Christina Hartwig

ExoY from Pseudomonas aeruginosa is a nucleotidyl cyclase with preference for cGMP and cUMP formation

In addition to the well known second messengers cAMP and cGMP, mammalian cells contain the cyclic pyrimidine nucleotides cCMP and cUMP. Soluble guanylyl cyclase and soluble adenylyl cyclase produce all four cNMPs. Several bacterial toxins exploit mammalian cyclic nucleotide signaling. The type III secretion protein ExoY from Pseudomonas aeruginosa induces severe lung damage and effectively produces cGMP. Here, we show that transfection of mammalian cells with ExoY or infection with ExoY-expressing P. aeruginosa not only massively increases cGMP but also cUMP levels. In contrast, the structurally related CyaA from Bordetella pertussis and edema factor from Bacillus anthracis exhibit a striking preference for cAMP increases. Thus, ExoY is a nucleotidyl cyclase with preference for cGMP and cUMP production. The differential effects of bacterial toxins on cNMP levels suggest that cUMP plays a distinct second messenger role.

The histamine H4-receptor (H4R) regulates eosinophilic inflammation in ovalbumin-induced experimental allergic asthma in mice

Via the histamine H4‐receptor (H4R), histamine promotes the pathogenesis of experimental allergic asthma in mice. Application of H4R antagonists during sensitization as well as during provocation reduces the severity of the disease. However, the specific cell types functionally expressing H4R in experimental allergic asthma have not been well characterized in vivo. In this study, we identified the cell type(s) responsible for H4R activity in experimental asthma and related physiological mechanisms. Using H4R‐deficient mice, we studied the role of H4R in the sensitization and effector phase. DCs lacking H4R expression during the in vitro sensitization reaction resulted in effector T cells unable to induce an entire eosinophilic inflammation in the lung upon adoptive transfer in vivo. Recipient mice lacking H4R expression, which were adoptively transferred with H4R+/+ T cells polarized in the presence of H4R+/+ DCs, showed reduced signs of inflammation and ameliorated lung function. Here, we provide in vivo evidence that in experimental asthma in mice the H4R specifically regulates activation of DCs during sensitization, while in the effector phase the H4R is active in cells involved in the activation of eosinophils, and possibly other cells. A putative therapy targeting the H4R may be an option for asthma patients developing IL‐5‐dependent eosinophilia.

The dual H3/4R antagonist thioperamide does not fully mimic the effects of the 'standard' H4R antagonist JNJ 7777120 in experimental murine asthma.

Histamine is detected in high concentrations in the airways during an allergic asthma response. In a murine model of allergic asthma, the histamine H4 receptor (H4R)-selective ligand JNJ 7777120 reduces asthma-like symptoms. A sole antagonistic function of JNJ 7777120 at the murine H4R has, however, been questioned in the literature. Therefore, in the present study, we aimed at analyzing the effects of JNJ 7777120 in comparison to that of the H3/4R-selective antagonist thioperamide. Experimental murine asthma was induced by sensitization and provocation of BALB/c mice with ovalbumine (OVA). JNJ 7777120, thioperamide, or JNJ 5207852, an H3R-selective antagonist which was used to dissect H3R- and H4R-mediated activities of thioperamide, were injected subcutaneously during sensitization and effects were analyzed after provocation. Pharmacokinetic analyses revealed shortest t1/2 values in both plasma and lung tissue and lowest maximal concentration in lung tissue for JNJ 7777120 in comparison to thioperamide and JNJ 5207852. Nevertheless, JNJ 7777120 reduced serum titers of allergen-specific (anti-OVA) IgE, inflammatory infiltrations in lung tissue, and eosinophilia in bronchoalveolar lavage fluid. In contrast, thioperamide reduced only eosinophilia in bronchoalveolar lavage fluid, while anti-OVA IgE concentrations and lung infiltrations remained unaffected. JNJ 5207852 had no effect on these parameters. JNJ 7777120 provides beneficial effects in experimental murine asthma, which, however, could only partially be mimicked by thioperamide, despite more favorable pharmacokinetics. Thus, whether these effects of JNJ 7777120 are entirely attributable to an antagonistic activity at the murine H4R or whether an agonistic activity is also involved has to be reconsidered.

