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Osteoarthritis (OA) is recognized as being a cellular senescence-linked disease. Intra-articular injections of glucocorticoids (GC) are frequently used in knee OA to treat synovial effusion but face controversies about toxicity. We investigated the influence of GC on cellular senescence hallmarks and senescence induction in fibroblast-like synoviocytes (FLS) from OA patients and mesenchymal stem cells (MSC). Methods: Cellular senescence was assessed via the proliferation rate, β-galactosidase staining, DNA damage and CKI expression (p21, p16INK4A). Experimental senescence was induced by irradiation. Results: The GC prednisolone did not induce an apparent senescence phenotype in FLS, with even higher proliferation, no accumulation of β-galactosidase-positive cells nor DNA damage and reduction in p21mRNA, only showing the enhancement of p16INK4A. Prednisolone did not modify experimental senescence induction in FLS, with no modulation of any senescence parameters. Moreover, prednisolone did not induce a senescence phenotype in MSC: despite high β-galactosidase-positive cells, no reduction in proliferation, no DNA damage and no CKI enhancement was observed. Conclusions: We provide reassuring in vitro data about the use of GC regarding cellular senescence involvement in OA: the GC prednisolone did not induce a senescent phenotype in OA FLS (the proliferation ratio was even higher) and in MSC and did not worsen cellular senescence establishment.

Osteoarthritis (OA) synovial membrane is mainly characterized by low-grade inflammation, hyperplasia with increased cell proliferation and fibrosis. We previously underscored a critical role for CEMIP in fibrosis of OA cartilage. However, its role in OA synovial membrane remains unknown. An in vitro model with fibroblast-like synoviocytes from OA patients and an in vivo model with collagenase-induced OA mice were used to evaluate CEMIP-silencing effects on inflammation, hyperplasia and fibrosis. Our results showed that i. CEMIP expression was increased in human and mouse inflamed synovial membrane; ii. CEMIP regulated the inflammatory response pathway and inflammatory cytokines production in vitro and in vivo; iii. CEMIP induced epithelial to mesenchymal transition pathway and fibrotic markers in vitro and in vivo; iv. CEMIP increased cell proliferation and synovial hyperplasia; v. CEMIP expression was increased by inflammatory cytokines and by TGF-β signaling; vi. anti-fibrotic drugs decreased CEMIP expression. All these findings highlighted the central role of CEMIP in OA synovial membrane development and underscored that targeting CEMIP could be a new therapeutic approach.

Simple Summary Muscle wasting during cancer is recognized as an independent predictor of mortality. The aim of this study was to characterize the changes in the muscle secretome associated with cancer cachexia to gain a better understanding of the mechanisms involved and to identify secreted proteins which may reflect this wasting process. Our study demonstrated that skeletal muscle is a source of several acute phase reactants during cancer cachexia that may hold the key to a cachexia-specific signature. Future work will have to determine whether some of these acute phase reactants contribute to and/or reflect the muscle atrophy caused by cancer, therefore representing potential therapeutic targets and/or biomarkers of cancer cachexia. Abstract Loss of skeletal muscle mass in cancer cachexia is recognized as a predictor of mortality. This study aimed to characterize the changes in the muscle secretome associated with cancer cachexia to gain a better understanding of the mechanisms involved and to identify secreted proteins which may reflect this wasting process. The changes in the muscle proteome of the C26 model were investigated by label-free proteomic analysis followed by a bioinformatic analysis in order to identify potentially secreted proteins. Multiple reaction monitoring and Western blotting were used to verify the presence of candidate proteins in the circulation. Our results revealed a marked increased muscular production of several acute phase reactants (APR: Haptoglobin, Serine protease inhibitor A3N, Complement C3, Serum amyloid A-1 protein) which are released in the circulation during C26 cancer cachexia. This was confirmed in other models of cancer cachexia as well as in cancer patients. Glucocorticoids and proinflammatory cytokines are responsible for an increased production of APR by muscle cells. Finally, their muscular expressions are strongly positively correlated with body weight loss as well as the muscular induction of atrogens. Our study demonstrates therefore a marked increased production of APR by the muscle in cancer cachexia.

