R. Gras

The composition of house dust mite is critical for mucosal barrier dysfunction and allergic sensitisation

Background House dust mite (HDM) allergens have been reported to increase airway epithelial permeability, thereby facilitating access of allergens and allergic sensitisation. Objectives The authors aimed to understand which biochemical properties of HDM are critical for epithelial immune and barrier responses as well as T helper 2-driven experimental asthma in vivo. Methods Three commercially available HDM extracts were analysed for endotoxin levels, protease and chitinase activities and effects on transepithelial resistance, junctional proteins and pro-inflammatory cytokine release in the bronchial epithelial cell line 16HBE and normal human bronchial cells. Furthermore, the effects on epithelial remodelling and airway inflammation were investigated in a mouse model. Results The different HDM extracts varied extensively in their biochemical properties and induced divergent responses in vitro and in vivo. Importantly, the Greer extract, with the lowest serine protease activity, induced the most pronounced effects on epithelial barrier function and CCL20 release in vitro. In vivo, this extract induced the most profound epithelial E-cadherin delocalisation and increase in CCL20, CCL17 and interleukin 5 levels, accompanied by the most pronounced induction of HDM-specific IgE, goblet cell hyperplasia, eosinophilic inflammation and airway hyper-reactivity. Conclusions This study shows the ability of HDM extracts to alter epithelial immune and barrier responses is related to allergic sensitisation but independent of serine/cysteine protease activity.

Indoleamine 2,3-dioxygenase–dependent tryptophan metabolites contribute to tolerance induction during allergen immunotherapy in a mouse model

BACKGROUND The tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) has been implicated in immune suppression and tolerance induction. OBJECTIVE We examined (1) whether IDO activity is required during tolerance induction by allergen immunotherapy or for the subsequent suppressive effects on asthma manifestations and (2) whether tryptophan depletion or generation of its downstream metabolites is involved. METHODS Ovalbumin (OVA)-sensitized and OVA-challenged BALB/c mice that display increased airway responsiveness to methacholine, serum OVA-specific IgE levels, bronchoalveolar eosinophilia, and TH2 cytokine levels were used as a model of allergic asthma. Sensitized mice received subcutaneous optimal (1 mg) or suboptimal (100 microg) OVA immunotherapy. RESULTS Inhibition of IDO by 1-methyl-DL-tryptophan during immunotherapy, but not during inhalation challenge, partially reversed the suppressive effects of immunotherapy on airway eosinophilia and TH2 cytokine levels, whereas airway hyperresponsiveness and serum OVA-specific IgE levels remained suppressed. Administration of tryptophan during immunotherapy failed to abrogate its beneficial effects toward allergic airway inflammation. Interestingly, administration of tryptophan or its metabolites, kynurenine, 3-hydroxykynurenine, and xanthurenic acid, but not 3-hydroxyanthranilinic acid, quinolinic acid, and kynurenic acid, during suboptimal immunotherapy potentiated the reduction of eosinophilia. These effects coincided with reduced TH2 cytokine levels in bronchoalveolar lavage fluid, but no effects on IgE levels were detected. CONCLUSION During immunotherapy, the tryptophan metabolites kynurenine, 3-hydroxykynurenine, and xanthurenic acid generated through IDO contribute to tolerance induction regarding TH2-dependent allergic airway inflammation.

Critical role of aldehydes in cigarette smoke-induced acute airway inflammation.

BackgroundCigarette smoking (CS) is the most important risk factor for COPD, which is associated with neutrophilic airway inflammation. We hypothesize, that highly reactive aldehydes are critical for CS-induced neutrophilic airway inflammation.MethodsBALB/c mice were exposed to CS, water filtered CS (WF-CS) or air for 5 days. Levels of total particulate matter (TPM) and aldehydes in CS and WF-CS were measured. Six hours after the last exposure, inflammatory cells and cytokine levels were measured in lung tissue and bronchoalveolar lavage fluid (BALF). Furthermore, Beas-2b bronchial epithelial cells were exposed to CS extract (CSE) or WF-CS extract (WF-CSE) in the absence or presence of the aldehyde acrolein and IL-8 production was measured after 24 hrs.ResultsCompared to CS, in WF-CS strongly decreased (CS; 271.1 ± 41.5 μM, WF-CS; 58.5 ± 8.2 μM) levels of aldehydes were present whereas levels of TPM were only slightly reduced (CS; 20.78 ± 0.59 mg, WF-CS; 16.38 ± 0.36 mg). The numbers of mononuclear cells in BALF (p<0.01) and lung tissue (p<0.01) were significantly increased in the CS- and WF-CS-exposed mice compared to air control mice. Interestingly, the numbers of neutrophils (p<0.001) in BALF and neutrophils and eosinophils (p<0.05) in lung tissue were significantly increased in the CS-exposed but not in WF-CS-exposed mice as compared to air control mice. Levels of the neutrophil and eosinophil chemoattractants KC, MCP-1, MIP-1α and IL-5 were all significantly increased in lung tissue from CS-exposed mice compared to both WF-CS-exposed and air control mice. Interestingly, depletion of aldehydes in WF-CS extract significantly reduced IL-8 production in Beas-2b as compared to CSE, which could be restored by the aldehyde acrolein.ConclusionAldehydes present in CS play a critical role in inflammatory cytokine production and neutrophilic- but not mononuclear airway inflammation.

