François Daubeuf

A Dissociated Glucocorticoid Receptor Modulator Reduces Airway Hyperresponsiveness and Inflammation in a Mouse Model of Asthma

The glucocorticoid receptor (GR) is a transcription factor able to support either target gene activation via direct binding to DNA or gene repression via interfering with the activity of various proinflammatory transcription factors. An improved therapeutic profile for combating chronic inflammatory diseases has been reported through selectively modulating the GR by only triggering its transrepression function. We have studied in this paper the activity of Compound A (CpdA), a dissociated GR modulator favoring GR monomer formation, in a predominantly Th2-driven asthma model. CpdA acted similarly to the glucocorticoid dexamethasone (DEX) in counteracting OVA-induced airway hyperresponsiveness, recruitment of eosinophils, dendritic cells, neutrophils, B and T cells, and macrophages in bronchoalveolar lavage fluid, lung Th2, Tc2, Th17, Tc17, and mast cell infiltration, collagen deposition, and goblet cell metaplasia. Both CpdA and DEX inhibited Th2 cytokine production in bronchoalveolar lavage as well as nuclear translocation of NF-κB and its subsequent recruitment onto the IκBα promoter in the lung. By contrast, DEX but not CpdA induces expression of the GR-dependent model gene MAPK phosphatase 1 in the lung, confirming the dissociative action of CpdA. Mechanistically, we demonstrate that CpdA inhibited IL-4–induced STAT6 translocation and that GR is essential for CpdA to mediate chemokine repression. In conclusion, we clearly show in this study the anti-inflammatory effect of CpdA in a Th2-driven asthma model in the absence of transactivation, suggesting a potential therapeutic benefit of this strategy.

Mast Cells Contribute to Bleomycin-Induced Lung Inflammation and Injury in Mice through a Chymase/Mast Cell Protease 4–Dependent Mechanism

Mast cells (MCs) are found in large numbers in lungs of patients with pulmonary fibrosis. However, the functions of MCs in lung fibrosis remain largely unknown. We assessed the role of MCs and MC protease 4 (MCPT4), the mouse counterpart of human MC chymase, in a mouse model of bleomycin (BLM)-induced lung injury. We found that levels of inflammation in the bronchoalveolar lavage and the lung, as well as levels of lung fibrosis, were reduced 7 d after intranasal delivery of BLM MC-deficient KitW-sh/W-sh mice compared with wild-type (WT) mice. Confirming the implication of MCs in these processes, we report that the levels of inflammation and fibrosis observed in KitW-sh/W-sh mice can be restored to those observed in WT mice after the adoptive transfer of bone marrow–derived cultured MCs into KitW-sh/W-sh mice. Additionally, we show that levels of inflammation and fibrosis are also reduced in MC chymase MCPT4-deficient mice as compared with WT mice at day 7, suggesting a role for MC-derived MCPT4 in these processes. Our results support the conclusion that MCs can contribute to the initial lung injury induced by BLM through release of the MCPT4 chymase.

Neutralizing endogenous chemokines with small molecules. Principles and potential therapeutic applications.

Abstract Regulation of cellular responses to external stimuli such as hormones, neurotransmitters, or cytokines is achieved through the control of all steps of the complex cascade starting with synthesis, going through maturation steps, release, distribution, degradation and/or uptake of the signalling molecule interacting with the target protein. One possible way of regulation, referred to as scavenging or neutralization of the ligand, has been increasingly studied, especially for small protein ligands. It shows innovative potential in chemical biology approaches as well as in disease treatment. Neutralization of protein ligands, as for example cytokines or chemokines can lead to the validation of signalling pathways under physiological or pathophysiological conditions, and in certain cases, to the development of therapeutic molecules now used in autoimmune diseases, chronic inflammation and cancer treatment. This review explores the field of ligand neutralization and tries to determine to what extent small chemical molecules could substitute for neutralizing antibodies in therapeutic approaches.