Introduction L’arthrose est maintenant reconnue comme une pathologie associee a la senescence cellulaire, caracterisee par un arret robuste de la proliferation cellulaire, l’apparition de degâts a l’ADN, l’expression d’une activite enzymatique metabolique comme la β-galactosidase acide ou encore l’expression d’inhibiteurs des proteines-kinases dependantes des cyclines (CKI) p16INK4A et p21, induisant un secretome catabolique et pro-inflammatoire. A ce jour, il n’y a pas de traitement curatif de l’arthrose et les infiltrations intra-articulaires de glucocorticoides restent toujours largement utilisees dans la gonarthrose pour lutter contre l’inflammation de la membrane synoviale. Toutefois, ces infiltrations restent debattues compte tenu d’une toxicite potentielle sur l’articulation et d’une possible augmentation des marqueurs de senescence par les glucocorticoides dans des etudes in vitro. L’objectif de ce travail est d’etudier l’influence des glucocorticoides sur les marqueurs de senescence et sur l’induction de la senescence dans des fibroblastes synoviaux (FS) issus de patients arthrosiques et dans des cellules souches mesenchymateuses (CSM). Materiels et methodes In vitro, les FS et les CSM sont stimules pendant 7 jours avec le glucocorticoide prednisolone a trois doses differentes (1,5 et 10 μM), puis etudies aux 8e et au 15e jours. Les marqueurs de senescence cellulaire sont etudies par les techniques suivantes : proliferation et viabilite cellulaire (BrdU et MTS), coloration β-galactosidase acide, lesions a l’ADN (immunofluorescence pour ɣH2AX) et expression (Western-Blot et RT-qPCR) des CKI p16INK4A et p21. La senescence est induite experimentalement par irradiation. p16INK4A est egalement etudie dans des biopsies de membrane synoviale par immunohistochimie (CINtec, Roche). Resultats (1) Nous confirmons la presence de cellules senescentes positives pour p16INK4A dans les membranes synoviales de patients arthrosiques. Ces cellules sont significativement plus presentes dans les stades avances (Kellgren & Lawrence grade 2-4 versus grade 0–1). (2) La prednisolone n’induit pas de phenotype senescent dans les FS, quelle que soit la dose utilisee : nous n’observons pas de reduction de la proliferation cellulaire (au contraire, le taux de proliferation est plus eleve dans les FS stimules par la prednisolone), sans accumulation significative de cellules positives pour la β-galactosidase acide ni apparition de lesions a l’ADN. Les taux d’ARNm codant pour p21 sont diminues, alors que seule une augmentation de p16INK4A est retrouvee. (3) La prednisolone n’aggrave pas l’induction experimentale de la senescence dans les FS apres irradiation : aucune modulation des parametres de senescence n’est observee. (4) La prednisolone n’induit pas de phenotype senescent dans les CSM : malgre l’augmentation du pourcentage de cellules positives pour la β-galactosidase apres stimulation par la prednisolone, nous n’observons aucune reduction de la proliferation cellulaire, aucune accumulation de degâts a l’ADN et pas d’augmentation d’expression des CKI p16INK4A et p21. Discussion La prednisolone n’induit pas de phenotype senescent dans les FS de patients arthrosiques et les CSM, avec au contraire un taux de proliferation plus eleve dans les FS apres stimulation par la prednisolone. Il n’y a pas d’effet deletere (mais pas non plus d’effet positif) sur l’etablissement de la senescence cellulaire apres induction experimentale dans les cellules arthrosiques. Conclusion Les cellules senescentes sont presentes dans la membrane synoviale arthrosique, mais nos donnees in vitro sont rassurantes quant a l’absence d’induction ou d’aggravation de la senescence cellulaire apres stimulation par un derive cortisone.