Cigarette smoke-induced necroptosis and DAMP release trigger neutrophilic airway inflammation in mice.

Recent data indicate a role for airway epithelial necroptosis, a regulated form of necrosis, and the associated release of damage-associated molecular patterns (DAMPs) in the development of chronic obstructive pulmonary disease (COPD). DAMPs can activate pattern recognition receptors (PRRs), triggering innate immune responses. We hypothesized that cigarette smoke (CS)-induced epithelial necroptosis and DAMP release initiate airway inflammation in COPD. Human bronchial epithelial BEAS-2B cells were exposed to cigarette smoke extract (CSE), and necrotic cell death (membrane integrity by propidium iodide staining) and DAMP release (i.e., double-stranded DNA, high-mobility group box 1, heat shock protein 70, mitochondrial DNA, ATP) were analyzed. Subsequently, BEAS-2B cells were exposed to DAMP-containing supernatant of CS-induced necrotic cells, and the release of proinflammatory mediators [C-X-C motif ligand 8 (CXCL-8), IL-6] was evaluated. Furthermore, mice were exposed to CS in the presence and absence of the necroptosis inhibitor necrostatin-1, and levels of DAMPs and inflammatory cell numbers were determined in bronchoalveolar lavage fluid. CSE induced a significant increase in the percentage of necrotic cells and DAMP release in BEAS-2B cells. Stimulation of BEAS-2B cells with supernatant of CS-induced necrotic cells induced a significant increase in the release of CXCL8 and IL-6, in a myeloid differentiation primary response gene 88-dependent fashion. In mice, exposure of CS increased the levels of DAMPs and numbers of neutrophils in bronchoalveolar lavage fluid, which was statistically reduced upon treatment with necrostatin-1. Together, we showed that CS exposure induces necrosis of bronchial epithelial cells and subsequent DAMP release in vitro, inducing the production of proinflammatory cytokines. In vivo, CS exposure induces neutrophilic airway inflammation that is sensitive to necroptosis inhibition.

TLR-2 Activation Induces Regulatory T Cells and Long-Term Suppression of Asthma Manifestations in Mice

Asthma is a chronic inflammatory disease of the airways characterized by variable airway obstruction and airway hyperresponsiveness (AHR). The T regulatory (Treg) cell subset is critically important for the regulation of immune responses. Adoptive transfer of Treg cells has been shown to be sufficient for the suppression of airway inflammation in experimental allergic asthma. Intervention strategies aimed at expanding the Treg cell population locally in the airways of sensitized individuals are therefore of high interest as a potential therapeutic treatment for allergic airway disease. Here, we aim to test whether long-term suppression of asthma manifestations can be achieved by locally expanding the Treg cell subset via intranasal administration of a TLR-2 agonist. To model therapeutic intervention aimed at expanding the endogenous Treg population in a sensitized host, we challenged OVA-sensitized mice by OVA inhalation with concomitant intranasal instillation of the TLR-2 agonist Pam3Cys, followed by an additional series of OVA challenges. Pam3Cys treatment induced an acute but transient aggravation of asthma manifestations, followed by a reduction or loss of AHR to methacholine, depending on the time between Pam3Cys treatment and OVA challenges. In addition, Pam3Cys-treatment induced significant reductions of eosinophils and increased numbers of Treg cells in the lung infiltrates. Our data show that, despite having adverse acute effects, TLR2 agonist treatment as a therapeutic intervention induces an expansion of the Treg cell population in the lungs and results in long-term protection against manifestation of allergic asthma upon subsequent allergen provocation. Our data indicate that local expansion of Tregs in allergic airway disease is an interesting therapeutic approach that warrants further investigation.