Discovery and characterization of an endogenous CXCR4 antagonist.

CXCL12-CXCR4 signaling controls multiple physiological processes and its dysregulation is associated with cancers and inflammatory diseases. To discover as-yet-unknown endogenous ligands of CXCR4, we screened a blood-derived peptide library for inhibitors of CXCR4-tropic HIV-1 strains. This approach identified a 16 amino acid fragment of serum albumin as an effective and highly specific CXCR4 antagonist. The endogenous peptide, termed EPI-X4, is evolutionarily conserved and generated from the highly abundant albumin precursor by pH-regulated proteases. EPI-X4 forms an unusual lasso-like structure and antagonizes CXCL12-induced tumor cell migration, mobilizes stem cells, and suppresses inflammatory responses in mice. Furthermore, the peptide is abundant in the urine of patients with inflammatory kidney diseases and may serve as a biomarker. Our results identify EPI-X4 as a key regulator of CXCR4 signaling and introduce proteolysis of an abundant precursor protein as an alternative concept for chemokine receptor regulation.

An antedrug of the CXCL12 neutraligand blocks experimental allergic asthma without systemic effect in mice.

Background: The chemokine CXCL12 and its receptor CXCR4 are widely distributed and contribute to the physiopathology of inflammation. Results: Recruitment of eosinophils in the inflamed airway is selectively attenuated by short lived antagonists that block CXCL12-mediated activation of CXCR4. Conclusion: CXCL12/CXCR4 signaling regulates local leukocyte-mediated inflammation. Significance: Antedrugs of neutraligands allow dissecting the physiological role of chemokines, especially when expression occurs in multiple tissues. The chemokine receptor CXCR4 and its chemokine CXCL12 are involved in normal tissue patterning but also in tumor cell growth and survival as well as in the recruitment of immune and inflammatory cells, as successfully demonstrated using agents that block either CXCL12 or CXCR4. In order to achieve selectivity in drug action on the CXCR4/CXCL12 pair, in particular in the airways, drugs should be delivered as selectively as possible in the treated tissue and should not diffuse in the systemic circulation, where it may reach undesired organs. To this end, we used a previously unexploited Knoevenagel reaction to create a short lived drug, or soft drug, based on the CXCL12-neutralizing small molecule, chalcone 4, which blocks binding of CXCL12 to CXCR4. We show that the compound, carbonitrile-chalcone 4, blocks the recruitment of eosinophils to the airways in ovalbumin-sensitized and challenged mice in vivo when administered directly to the airways by the intranasal route, but not when administered systemically by the intraperitoneal route. We show that the lack of effect at a distant site is due to the rapid degradation of the molecule to inactive fragments. This approach allows selective action of the CXCL12 neutraligands although the target protein is widely distributed in the organism.

Performing Bronchoalveolar Lavage in the Mouse

Bronchoalveolar lavage (BAL) is a simple technique commonly used in humans to sample the contents of the epithelial lining fluid and determine the cellular and molecular composition of the pulmonary airways. In murine models, BAL makes it possible to sample immunological and inflammatory cell populations; it is indispensable for studying cell influx in disease models of the airways such as asthma and COPD. Cell counts can be combined with methods such as ELISA, immunoblot, immunohistochemistry, quantitative polymerase chain reaction, and HPLC to assess such inflammatory components as cytokines, growth factors, analytes, and receptors expressed at the cell membrane. Performing BAL in a reproducible manner is a hallmark of airway research in the mouse. Several procedures may be implemented. This unit describes a basic, rapid, inexpensive, and highly reproducible procedure to collect BAL fluid and cells that can be counted efficiently and reproducibly. Curr. Protoc. Mouse Biol. 2:167‐175

The AGC Kinase Inhibitor H89 Attenuates Airway Inflammation in Mouse Models of Asthma

