Human studies demonstrated that allergen-specific immunotherapy (IT) represents an effective treatment for allergic diseases. IT involves repeated administration of the sensitizing allergen, indicating a crucial contribution of T cells to its medicinal benefit. However, the underlying mechanisms of IT, especially in a chronic disease, are far from being definitive. In the current study, we sought to elucidate the suppressive mechanisms of IT in a mouse model of chronic allergic asthma. OVA-sensitized mice were challenged with OVA or PBS for 4 wk. After development of chronic airway inflammation, mice received OVA-specific IT or placebo alternately to airway challenge for 3 wk. To analyze the T cell–mediated mechanisms underlying IT in vivo, we elaborated the role of T-bet–expressing Th1 cells, T cell–derived IL-10, and Ag-specific thymic as well as peripherally induced Foxp3+ regulatory T (Treg) cells. IT ameliorated airway hyperresponsiveness and airway inflammation in a chronic asthma model. Of note, IT even resulted in a regression of structural changes in the airways following chronic inhaled allergen exposure. Concomitantly, IT induced Th1 cells, Foxp3+, and IL-10–producing Treg cells. Detailed analyses revealed that thymic Treg cells crucially contribute to the effectiveness of IT by promoting IL-10 production in Foxp3-negative T cells. Together with the peripherally induced Ag-specific Foxp3+ Treg cells, thymic Foxp3+ Treg cells orchestrate the curative mechanisms of IT. Taken together, we demonstrate that IT is effective in a chronic allergic disease and dependent on IL-10 and thymic as well as peripherally induced Ag-specific Treg cells.

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.

Mast cells are able to trigger life-saving immune responses in murine models for acute inflammation. In such settings, several lines of evidence indicate that the rapid and protective recruitment of neutrophils initiated by the release of mast cell-derived pro-inflammatory mediators is a key element of innate immunity. Herein, we investigate the impact of mast cells on critical parameters of neutrophil effector function. In the presence of activated murine bone marrow-derived mast cells, neutrophils freshly isolated from bone marrow rapidly lose expression of CD62L and up-regulate CD11b, the latter being partly driven by mast cell-derived TNF and GM-CSF. Mast cells also strongly enhance neutrophil phagocytosis and generation of reactive oxygen species. All these phenomena partly depend on mast cell-derived TNF and to a greater extend on GM-CSF. Furthermore, spontaneous apoptosis of neutrophils is greatly diminished due to the ability of mast cells to deliver antiapoptotic GM-CSF. Finally, we show in a murine model for acute lung inflammation that neutrophil phagocytosis is impaired in mast cell-deficient Kit (W-sh) /Kit (W-sh) mice but can be restored upon mast cell engraftment. Thus, a previously underrated feature of mast cells is their ability to boost neutrophil effector functions in immune responses.

IL-6 is a pleiotropic cytokine which induces Th2 type cells. IL-6 binds to IL-6R alpha chain and then induces gp130 dimerization resulting in the intracellular activation of Janus kinase and signal transducers and activators of transcription (STAT) pathways. We previously demonstrated that patients affected by allergic asthma have an increased level of soluble IL-6R which correlates with Th2 CD4+ T-cells in their airways. In a murine model of this disease, we also demonstrated that IL-6 inhibits the development of T-regulatory cells (Tregs) and local blockade of IL-6R resulted in the expansion of Tregs as well as Th1 cells in the lung. T-bet is a Th1-specific transcriptional factor. Mice lacking T-bet show increased asthmatic symptoms. We thus want to study the influence of T-bet on IL-6 mediated inhibition of Tregs. To this aim an antibody against IL-6R was applied to T-bet deficient mice (T-bet (-/-)) during allergen challenge. In preliminary studies we found that local blockade of IL-6R in T-bet (-/-) mice resulted in a reduced AHR and decreased lung eosinophilia. Furthermore we observed selective expansion of the CD4+ CD25+ Foxp3+ Tregs in the draining lymph-nodes taken from anti IL-6R-antibody treated T-bet (-/-) mice, compared to T-bet (-/-) untreated mice. These results suggest that local blockade of IL-6R in the absence of T-bet in allergic asthma could result in amelioration of the disease hallmarks because of inducing Tregs in the local draining lymph nodes.

Clinical studies have demonstrated that repeated subcutaneous (s.c.) administrations of allergen are effective in the treatment of allergic diseases. It has been suggested, that the success of this treatment, also known as allergen specific immunotherapy (IT), relates to a shift of the T helper 2 (Th2) to T helper 1 (Th1) cells, a blockade of specific antibodies and/or the induction of regulatory T cells (Treg). The T box transcription factor (T-bet) is crucial for the Th1 development and previous studies demonstrated that T-bet deficient mice (T-bet KO) develop a severe asthmatic phenotype. Thus we address the question of whether T-bet is necessary for the effectiveness of IT. T-bet KO mice were analyzed in a murine model of allergic airway disease and IT. OVA-sensitized T-bet KO or BALB/c mice were treated with 3 s.c. injections of OVA (1000µg/injection) prior to airway challenge. This treatment was effective in suppressing the allergen-induced asthma manifestations such as airway hyperresponsiveness (AHR), serum IgE, lung eosinophilia and airway inflammation in sensitized and challenged wildtype mice. Consistently Th2 cytokines were reduced in the lung of IT treated mice. IT treated T-bet KO mice showed an alleviated airway disease following IT to a comparable degree as that observed in the wildtype mice. In summary, the present study shows that IT is not dependent on T-bet and that this treatment effectively suppresses the development of AHR and airway inflammation.