International audience; Living in a microbe-rich environment reduces the risk of developing asthma. Exposure of humans or mice to unmethylated CpG DNA (CpG) from bacteria reproduces these protective effects, suggesting a major contribution of CpG to microbe-induced asthma resistance. However, how CpG confers protection remains elusive. We found that exposure to CpG expanded regulatory lung interstitial macrophages (IMs) from monocytes infiltrating the lung or mobilized from the spleen. Trafficking of IM precursors to the lung was independent of CCR2, a chemokine receptor required for monocyte mobilization from the bone marrow. Using a mouse model of allergic airway inflammation, we found that adoptive transfer of IMs isolated from CpG-treated mice recapitulated the protective effects of CpG when administered before allergen sensitization or challenge. IM-mediated protection was dependent on IL-10, given that Il10(-/-) CpG-induced IMs lacked regulatory effects. Thus, the expansion of regulatory lung IMs upon exposure to CpG might underlie the reduced risk of asthma development associated with a microbe-rich environment.

Increases in eosinophil numbers are associated with infection and allergic diseases, including asthma, but there is also evidence that eosinophils contribute to homeostatic immune processes. In mice, the normal lung contains resident eosinophils (rEos), but their function has not been characterized. Here, we have reported that steady-state pulmonary rEos are IL-5-independent parenchymal Siglec-FintCD62L+CD101lo cells with a ring-shaped nucleus. During house dust mite-induced airway allergy, rEos features remained unchanged, and rEos were accompanied by recruited inflammatory eosinophils (iEos), which were defined as IL-5-dependent peribronchial Siglec-FhiCD62L-CD101hi cells with a segmented nucleus. Gene expression analyses revealed a more regulatory profile for rEos than for iEos, and correspondingly, mice lacking lung rEos showed an increase in Th2 cell responses to inhaled allergens. Such elevation of Th2 responses was linked to the ability of rEos, but not iEos, to inhibit the maturation, and therefore the pro-Th2 function, of allergen-loaded DCs. Finally, we determined that the parenchymal rEos found in nonasthmatic human lungs (Siglec-8+CD62L+IL-3Rlo cells) were phenotypically distinct from the iEos isolated from the sputa of eosinophilic asthmatic patients (Siglec-8+CD62LloIL-3Rhi cells), suggesting that our findings in mice are relevant to humans. In conclusion, our data define lung rEos as a distinct eosinophil subset with key homeostatic functions.

Background: Overall clinical outcome for advanced lung cancer remains very disappointing despite recent advances in treatment. Curcumin has been reported as potentially active against cancer. Methods: Owing to poor curcumin solubility, we have used cyclodextrins (CD) as an excipient allowing a considerable increase of aqueous solubility and bioavailability of curcumin. The effects of solubilised curcumin have been evaluated in cell cultures as well as in an in vivo orthotopic lung tumour mouse model. Results: Cell proliferation was reduced while apoptosis rates were increased when lung epithelial tumour cells were cultured in the presence of curcumin–CD complexes. For in vivo experiments, cells were grafted into lungs of C57Bl/6 mice treated by an oral administration of a non-soluble form of curcumin, CDs alone or curcumin–CD complexes, combined or not with gemcitabine. The size of orthotopically implanted lung tumours was reduced upon curcumin complex administration as compared with treatments with placebo or non-solubilised curcumin. Moreover, curcumin potentiated the gemcitabine-mediated antitumour effects. Conclusion: Our data demonstrate that curcumin, when given orally in a CD-solubilised form, reduces lung tumour size in vivo. In vitro experiments show impaired tumour cell proliferation and increased cell apoptosis. Moreover, our data underline a potential additive effect of curcumin with gemcitabine thus providing an efficient therapeutic option for antilung cancer therapy.