GITR signaling potentiates airway hyperresponsiveness by enhancing Th2 cell activity in a mouse model of asthma

BackgroundAllergic asthma is characterized by airway hyperresponsiveness (AHR) and allergic inflammation of the airways, driven by allergen-specific Th2 cells. The asthma phenotypes and especially AHR are sensitive to the presence and activity of regulatory T (Treg) cells in the lung. Glucocorticoid-induced tumor necrosis factor receptor (GITR) is known to have a co-stimulatory function on effector CD4+ T cells, rendering these cells insensitive to Treg suppression. However, the effects of GITR signaling on polarized Th1 and Th2 cell effector functions are not well-established. We sought to evaluate the effect of GITR signaling on fully differentiated Th1 and Th2 cells and to determine the effects of GITR activation at the time of allergen provocation on AHR and airway inflammation in a Th2-driven mouse model of asthma.MethodsCD4+CD25- cells were polarized in vitro into Th1 and Th2 effector cells, and re-stimulated in the presence of GITR agonistic antibodies to assess the effect on IFNγ and IL-4 production. To evaluate the effects of GITR stimulation on AHR and allergic inflammation in a mouse asthma model, BALB/c mice were sensitized to OVA followed by airway challenges in the presence or absence of GITR agonist antibodies.ResultsGITR engagement potentiated cytokine release from CD3/CD28-stimulated Th2 but not Th1 cells in vitro. In the mouse asthma model, GITR triggering at the time of challenge induced enhanced airway hyperresponsiveness, serum IgE and ex vivo Th2 cytokine release, but did not increase BAL eosinophilia.ConclusionGITR exerts a differential effect on cytokine release of fully differentiated Th1 and Th2 cells in vitro, potentiating Th2 but not Th1 cytokine production. This effect on Th2 effector functions was also observed in vivo in our mouse model of asthma, resulting in enhanced AHR, serum IgE responses and Th2 cytokine production. This is the first report showing the effects of GITR activation on cytokine production by polarized primary Th1 and Th2 populations and the relevance of this pathway for AHR in mouse models for asthma. Our data provides crucial information on the mode of action of the GITR signaling, a pathway which is currently being considered for therapeutic intervention.

Suppression of Th2-Driven Airway Inflammation by Allergen Immunotherapy Is Independent of B Cell and Ig Responses in Mice

Allergen-specific immunotherapy (IT) uniquely renders long-term relief from allergic symptoms and is associated with elevated serum levels of allergen-specific IgG and IgA. The allergen-specific IgG response induced by IT treatment was shown to be critical for suppression of the immediate phase of the allergic response in mice, and this suppression was partially dependent on signaling through FcγRIIB. To investigate the relevance of the allergen-specific IgG responses for suppression of the Th2-driven late-phase allergic response, we performed IT in a mouse model of allergic asthma in the absence of FcγRIIB or FcγRI/FcγRIII signaling. We found that suppression of Th2 cell activity, allergic inflammation, and allergen-specific IgE responses is independent of FcγRIIB and FcγRI/FcγRIII signaling. Moreover, we show that the IT-induced allergen-specific systemic IgG or IgA responses and B cell function are dispensable for suppression of the late-phase allergic response by IT treatment. Finally, we found that the secretory mucosal IgA response also is not required for suppression of the Th2-driven allergic inflammation by IT. These data are in contrast to the suppression of the immediate phase of the allergic response, which is critically dependent on the induced allergen-specific serum IgG response. Hence, IT-induced suppression of the immediate and late phases of the allergic response is governed by divergent and independent mechanisms. Our data show that the IT-induced suppression of the Th2 cell-dependent late-phase allergic response is independent of the allergen-specific IgG and IgA responses that are associated with IT treatment.

