresponsiveness and reduced the inflammatory immune response. In contrast, treatment had no effect on inflammation and airway hyperresponsiveness in mice that received CD25-depleted PBMCs, demonstrating Treg-cell dependency of disease prevention. CONCLUSION: Allergic airway inflammation can be prevented by stimulation of human Treg cells by CD4. These results suggest a clinical potential of Treg-cell activation by high-affinity CD4 ligands in allergic diseases.

Protection from graft-versus-host disease by HIV-1 envelope protein gp120-mediated activation of human CD4+CD25+ regulatory T cells.

Naturally occurring CD4(+)CD25(+) regulatory T cells (Tregs) represent a unique T-cell lineage that is endowed with the ability to actively suppress immune responses. Therefore, approaches to modulate Treg function in vivo could provide ways to enhance or reduce immune responses and lead to novel therapies. Here we show that the CD4 binding human immunodeficiency virus-1 envelope glycoprotein gp120 is a useful and potent tool for functional activation of human Tregs in vitro and in vivo. Gp120 activates human Tregs by binding and signaling through CD4. Upon stimulation with gp120, human Tregs accumulate cyclic adenosine monophosphate (cAMP) in their cytosol. Inhibition of endogeneous cAMP synthesis prevents gp120-mediated Treg activation. Employing a xenogeneic graft versus host disease model that has been shown to be applicable for the functional analysis of human Tregs in vivo, we further show that a single dose of gp120 is sufficient to prevent lethal graft versus host disease and that the tolerizing effect of gp120 is strictly dependent on the presence of human Tregs and their up-regulation of cAMP upon gp120-mediated activation. Our findings demonstrate that stimulation via the CD4 receptor represents a T-cell receptor-independent Treg activating pathway with potential to induce immunologic tolerance in vivo.

Inhibition of cAMP degradation improves regulatory T cell-mediated suppression.

Naturally occurring regulatory T cells (nTreg cells) are crucial for the maintenance of peripheral tolerance. We have previously shown that a key mechanism of their suppressive action is based on a contact-dependent transfer of cAMP from nTreg cells to responder T cells. Herein, we further elucidate the important role of cAMP for the suppressive properties of nTreg cells. Prevention of cAMP degradation by application of the phosphodiesterase 4 inhibitor rolipram led to strongly increased suppressive potency of nTreg cells for Th2 cells in vitro and in vivo. Detailed analyses revealed that rolipram caused, in the presence of nTreg cells, a synergistic increase of cAMP in responder Th2 cells. In vivo, the application of nTreg cells in a strictly Th2-dependent preclinical model of asthma had only a marginal effect. However, the additional treatment with rolipram led to a considerable reduction of airway hyperresponsiveness and inflammation in a prophylactic as well as in a therapeutic model. This amelioration was correlated with enhanced cAMP-levels in lung Th2 cells in vivo. Collectively, these data support our observation that cAMP has a key function for nTreg cell-based suppression and they clearly demonstrate that the effect of cAMP on T responder cells can be greatly enhanced upon application of phosphodiesterase 4 inhibitors.

Mast cells induce migration of dendritic cells in a murine model of acute allergic airway disease.

BACKGROUND: The migration of dendritic cells (DCs) from the lungs to the regional lymph nodes is necessary for the development of allergic airway disease. Following activation, mast cells release a variety of stored or de novo-produced inflammatory mediators, several of them being capable of activating DCs. In this study, the role of mast cells on DC migration from the lungs to the thoracic lymph nodes was investigated in sensitized mice. METHODS: Mast cell-deficient mice (Kit(W-sh/W-sh)) and their wild-type counterparts were sensitized intraperitoneally with ovalbumine (OVA) in saline and challenged by a single intranasal administration of OVA labeled with a fluorescent dye (OVA-Alexa). RESULTS: Following challenge, the relative and absolute amount of OVA- Alexa-positive DCs was clearly increased in sensitized wild-type mice compared to nonsensitized mice. In contrast, sensitized Kit(W-sh/W-sh) showed no increase in OVA-Alexa-positive DCs compared to nonsensitized mast cell-deficient animals. In sensitized Kit(W-sh/W-sh) mice reconstituted with bone marrow-derived mast cells (BMMCs), the number of OVA- Alexa-positive DCs was comparable to that in sensitized wild-type animals. However, transfer of allergen-exposed BMMCs to sensitized mice prior to airway challenge augmented airway inflammation similarly in wild-type and mast cell-deficient mice. In line with this, sensitization with allergen-pulsed DCs induced allergic airway disease independently of mast cells. CONCLUSIONS: This study shows an interaction between mast cells and DCs following allergen challenge in sensitized hosts. However, the function of mast cells can be bypassed in models utilizing activated allergen-exposed DCs to initiate the development of allergic airway disease.

Helicobacter pylori infection prevents allergic asthma in mouse models through the induction of regulatory T cells.

Atopic asthma is a chronic disease of the airways that has taken on epidemic proportions in the industrialized world. The increase in asthma rates has been linked epidemiologically to the rapid disappearance of Helicobacter pylori, a bacterial pathogen that persistently colonizes the human stomach, from Western societies. In this study, we have utilized mouse models of allergic airway disease induced by ovalbumin or house dust mite allergen to experimentally examine a possible inverse correlation between H. pylori and asthma. H. pylori infection efficiently protected mice from airway hyperresponsiveness, tissue inflammation, and goblet cell metaplasia, which are hallmarks of asthma, and prevented allergen-induced pulmonary and bronchoalveolar infiltration with eosinophils, Th2 cells, and Th17 cells. Protection against asthma was most robust in mice infected neonatally and was abrogated by antibiotic eradication of H. pylori. Asthma protection was further associated with impaired maturation of lung-infiltrating dendritic cells and the accumulation of highly suppressive Tregs in the lungs. Systemic Treg depletion abolished asthma protection; conversely, the adoptive transfer of purified Treg populations was sufficient to transfer protection from infected donor mice to uninfected recipients. Our results thus provide experimental evidence for a beneficial effect of H. pylori colonization on the development of allergen-induced asthma.

IL-22 is produced by innate lymphoid cells and limits inflammation in allergic airway disease.

Interleukin (IL)-22 is an effector cytokine, which acts primarily on epithelial cells in the skin, gut, liver and lung. Both pro- and anti-inflammatory properties have been reported for IL-22 depending on the tissue and disease model. In a murine model of allergic airway inflammation, we found that IL-22 is predominantly produced by innate lymphoid cells in the inflamed lungs, rather than TH cells. To determine the impact of IL-22 on airway inflammation, we used allergen-sensitized IL-22-deficient mice and found that they suffer from significantly higher airway hyperreactivity upon airway challenge. IL-22-deficiency led to increased eosinophil infiltration lymphocyte invasion and production of CCL17 (TARC), IL-5 and IL-13 in the lung. Mice treated with IL-22 before antigen challenge displayed reduced expression of CCL17 and IL-13 and significant amelioration of airway constriction and inflammation. We conclude that innate IL-22 limits airway inflammation, tissue damage and clinical decline in allergic lung disease.