Among proteases, metalloproteases are implicated in tissue remodeling, as shown in numerous diseases including allergy. ADAMs (A Disintegrin And Metalloprotease) metalloproteases are implicated in physiologic processes such as cytokine and growth factor shedding, cell migration, adhesion, or repulsion. Our aim was to measure ADAM-12 expression in airway epithelium and to define its role during the allergic response. To raise this question, we analyzed the ADAM-12 expression ex vivo after allergen exposure in patients with allergic rhinitis and in vitro in cultured primary human airway epithelial cells (AEC). Clones of BEAS-2B cells transfected with the full-length form of ADAM-12 were generated to study the consequences of ADAM-12 up-regulation on AEC function. After allergen challenge, a strong increase of ADAM-12 expression was observed in airway epithelium from patients with allergic rhinitis but not from control subjects. In contrast with the other HB-epidermal growth factor sheddases, ADAM-10 and -17, TNF-alpha in vitro increased the expression of ADAM-12 by AEC, an effect amplified by IL-4 and IL-13. Up-regulation of ADAM-12 in AEC increased the expression of alpha3 and alpha4 integrins and to the modulation of cell migration on fibronectin but not on collagen. Moreover, overexpression of ADAM-12 in BEAS-2B enhanced the secretion of CXCL1 and CXCL8 and their capacity to recruit neutrophils. CD47 was strongly decreased by ADAM-12 overexpression, a process associated with a reduced adhesion of neutrophils. These effects were mainly dependent on epidermal growth factor receptor activation. In summary, ADAM-12 is produced during allergic reaction by AEC and might increase neutrophil recruitment within airway mucosa.

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) anchorage-independent 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 β (TGF-β), and interleukin-1β (IL-1β). Neutralizing antibodies against TGF-β and IL-1β 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. [Cancer Res 2008;68(22):9541–50]

A disintegrin and metalloproteinases (ADAMs) are a recently discovered family of proteins that share the metalloproteinase domain with matrix metalloproteinases (MMPs). Among this family, structural features distinguish the membrane-anchored ADAMs and the secreted ADAMs with thrombospondin motifs referred to as ADAMTSs. By acting on a large panel of membrane-associated and extracellular substrates, they control several cell functions such as adhesion, fusion, migration and proliferation. The current review addresses the contribution of these proteinases in the positive and negative regulation of cancer progression as mainly mediated by the regulation of growth factor activities and integrin functions.

Introduction: As opposed to their well-characterized contributions to inflammatory processes, tissue eosinophils are now also thought to contribute to immune homeostasis at mucosal sites such as the gut. Yet, whether such steady-state eosinophils exist in the lung is currently unclear. Methods: We combined flow cytometry phenotyping and sorting, immuno-histochemistry, microscopy and microarray analyses to identify and characterize lung eosinophil populations in naive versus host dust mite (HDM)-treated mice. We then used eosinophil-deficient transgenic mice as well as models of allergic airway sensitization to assess their functions. Results: In the lung of naive and HDM-treated mice, we identified resident eosinophils (rEOS) as eosinophilic cells with a distinct phenotype surface (SiglecF + CD125 int ), morphology (donut shape nucleus), localization (in parenchyma) and transcriptional signature than lung inflammatory eosinophils (iEOS) found in HDM-treated mice (SiglecF high CD125 int cells, polylobuled nucleus, around bronchi). We showed that mice deficient for eosinophils (ΔdblGATA mice) are deficient in rEos and are more sensitive to HDM-induced allergic airway sensitization than control mice, suggesting a role for rEOS in inhibiting allergen-induced sensitization. Moreover, dendritic cell(DC)-rEos co-culture models suggest that rEos may act directly on DCs to prevent development of airway allergy. Conclusion: We report on a previously unknown mouse lung-rEOS population which is distinct from lung iEOS and exhibit negative immunoregulatory roles according to experimental data from mouse models. We are now investigating the potential presence of such rEos in the human lung.