Pim1 kinase protects airway epithelial cells from cigarette smoke-induced damage and airway inflammation

Exposure to cigarette smoke (CS) is the main risk factor for developing chronic obstructive pulmonary disease and can induce airway epithelial cell damage, innate immune responses, and airway inflammation. We hypothesized that cell survival factors might decrease the sensitivity of airway epithelial cells to CS-induced damage, thereby protecting the airways against inflammation upon CS exposure. Here, we tested whether Pim survival kinases could protect from CS-induced inflammation. We determined expression of Pim kinases in lung tissue, airway inflammation, and levels of keratinocyte-derived cytokine (KC) and several damage-associated molecular patterns in bronchoalveolar lavage in mice exposed to CS or air. Human bronchial epithelial BEAS-2B cells were treated with CS extract (CSE) in the presence or absence of Pim1 inhibitor and assessed for loss of mitochondrial membrane potential, induction of cell death, and release of heat shock protein 70 (HSP70). We observed increased expression of Pim1, but not of Pim2 and Pim3, in lung tissue after exposure to CS. Pim1-deficient mice displayed a strongly enhanced neutrophilic airway inflammation upon CS exposure compared with wild-type controls. Inhibition of Pim1 activity in BEAS-2B cells increased the loss of mitochondrial membrane potential and reduced cell viability upon CSE treatment, whereas release of HSP70 was enhanced. Interestingly, we observed release of S100A8 but not of double-strand DNA or HSP70 in Pim1-deficient mice compared with wild-type controls upon CS exposure. In conclusion, we show that expression of Pim1 protects against CS-induced cell death in vitro and neutrophilic airway inflammation in vivo. Our data suggest that the underlying mechanism involves CS-induced release of S100A8 and KC.

Genetic variation associates with susceptibility for cigarette smoke-induced neutrophilia in mice

Neutrophilic airway inflammation is one of the major hallmarks of chronic obstructive pulmonary disease and is also seen in steroid resistant asthma. Neutrophilic airway inflammation can be induced by different stimuli including cigarette smoke (CS). Short-term exposure to CS induces neutrophilic airway inflammation in both mice and humans. Since not all individuals develop extensive neutrophilic airway inflammation upon smoking, we hypothesized that this CS-induced innate inflammation has a genetic component. This hypothesis was addressed by exposing 30 different inbred mouse strains to CS or control air for 5 consecutive days, followed by analysis of neutrophilic lung inflammation. By genomewide haplotype association mapping, we identified four susceptibility genes with a significant association to lung tissue levels of the neutrophil marker myeloperoxidase under basal conditions and an additional five genes specifically associated with CS-induced tissue MPO levels. Analysis of the expression levels of the susceptibility genes by quantitative RT-PCR revealed that three of the four genes associated with CS-induced tissue MPO levels had CS-induced changes in gene expression levels that correlate with CS-induced airway inflammation. Most notably, CS exposure induces an increased expression of the coiled-coil domain containing gene, Ccdc93, in mouse strains susceptible for CS-induced airway inflammation whereas Ccdc93 expression was decreased upon CS exposure in nonsusceptible mouse strains. In conclusion, this study shows that CS-induced neutrophilic airway inflammation has a genetic component and that several genes contribute to the susceptibility for this response.

Identification of the Mhc Region as an Asthma Susceptibility Locus in Recombinant Congenic Mice

Mouse models of allergic asthma are characterized by airway hyperreactivity (AHR), Th2-driven eosinophilic airway inflammation, high allergen-specific IgE (anti-OVA IgE) levels in serum, and airway remodeling. Because asthma susceptibility has a strong genetic component, we aimed to identify new asthma susceptibility genes in the mouse by analyzing the asthma phenotypes of the Leishmania major resistant (lmr) recombinant congenic (RC) strains. The lmr RC strains are derived from C57BL/6 and BALB/c intercrosses and carry congenic loci on chromosome 17 (lmr1) and 9 (lmr2) in both backgrounds. Whereas the lmr2 locus on chromosome 9 contributes to a small background-specific effect on anti-OVA IgE and AHR, the lmr1 locus on chromosome 17 mediates a strong effect on Th2-driven eosinophilic airway inflammation and background-specific effects on anti-OVA IgE and AHR. The lmr1 locus contains almost 600 polymorphic genes. To narrow down this number of candidate genes, we performed genome-wide transcriptional profiling on lung tissue from C.lmr1 RC mice and BALB/c control mice. We identified a small number of differentially expressed genes located within the congenic fragment, including a number of Mhc genes, polymorphic between BALB/c and C57Bl/6. The analysis of asthma phenotypes in the C.B10-H2b RC strain, carrying the C57Bl/6 haplotype of the Mhc locus in a BALB/c genetic background, reveals a strikingly similar asthma phenotype compared with C.lmr1, indicating that the differentially expressed genes located within the C.B10-H2b congenic fragment are the most likely candidate genes to contribute to the reduced asthma phenotypes associated with the C57Bl/6 allele of lmr1.