Delineating the Role of Histamine-1- and -4-Receptors in a Mouse Model of Th2-Dependent Antigen-Specific Skin Inflammation

Background Histamine drives pruritus in allergic skin diseases which clinically constitutes a most disruptive symptom. Skin pathology in allergic skin diseases is crucially influenced by different T-helper subsets. However, the contribution of different histamine-receptors to T-helper cell dependent skin pathology has not been definitively answered. Models which can specifically address the functional role of T-helper subsets and the mediators involved are therefore valuable to gain further insights into molecular pathways which contribute to allergic skin disease. They might also be helpful to probe amendable therapeutic interventions such as histamine-receptor antagonism. Objective Establishing an adoptive transfer model for antigen-specific Th cells, we aimed to delineate the role of histamine H1- and H4-receptors in Th2-dependent skin inflammation. Methods In-vitro differentiated and OVA primed Th2 cells were adoptively transferred into congenic recipient mice. In vivo treatment with specific histamine H1- and H4-receptor antagonists was performed to analyze the contribution of these histamine-receptors to Th2-dependent skin pathology in our model. Analysis four days after epicutaneous challenge comprised skin histology, flow cytometric detection of transferred T-helper cells and analysis of antigen-cytokine profiles in skin-draining lymph nodes. Results Use of specific H1- and H4-receptor antagonists revealed a crucial role for H1- and H4-receptors for Th2 migration and cytokine secretion in a Th2-driven model of skin inflammation. While H1- and H4-receptor antagonists both reduced Th2 recruitment to the site of challenge, local cytokine responses in skin-draining lymph nodes were only reduced by the combined application of H1- and H4-receptor antagonists and mast cell counts remained altogether unchanged by either H1R-, H4R- or combined antagonism. Conclusion Our model demonstrates a role for H1- and H4-receptors in Th2 cell infiltration and cytokine secretion in allergic skin diseases and suggests further studies to evaluate these findings for therapeutic approaches.

Endogenous IL-18 in experimentally induced asthma affects cytokine serum levels but is irrelevant for clinical symptoms

T cells and T cell derived cytokines are involved in the complex pathogenesis of asthma. The role of the cytokine IL-18 however, is not clearly defined so far. On the one hand side IL-18 induces Th1-type cytokines and thereby might counter-regulate Th2-mediated allergic asthma. On the other hand IL-18 also bears pro-inflammatory effects possibly enhancing experimental asthma. In order to elucidate the role of IL-18 in allergic pulmonary inflammation typical symptoms were compared after induction of experimental asthma in IL-18(-/-) and in wild type mice. Asthma was induced using ovalbumin (OVA) as allergen for sensitization and challenge. Sham sensitized and OVA challenged mice served as controls. Bronchoalveolar lavage-fluid cytology, leukocyte infiltration in lung tissues, serum levels of OVA-specific IgE and cytokines, and lung function were analyzed. Clear differences could be observed between control and asthmatic mice, both in wild type and IL-18(-/-) animals. Surprisingly, no differences were found between asthmatic wild type and IL-18(-/-) mice. Thus, in contrast to conflicting data in the literature IL-18 did not suppress or enhance the pulmonary allergic immune response in a murine experimental model of asthma.

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.

Impaired Lung Dendritic Cell Migration and T Cell Stimulation Induced by Immunostimulatory Oligonucleotides Contribute to Reduced Allergic Airway Inflammation

CpG-containing oligonucleotides (CpG) have been shown to reduce key features of allergic airway inflammation in mouse models. Given the inhibitory effects of CpG treatment on Ag presentation of subsequently encountered Ags via MHC class I and II molecules by dendritic cells (DC), we hypothesized that intranasal CpG treatment would lead to reduced Ag-specific T cell stimulation in the lung-draining lymph nodes, thereby reducing the inflammatory response in sensitized mice. Intranasal CpG administration led to phenotypic maturation of lung and mediastinal lymph node DC as determined by expression of MHC class II, CD80, and CD86. This was accompanied by a significant reduction in the proliferation of adoptively transferred Ag-specific CD4+ and CD8+ T cells in mediastinal lymph nodes, when CpG was given before inhalative OVA challenges. DC obtained from mediastinal lymph nodes of CpG-treated mice before OVA inhalation led to reduced T cell stimulation via MHC class I and II molecules. In addition, CpG diminished airway eosinophilia and pulmonary infiltration after sensitization or following adoptive transfer of Ag-specific Th2 cells. These results were explained by reduced CCL21 expression and inhibition of lung DC migration following CpG administration, which could be restored by transfer of bone marrow-derived DC, because CpG had no major impact on the constitutive MHC class II Ag presentation of protein-derived Ag by lung tissue-derived DC. We conclude that CpG treatment can effectively impair the DC-mediated Ag transport from the lungs to the lymph nodes, resulting in reduced T cell activation and blunted airway inflammation.