Introduction: To ensure appropriate responses to foreign components present in the environment, the lung is endowed with an important network of immune cells. Among these cells, macrophages are tissue-resident phagocytes that play an essential role in the onset and the regulation of immune responses. If alveolar macrophages (AMs) were described decades ago, the last few years have seen the outcome of a new understanding of their ontogeny but also the characterization of a new macrophage subset, the interstitial macrophages (IMs). These discoveries highlight the complexity of the monocyte/macrophage axis in the lung. Yet, precise description of the whole monocyte/macrophage network in the lung is lacking. Materials and Methods: Lung-resident monocytes/macrophages were selected as F4/80 + tissue-resident cells using different approaches, such as intravenous injections of clodronate liposomes and fluorochrome-associated antibodies or immunohistochemistry. We studied their origin at the steady state and under stress condition using transgenic animals and bone marrow chimerism. To further assess their function, local stimulation with various TLR ligands were used. Results: In addition to AMs and IMs, we identified a Ly6c + tissue-resident monocyte which does not derive from classical blood monocytes. Such tissue monocytes are able to proliferate in vivo upon stimulation with some, but not all TLR ligands to induce a new population of immunosuppressive lung macrophages. Conclusions: This study identifies the existence of a local pool of lung-resident monocytes able to proliferate and generate new immunosuppressive lung macrophages in response to specific environmental stimuli.

ADAMs (a disintegrin and metalloprotease) constitute a family of cell surface proteins containing disintegrin and metalloprotease domains which associate features of adhesion molecules and proteases. ADAMTSs (a disintegrin and metalloprotease with thrombospondin motifs) bear thrombospondin type I motifs in C-terminal extremity, and most of them are secreted proteins. Because genetic studies have shown that ADAM-33 gene polymorphisms are associated with asthma, we designed this study to assess mRNA expression profile of several ADAM and ADAMTS proteases in sputum from patients with asthma and to investigate the relationship between expression of these proteases and asthma-associated inflammation and airway obstruction. mRNA expression profile of selected ADAM and ADAMTS proteinases (ADAM-8, -9, -10, -12, -15, -17, and -33; ADAMTS-1, -2, -15, -16, -17, -18, and -19), their physiological inhibitors TIMP-1 and TIMP-3, and RECK, a membrane-anchored MMP activity regulator, was obtained by RT-PCR analysis performed on cells collected by sputum induction from 21 patients with mild to moderate asthma and 17 healthy individuals. mRNA levels of ADAM-8, ADAM-9, ADAM-12, TIMP-1, and TIMP-3 were significantly increased, whereas mRNA levels coding for ADAMTS-1, ADAMTS-15, and RECK were significantly decreased in patients with asthma compared with control patients. ADAM-8 expression was negatively correlated with the forced expiratory volume at the first second (FEV1) (r = −0.57, P < 0.01), whereas ADAMTS-1 and RECK expressions were positively correlated to FEV1 (r = 0.45, P < 0.05, and r = 0.55, P = 0.01, respectively). We conclude that expression of ADAMs and ADAMTSs and their inhibitors is modulated in airways from patients with asthma and that these molecules may play a role in the pathogenesis of asthma.

International audience; A Disintegrin and Metalloprotease (ADAM) are transmembrane proteases displaying multiple functions. ADAM with ThromboSpondin-like motifs (ADAMTS) are secreted proteases characterised by thrombospondin (TS) motifs in their C-terminal domain. The aim of this work was to evaluate the expression pattern of ADAMs and ADAMTS in non small cell lung carcinomas (NSCLC) and to investigate the possible correlation between their expression and cancer progression. Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot and immunohistochemical analyses were performed on NSCLC samples and corresponding nondiseased tissue fragments. Among the ADAMs evaluated (ADAM-8, -9, -10, -12, -15, -17, ADAMTS-1, TS-2 and TS-12), a modulation of ADAM-12 and ADAMTS-1 mRNA expression was observed. Amounts of ADAM-12 mRNA transcripts were increased in tumour tissues as compared to the corresponding controls. In sharp contrast, ADAMTS-1 mRNA levels were significantly lower in tumour tissues when compared to corresponding nondiseased lung. These results were corroborated at the protein level by Western blot and immunohistochemistry. A positive correlation was observed between the mRNA levels of ADAM-12 and those of two vascular endothelial growth factor (VEGF)-A isoforms (VEGF-A(165) and VEGF-A(121)). Taken together, these results providing evidence for an overexpression of ADAM-12 and a lower expression of ADAMTS-1 in non-small-cell lung cancer suggest that these proteases play different functions in cancer progression.