Background H89 is a potent inhibitor of Protein Kinase A (PKA) and Mitogen- and Stress-Activated protein Kinase 1 (MSK1) with some inhibitory activity on other members of the AGC kinase family. H89 has been extensively used in vitro but its anti-inflammatory potential in vivo has not been reported to date. To assess the anti-inflammatory properties of H89 in mouse models of asthma. Methodology/Principal Findings Mice were sensitized intraperitoneally (i.p.) to ovalbumin (OVA) with or without alum, and challenged intranasally with OVA. H89 (10 mg/kg) or vehicle was given i.p. two hours before each OVA challenge. Airway hyperresponsiveness (AHR) was assessed by whole-body barometric plethysmography. Inflammation was assessed by the total and differential cell counts and IL-4 and IL-5 levels in bronchoalveolar lavage (BAL) fluid. Lung inflammation, mucus production and mast cell numbers were analyzed after histochemistry. We show that treatment with H89 reduces AHR, lung inflammation, mast cell numbers and mucus production. H89 also inhibits IL-4 and IL-5 production and infiltration of eosinophils, neutrophils and lymphocytes in BAL fluid. Conclusions/Significance Taken together, our findings implicate that blockade of AGC kinases may have therapeutic potential for the treatment of allergic airway inflammation.

A strategy to discover decoy chemokine ligands with an anti-inflammatory activity

Excessive signaling by chemokines has been associated with chronic inflammation or cancer, thus attracting substantial attention as promising therapeutic targets. Inspired by chemokine-clearing molecules shaped by pathogens to escape the immune system, we designed a generic screening assay to discover chemokine neutralizing molecules (neutraligands) and unambiguously distinguish them from molecules that block the receptor (receptor antagonists). This assay, called TRIC-r, combines time-resolved intracellular calcium recordings with pre-incubation of bioactive compounds either with the chemokine or the receptor-expressing cells. We describe here the identification of high affinity neutraligands of CCL17 and CCL22, two chemokines involved in the Th2-type of lung inflammation. The decoy molecules inhibit in vitro CCL17- or CCL22-induced intracellular calcium responses, CCR4 endocytosis and human T cell migration. In vivo, they inhibit inflammation in a murine model of asthma, in particular the recruitment of eosinophils, dendritic cells and CD4+T cells. Altogether, we developed a successful strategy to discover as new class of pharmacological tools to potently control cell chemotaxis in vitro and in vivo.

NMR HRMAS spectroscopy of lung biopsy samples: Comparison study between human, pig, rat, and mouse metabolomics

Using the metabolomics by NMR high‐resolution magic angle spinning spectroscopy, we assessed the lung metabolome of various animal species in order to identify the animal model that could be substituted to human lung in studies on fresh lung biopsies.

Eosinophils and the Ovalbumin Mouse Model of Asthma

Mouse models of asthma are essential to understand asthma pathogenesis and eosinophil recruitment in the airways, and to develop new therapeutic strategies. Animal models try to mimic features of the human disease including airway hyperresponsiveness (AHR), eosinophilic inflammation, and remodeling, which are the typical asthma-related characteristics. The mouse is now the species of choice for asthma research due to the availability of transgenic animals and a wide array of specific reagents and techniques available. Cellular responses may be studied with innovative imaging and flow cytometry methods while lung mechanics may be precisely measured by the forced oscillation technique, and airway responsiveness approached by barometric plethysmography in conscious and unconstrained animals. Here, we describe procedures to generate acute models of hypereosinophilic asthma in mice, with ovalbumin (OVA) as the allergen. The presented allergic asthma models offer a large and reproducible eosinophil recruitment, measured in the bronchoalveolar lavage (BAL), accompanied with AHR, inflammation, and remodeling, and are particularly suited to assess the activity of drug candidates. We here present the classical 21-day allergic asthma model to OVA, and adjustments for a rapid 8-day model of airway allergic hypereosinophilia, and a more chronic 57-day model suitable for C57BL/6 mice to develop AHR together with airway eosinophilic inflammation and remodeling.