Impact of boostering for the strength of asthma parameters and dendritic cell numbers in a C57BL/6 model of allergic airway inflammation

BACKGROUND Murine models assist in elucidating the pathogenesis of allergic asthma and evaluation of new therapeutic strategies. We aimed to assess the requirement of boostering needed in the BL/6 murine asthma model and its influence on DC populations in lungs and bronchial lymph nodes. METHODS AND RESULTS Two injections of OVA+alum - one sensitization and one booster - followed by two aerosol challenges were sufficient to induce a distinct asthma-like inflammation in BL/6 mice, including significant increased immunoglobulin (IgE) level, influx of eosinophils in the airway lumen, and evident histopathology. Using this protocol, CD11chighMHC-II+ DC counts in lungs and lymph nodes doubled with no changes of CD8+ DC in the lungs but increase in lung-draining lymph nodes. CONCLUSIONS Given the site-specific changes of dendritic cell (DC) subpopulations during allergic asthma we propose a distinct regulation of antigen transport and antigen presentation in the murine asthma model.

Surfactant protein D modulates pulmonary clearance of pollen starch granules.

ABSTRACT Pollen starch granules (PSGs) are allergen particles that get into contact with pulmonary surfactant and phagocytes in the terminal airways. In this study, the effects of surfactant protein D (SP-D) on the interaction of PSGs with phagocytes and on the pulmonary clearance of PSGs were determined. Fluorescently labeled PSGs were incubated in vitro with murine lung macrophages or dendritic cells (DCs) ± recombinant rat SP-D (rrSP-D). In addition, the effect of SP-D on uptake of PSGs by lung macrophages and DCs was studied in vivo. Furthermore, PSGs were instilled in Balb/c mice and the effects of SP-D on total lung clearance were assessed by optical imaging. SP-D treatment increased the number of PSG-positive macrophages and DCs in vitro. Furthermore, SP-D accelerated uptake/binding by alveolar macrophages and reduced the number of PSG-positive tissue macrophages and DCs at 24 hours. However, SP-D did not affect total lung clearance of PSGs and it did not enhance the T-cell proliferation induced by PSG-positive DCs. In conclusion, SP-D increased PSG-positive cells in vitro and accelerated PSG binding/uptake in vivo. The observed effects were limited to cellular clearance mechanisms and did not affect the total clearance of PSGs from the lung.

Pseudomonas aeruginosa ExoY, a cyclic GMP- and cyclic UMP-generating nucleotidyl cyclase

Background In previous studies we showed that in addition to the cyclic nucleotides (cNMPs) cAMP and cGMP, mammalian cells also contain cCMP and cUMP [for review see [1], [2]]. cCMP and cUMP are generated by the bacterial exotoxins CyaA from Bordetella pertussis and edema factor (EF) from Bacillus anthracis as well as nitric oxide (NO)stimulated soluble guanylyl cyclase (sGC). cCMP and cUMP activate cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG) and cyclic nucleotide-regulated ion channels (HCN channels). cUMP, but not cCMP, is degraded by several known phosphodiesterases (PDEs). cCMP and cUMP are also exported from cells via multidrug resistance proteins (MRPs 4 and 5). These findings suggest that cCMP and cUMP constitute second messenger molecules with distinct signalling properties. Pseudomonas aeruginosa is an important pathogenic bacterium, specifically in patients with cystic fibrosis. P. aeruginosa injects effector proteins into host cells via the type III secretion system and, thereby, manipulates their functions. One of the effector proteins of P. aeruginosa is ExoY. ExoY possesses structural similarity with CyaA and EF in the catalytic domain and was assumed to constitute an adenylyl cyclase (AC) [3]. However, the cAMP-forming capacity of ExoY is rather low. More recently, it has been shown that ExoY is capable of producing large quantities of cGMP [4]. On this background, we examined the question whether ExoY also exhibits cytidylyland uridylyl cyclase activity. Methods We transfected mammalian cells with a plasmid encoding ExoY or a catalytically inactive ExoY mutant (ExoY-M). We also infected cells with P. aeruginosa encoding ExoY or ExoY-M. In addition, we infected mice intratracheally with P. aeruginosa. We determined cNMP concentrations with highly sensitive and specific HPLC-MS/MS and HPLC-MS/TOF methods. We assessed cell morphology, apoptosis and label-free dynamic mass distribution, constituting a holistic and unbiased assay for cell activation.