Inhalation aerosols offer a targeted therapy for respiratory diseases. However, the therapeutic efficacy of inhaled biopharmaceuticals is limited by the rapid clearance of macromolecules in the lungs. The aim of this research was to study the effects of the PEGylation of antibody fragments on their local residence time after administration to the respiratory tract. We demonstrate that the conjugation of a two-armed 40-kDa polyethylene glycol (PEG) chain to anti-interleukin-17A (IL-17A) F(ab')2 and anti-IL-13 Fab' greatly prolonged the presence of these fragments within the lungs of mice. The content of PEGylated antibody fragments within the lungs plateaued up to 4hours post-delivery, whereas clearance of unconjugated proteins started immediately after administration. Forty-eight hours post-delivery, F(ab')2 and Fab' content in the lungs had decreased to 10 and 14 % of the dose initially deposited, respectively. However, this value was 40 % for both PEG40-F(ab')2 and PEG40-Fab'. The prolonged pulmonary residency of the anti-IL-17A PEG40-F(ab')2 translated into an improved efficacy in reducing lung inflammation in a murine model of house dust mite-induced lung inflammation. We demonstrate that PEGylated proteins were principally retained within the lung lumen rather than the nasal cavities or lung parenchyma. In addition, we report that PEG increased pulmonary retention of antibody fragments through mucoadhesion and escape from alveolar macrophages rather than increased hydrodynamic size or improved enzymatic stability. The PEGylation of proteins might find broad application in the local delivery of therapeutic proteins to diseased airways.

IL-13 is a prototypic T helper type 2 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. 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. BALB/c mice were treated by PBS, anti-IL-13 Fab', or A33 Fab' fragment and subjected to ovalbumin exposure for 1 and 5 weeks (short-term and long-term protocols). Our data demonstrate a significant antiasthma effect after nebulization of anti-IL-13 Fab' in a model of asthma driven by allergen exposure as compared with saline and nonimmune Fab fragments. In short- and long-term protocols, administration of the anti-IL-13 Fab' by inhalation significantly decreased bronchial responsiveness to methacholine, bronchoalveolar lavage fluid eosinophilia, inflammatory cell infiltration in lung tissue, and many features of airway remodeling. Levels of proinflammatory mediators and matrix metalloprotease were significantly lower in lung parenchyma of mice treated with anti-IL-13 Fab'. 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.

A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) constitute a family of endopeptidases related to matrix metalloproteinases. These proteinases have been largely implicated in tissue remodeling associated with pathological processes. Among them, ADAMTS12 was identified as an asthma-associated gene in a human genome screening program. However, its functional implication in asthma is not yet documented. The present study aims at investigating potential ADAMTS-12 functions in experimental models of allergic airways disease. Two different in vivo protocols of allergen-induced airways disease were applied to the recently generated Adamts12-deficient mice and corresponding wild-type mice. In this study, we provide evidence for a protective effect of ADAMTS-12 against bronchial inflammation and hyperresponsiveness. In the absence of Adamts12, challenge with different allergens (OVA and house dust mite) led to exacerbated eosinophilic inflammation in the bronchoalveolar lavage fluid and in lung tissue, along with airway dysfunction assessed by increased airway responsiveness following methacholine exposure. Furthermore, mast cell counts and ST2 receptor and IL-33 levels were higher in the lungs of allergen-challenged Adamts12-deficient mice. The present study provides, to our knowledge, the first experimental evidence for a contribution of ADAMTS-12 as a key mediator in airways disease, interfering with immunological processes leading to inflammation and airway hyperresponsiveness.

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 weight20 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.

