MMP-19 Deficiency Promotes Tenascin-C Accumulation and Allergen-induced Airway Inflammation.

Matrix metalloproteinases (MMPs) recently appeared as key regulators of inflammation, allowing recruitment and clearance of inflammatory cells and modifying the biological activity of many peptidic mediators by cleavage. MMP-19 is a newly described MMP and preferentially cleaves matrix proteins such as collagens and tenascin-C. The role of MMP-19 in asthma has not been described to date. The purpose of the present study was to assess MMP-19 expression in a murine asthma model and to address biological effects of MMP-19 deficiency in mice. Allergenexposed wild-type (WT) mice displayed an increased expression of MMP-19 mRNA and an increased number of MMP-19-positive cells in the lungs detected by immunohistochemistry. After allergen challenge of MMP-19 knockout (MMP-19-/-) mice, an exacerbated eosinophilic inflammation was detected in bronchoalveolar lavage fluid and bronchial tissue along with an increased airway responsiveness to methacholine. A shift towards increased Th2-driven inflammation in MMP-19-/- mice was demonstrated by 1) increased numbers of cells expressing the IL-33 receptor T1/ST2 in lung parenchyma, 2) increased IgG1 levels in serum and 3) higher levels of IL-13 and CCL11 in lung extracts. Tenascin-C was found accumulated in peribronchial areas of MMP-19-/- after allergen challenges as assessed by Western blot and immunohistochemistry analysis. We conclude that MMP-19 is a new mediator in asthma, preventing tenascin-C accumulation and directly or indirectly controlling Th2-driven airway eosinophilia and airway hyperreactivity. Our data suggest that MMP-19 might act on Th2 inflammation homeostasis through preventing tenascin protein accumulation

Inflammatory signatures for eosinophilic versus neutrophilic allergic pulmonary inflammation reveal critical regulatory checkpoints.

Contrarily to the Th-2-bias and eosinophil-dominated bronchial inflammation encountered in most asthmatics, other patients may exhibit neutrophil-predominant asthma sub-phenotypes along with Th-1 and Th-17 cells. However, the etiology of many neutrophil-dominated asthma sub-phenotypes remains ill-understood, in part due to a lack of appropriate experimental models. To better understand the distinct immune-pathological features of eosinophilic versus neutrophilic asthma types, we developed an Ovalbumin (OVA)-based mouse model of neutrophil-dominated allergic pulmonary inflammation. Consequently, we probed for particular inflammatory signatures and checkpoints underlying the immune-pathology in this new model as well as in a conventional, eosinophil-dominated asthma model. Briefly, mice were OVA-sensitized using either aluminium hydroxide (alum) or Complete Freund's (CFA)-adjuvants followed by OVA aerosol challenge. T-cell, granulocyte and inflammatory mediator profiles were determined along with alveolar macrophage genome-wide transcriptome profiling. In contrast to the Th-2-dominated phenotype provoked by alum, OVA/CFA-adjuvant-based sensitization followed by allergen challenge elicited a pulmonary inflammation that was poorly controlled by dexamethasone, and in which Th-1 and Th-17 cells additionally participated. Analysis of the overall pulmonary and alveolar macrophage inflammatory mediator profiles revealed remarkable similarities between both models. Nevertheless, we observed pronounced differences in the IL-12/IFN-γ axis and its control by IL-18 and IL-18 Binding Protein (BP), but also in macrophage arachidonic acid metabolism and expression of T-cell instructive ligands. These differential signatures, superimposed onto a generic inflammatory signature, denote distinctive inflammatory checkpoints potentially involved in orchestrating neutrophil-dominated asthma. Key words: neutrophil-predominant asthma, allergic inflammation, alveolar macrophage, transcriptome, mouse models

Nebulized Anti-IL-13 Monoclonal Antibody Fab' Fragment Reduces Allergen-Induced Asthma

Rationale: Interleukin-13 (IL-13) is a prototypic Th2 cytokine and a central mediator of the complex cascade of events leading to asthmatic phenotype. Indeed, IL-13 plays key roles in IgE synthesis, bronchial hyperresponsiveness, mucus hypersecretion, subepithelial fibrosis and eosinophil infiltration. Objectives: We assessed the potential efficacy of inhaled anti-IL-13 monoclonal antibody Fab' fragment on allergen-induced airway inflammation, hyperresponsiveness and remodeling in an experimental model of allergic asthma. Anti-IL-13 Fab' was administered to mice as a liquid aerosol generated by inExpose® inhalation system in a tower allowing a nose-only exposure. Methods: BALB/c mice were treated by PBS, anti-IL-13 Fab' or A33 Fab' fragment and subjected to ovalbumin (OVA) exposure for 1 and 5 weeks (short term (ST) and long term (LT) protocols). Measurements and Main Results: Our data demonstrate a significant anti-asthma effect following nebulization of anti-IL-13 Fab' in a model of asthma driven by allergen exposure as compared to saline and non-immune Fab fragments. In short and long terms protocols, administration of the anti-IL-13 Fab' by inhalation significantly decreased bronchial responsiveness to methacholine, BALF eosinophilia, inflammatory cell infiltration in lung tissue, and many features of airway remodeling. Levels of pro-inflammatory mediators and matrix metalloprotease levels were significantly lower in lung parenchyma of mice treated with anti-IL-13 Fab'. Conclusions: These data demonstrate that an inhaled anti-IL-13 Fab' significantly reduces airway inflammation, hyperresponsiveness and remodeling. Specific neutralization of IL-13 in the lungs using an inhaled anti-IL-13 Fab' could represent a novel and effective therapy for the treatment of asthma

ADAMTS-1 metalloproteinase induces a stromal reaction and propotes tumor development in mice.

ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin motifs), the first described member of the ADAMTS family, is differentially expressed in various tumors. However, its exact role in tumor development and progression is still unclear. The aim of this study was to investigate the effects of ADAMTS-1 transfection in a bronchial epithelial tumor cell line (BZR) and its potential to modulate tumor development. ADAMTS-1 overexpression did not affect in vitro cell properties such as (a) proliferation in two-dimensional culture, (b) proliferation in three-dimensional culture, (c) anchorageindependent growth in soft agar, (d) cell migration and invasion in modified Boyden chamber assay, (e) angiogenesis in the aortic ring assay, and (f) cell apoptosis. In contrast, ADAMTS-1 stable transfection in BZR cells accelerated the in vivo tumor growth after s.c. injection into severe combined immunodeficient mice. It also promoted a stromal reaction characterized by myofibroblast infiltration and excessive matrix deposition. These features are, however, not observed in tumors derived from cells overexpressing a catalytically inactive mutant of ADAMTS-1. Conditioned media from ADAMTS-1–overexpressing cells display a potent chemotactic activity toward fibroblasts. ADAMTS-1 overexpression in tumors was associated with increased production of matrix metalloproteinase-13, fibronectin, transforming growth factor B (TGF-B), and interleukin-1B (IL-1B). Neutralizing antibodies against TGF-B and IL-1B blocked the chemotactic effect of medium conditioned by ADAMTS-1–expressing cells on fibroblasts, showing the contribution of these factors in ADAMTS-1–induced stromal reaction. In conclusion, we propose a new paradigm for catalytically active ADAMTS-1 contribution to tumor development, which consists of the recruitment of fibroblasts involved in tumor growth and tumor-associated stroma remodeling

Potential Therapeutic Target Discovery by 2D-DIGE Proteomic Analysis in Mouse Models of Asthma

As asthma physiopathology is complex and not fully understood to date; it is expected that new key mediators are still to be unveiled in this disease. The main objective of this study was to discover potential new target proteins with a molecular weight >20 kDa by using two-dimensional differential in-gel electrophoresis (2D-DIGE) on lung parenchyma extracts from control or allergen-exposed mice (ovalbumin). Two different mouse models leading to the development of acute airway inflammation (5 days allergen exposure) and airway remodeling (10 weeks allergen exposure) were used. This experimental setting allowed the discrimination of 33 protein spots in the acute inflammation model and 31 spots in the remodeling model displaying a differential expression. Several proteins were then identified by MALDI-TOF/TOF MS. Among those differentially expressed proteins, PDIA6, GRP78, Annexin A6, hnRPA3, and Enolase display an increased expression in lung parenchyma from mice exposed to allergen for 5 days. Conversely, Apolipoprotein A1 was shown to be decreased after allergen exposure in the same model. Analysis on lung parenchyma of mice exposed to allergens for 10 weeks showed decreased calreticulin levels. Changes in the levels of those different mediators were confirmed by Western blot and immunohistochemical analysis. Interestingly, alveolar macrophages isolated from lungs in the acute inflammation model displayed enhanced levels of GRP78. Moreover, intratracheal instillation of anti-GRP78 siRNA in allergen-exposed animals led to a decrease in eosinophilic inflammation and bronchial hyperresponsiveness. This study unveils new mediators of potential importance that are up- and down-regulated in asthma. Among up-regulated mediators, GRP-78 appears as a potential new therapeutic target worthy of further investigations

Mouse models of asthma: a comparison between C57BL/6 and BALB/c strains regarding bronchial responsiveness, inflammation, and cytokine production

Objective Animal models of asthma mimic major features of human disease. Since the genetic background of experimental animals might affect hyperresponsiveness and inflammation, we studied its potential influence and the mechanisms leading to differences in strains. Methods We applied a mouse model of allergic asthma to BALB/c and C57BL/6 mice. Results BALB/c mice displayed greater levels of airway reactivity to methacholine than C57BL/6 mice. Moreover, BALB/c mice exhibited higher numbers of mast cells in lung tissue when compared to C57BL/6. On the contrary, eosinophil and neutrophil counts in bronchoalveolar lavage fluid (BALF) as well as peribronchial eosinophilia were greater in C57BL/6. IL (Interleukin)-4, IL-5, IL-13, and CCL11 levels measured in whole-lung extracts were higher in BALB/c, while, in sharp contrast, CCL11 and CCL5 levels were higher in BALF of C57BL/6 mice. Conclusions We observed phenotypic differences between C57BL/6 and BALB/c mice in an asthma model with different distributions of pro-inflammatory cytokines and inflammatory cells