Interleukin-13 (IL-13) is a prototypic Th2 cytokine and a potential cornerstone of asthma pathology. IL-13 is involved in IgE synthesis, bronchial hyperresponsiveness, mucus hypersecretion, subepithelial fibrosis and eosinophil infiltration. We assessed the potential efficacy of an inhaled high affinity monoclonal antibody (mAb) Fab' fragment neutralizing IL-13 against allergen-induced inflammation and hyperresponsiveness. BALB/c mice were subjected to ovalbumin (OVA) exposure for 1, 5, and 10 weeks referred to as short term (ST), intermediate term (IT) and long term (LT) protocols respectively. The antibody was administered as an aerosol generated by inExpose® in a tower allowing a nose-only exposure. In a one-week OVA-exposure model (ST), we assessed the effectiveness of different doses of anti-IL-13 (0.5 to 5 mg/ml). We report a dose-dependent increase of the anti-inflammatory effect reaching a maximum at a 5 mg/ml. Airway responsiveness to methacholine was measured by using the flexiVent® system. In the different protocols used in this study, administration of the anti-IL-13 Fab' by inhalation significantly decreased bronchial responsiveness to methacholine, BALF eosinophilia, inflammatory cell infiltration in lung tissue, mucus cell upregulation, peribronchial collagen deposition and smooth muscle hyperplasia. After 1 and 5 weeks of allergen exposure (ST and IT), levels of pro-inflammatory mediators IL-13, IL-4, IL-5, CCL-11, MMP-2 and MMP-9 were significantly lower in lung parenchyma when mice were treated by the IL-13 neutralizing antibody. In conclusion, our data generated in a rodent model suggest that inhaled anti-IL-13 Fab' could represent a novel and effective therapy for the treatment of asthma.

Matrix metalloproteinases (MMPs) recently appeared as key regulators of inflammation, allowing the recruitment and clearance of inflammatory cells and modifying the biological activity of many peptide mediators by cleavage. MMP-19 is newly described, and it preferentially cleaves matrix proteins such as collagens and tenascin-C. The role of MMP-19 in asthma has not been described to date. The present study sought to assess the expression of MMP-19 in a murine asthma model, and to address the biological effects of MMP-19 deficiency in mice. Allergen-exposed, wild-type mice displayed increased expression of MMP-19 mRNA and an increased number of MMP-19-positive cells in the lungs, as detected by immunohistochemistry. After an allergen challenge of MMP-19 knockout (MMP-19(-/-)) mice, exacerbated eosinophilic inflammation was detected in bronchoalveolar lavage fluid and bronchial tissue, along with increased airway responsiveness to methacholine. A shift toward increased T helper-2 lymphocyte (Th2)-driven inflammation in MMP-19(-/-) mice was demonstrated by (1) increased numbers of cells expressing the IL-33 receptor T(1)/ST(2) in lung parenchyma, (2) increased IgG(1) levels in serum, and (3) higher levels of IL-13 and eotaxin-1 in lung extracts. Tenascin-C was found to accumulate in peribronchial areas of MMP-19(-/-) after allergen challenges, as assessed by Western blot and immunohistochemistry analyses. 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 may act on Th2 inflammation homeostasis by preventing the accumulation of tenascin protein.

Asthma is a complex inflammatory disease of airways. A network of reciprocal interactions between inflammatory cells, peptidic mediators, extracellular matrix components, and proteases is thought to be involved in the installation and maintenance of asthma-related airway inflammation and remodeling. To date, new proteic mediators displaying significant activity in the pathophysiology of asthma are still to be unveiled. The main objective of this study was to uncover potential target proteins by using surface-enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS) on lung samples from mouse models of allergen-induced airway inflammation and remodeling. In this model, we pointed out several protein or peptide peaks that were preferentially expressed in diseased mice as compared to controls. We report the identification of different five proteins: found inflammatory zone 1 or RELM alpha (FIZZ-1), calcyclin (S100A6), clara cell secretory protein 10 (CC10), Ubiquitin, and Histone H4.

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.

Objectives: The ADAMs (a disintegrin and metalloproteinase) enzymes compose a family of membrane‐bound proteins characterized by their multi‐domain structure and ADAM‐12 expression is elevated in human non‐small cell lung cancers. The aim of this study was to investigate the roles played by ADAM‐12 in critical steps of bronchial cell transformation during carcinogenesis. Materials and methods: To assess the role of ADAM‐12 in tumorigenicity, BEAS‐2B cells were transfected with a plasmid encoding human full‐length ADAM‐12 cDNA, and then the effects of ADAM‐12 overexpression on cell behaviour were explored. Treatment of clones with heparin‐binding epidermal growth factor (EGF)‐like growth factor (HB‐EGF) neutralizing antibodies as well as an EGFR inhibitor allowed the dissection of mechanisms regulating cell proliferation and apoptosis. Results: Overexpression of ADAM‐12 in BEAS‐2B cells promoted cell proliferation. ADAM‐12 overexpressing clones produced higher quantities of HB‐EGF in their culture medium which may rely on membrane‐bound HB‐EGF shedding by ADAM‐12. Targeting HB‐EGF activity with a neutralizing antibody abrogated enhanced cell proliferation in the ADAM‐12 overexpressing clones. In sharp contrast, targeting of amphiregulin, EGF or transforming growth factor‐α failed to influence cell proliferation; moreover, ADAM‐12 transfectants were resistant to etoposide‐induced apoptosis and the use of a neutralizing antibody against HB‐EGF activity restored rates of apoptosis to be similar to controls.Conclusions: ADAM‐12 contributes to enhancing HB‐EGF shedding from plasma membranes leading to increased cell proliferation and reduced apoptosis in this bronchial epithelial cell line.

Introduction Asthma is an inflammatory disease of the airways leading to branchial morphological changes. Matrix metalloprotei-nases (MMPs) are thought to be responsible for a significant part of the allergen-driven inflammation and airway remodelling. The aim of the present study was to assess the effect of doxycycline, a potent MMP inhibitor, given by inhalation on airway inflammation, res-ponsiveness and remodelling. Methods BALB/c mice were sensitized at day 1 and 8 and exposed to aerosolized ovalbumin (OVA) from day 21 to 27 (short term exposure STE) or 5 days/odd weeks from day 22 to 96 (long term exposure LTE). Mice were exposed 30’ before each allergen inhalation to placebo (PBS), excipient alone, doxycycline, or fluti-casone. ResultS In STE model, doxycycline and fluticasone (used as a reference therapy) decreased the allergen-induced eosinophilic inflammation, methacholine-induced airway responsiveness, and peribronchial inflammation. In the LTE model, doxycycline and fluticasone decreased significantly the glandular hyperplasia, the airway wall thickness, and the collagen deposition in the bronchi which are major features of airway remodelling. Doxycycline inhalation did induce a huge increase in IL-10 levels and a decrease in IL-5 and IL-13 levels and decreased the proportion of activated MMP-9 measured in the lung extracts. Conclusion Inhaled doxycycline decreases the allergen-induced airway inflammation and responsiveness and prevents the occurrence of a branchial remodelling in a mouse model of asthma.

Lungs are exposed to the outside environment and therefore to toxic and infectious agents or allergens. This may lead to permanent activation of innate immune response elements. A Disintegrin And Metalloproteinases (ADAMs) and ADAMs with Thrombospondin motifs (ADAMTS) are proteinases closely related to Matrix Metalloproteinases (MMPs). These multifaceted molecules bear metalloproteinase and disintegrin domains endowing them with features of both proteinases and adhesion molecules. Proteinases of the ADAM family are associated to various physiological and pathological processes and display a wide spectrum of biological effects encompassing cell fusion, cell adhesion, "shedding process", cleavage of various substrates from the extracellular matrix, growth factors or cytokines... This review will focus on the putative roles of ADAM/ADAMTS proteinases in airway diseases such as asthma and COPD.