Differential regulation of lung homeostasis and silicosis by the TAM receptors MerTk and Axl.

Introduction: TAM receptor-mediated efferocytosis plays an important function in immune regulation and may contribute to antigen tolerance in the lungs, a site with continuous cellular turnover and generation of apoptotic cells. Some studies have identified failures in efferocytosis as a common driver of inflammation and tissue destruction in lung diseases. Our study is the first to characterize the in vivo function of the TAM receptors, Axl and MerTk, in the innate immune cell compartment, cytokine and chemokine production, as well as the alveolar macrophage (AM) phenotype in different settings in the airways and lung parenchyma. Methods: We employed MerTk and Axl defective mice to induce acute silicosis by a single exposure to crystalline silica particles (20 mg/50 µL). Although both mRNA levels of Axl and MerTk receptors were constitutively expressed by lung cells and isolated AMs, we found that MerTk was critical for maintaining lung homeostasis, whereas Axl played a role in the regulation of silica-induced inflammation. Our findings imply that MerTk and Axl differently modulated inflammatory tone via AM and neutrophil recruitment, phenotype and function by flow cytometry, and TGF-ß and CXCL1 protein levels, respectively. Finally, Axl expression was upregulated in both MerTk-/- and WT AMs, confirming its importance during inflammation. Conclusion: This study provides strong evidence that MerTk and Axl are specialized to orchestrate apoptotic cell clearance across different circumstances and may have important implications for the understanding of pulmonary inflammatory disorders as well as for the development of new approaches to therapy.

Annexin-A1-Derived Peptide Ac2-26 Suppresses Allergic Airway Inflammation and Remodelling in Mice.

Annexin-A1 (AnxA1) and its N-terminal derived peptide Ac2-26 regulate the inflammatory response in several experimental models of disorders. This study evaluated the effect of endogenous AnxA1 and its N-terminal peptide Acetyl 2-26 (Ac2-26) on allergic asthma triggered by house dust mite (HDM) extract in mice. ANXA1-/- and wildtype (WT) mice were exposed to intranasal instillation of HDM every other day for 3 weeks, with analyses performed 24 h following the last exposure. Intranasal administration of peptide Ac2-26 was performed 1 h before HDM, beginning 1 week after the initial antigen application. ANXA1-/- mice stimulated with HDM showed marked exacerbations of airway hyperreactivity (AHR), eosinophil accumulation, subepithelial fibrosis, and mucus hypersecretion, all parameters correlating with overexpression of cytokines (IL-4, IL-13, TNF-α, and TGF-ß) and chemokines (CCL11/eotaxin-1 and CCL2/MCP-1). Intranasal treatment with peptide Ac2-26 decreased eosinophil infiltration, peribronchiolar fibrosis, and mucus exacerbation caused by the allergen challenge. Ac2-26 also inhibited AHR and mediator production. Collectively, our findings show that the AnxA1-derived peptide Ac2-26 protects against several pathological changes associated with HDM allergic reaction, suggesting that this peptide or related AnxA1-mimetic Ac2-26 may represent promising therapeutic candidates for the treatment of allergic asthma.

Intranasal Flunisolide Suppresses Pathological Alterations Caused by Silica Particles in the Lungs of Mice.

Silicosis is an occupational disease triggered by the inhalation of fine particles of crystalline silica and characterized by inflammation and scarring in the form of nodular lesions in the lungs. In spite of the therapeutic arsenal currently available, there is no specific treatment for the disease. Flunisolide is a potent corticosteroid shown to be effective for controlling chronic lung inflammatory diseases. In this study, the effect of flunisolide on silica-induced lung pathological changes in mice was investigated. Swiss-Webster mice were injected intranasally with silica particles and further treated with flunisolide from day 21 to 27 post-silica challenge. Lung function was assessed by whole body invasive plethysmography. Granuloma formation was evaluated morphometrically, collagen deposition by Picrus sirius staining and quantitated by Sircol. Chemokines and cytokines were evaluated using enzyme-linked immunosorbent assay. The sensitivity of lung fibroblasts was also examined in in vitro assays. Silica challenge led to increased leukocyte numbers (mononuclear cells and neutrophils) as well as production of the chemokine KC/CXCL-1 and the cytokines TNF-α and TGF-ß in the bronchoalveolar lavage. These alterations paralleled to progressive granuloma formation, collagen deposition and impairment of lung function. Therapeutic administration of intranasal flunisolide inhibited granuloma and fibrotic responses, noted 28 days after silica challenge. The upregulation of MIP-1α/CCL-3 and MIP-2/CXCL-2 and the cytokines TNF-α and TGF-ß, as well as deposition of collagen and airway hyper-reactivity to methacholine were shown to be clearly sensitive to flunisolide, as compared to silica-challenge untreated mice. Additionally, flunisolide effectively suppressed the responses of proliferation and MCP-1/CCL-2 production from IL-13 stimulated lung fibroblasts from silica- or saline-challenged mice. In conclusion, we report that intranasal treatment with the corticosteroid flunisolide showed protective properties on pathological features triggered by silica particles in mice, suggesting that the compound may constitute a promising strategy for the treatment of silicosis.

Human Mesenchymal Stem Cell Therapy Reverses Su5416/Hypoxia-Induced Pulmonary Arterial Hypertension in Mice.

Aims: Pulmonary arterial hypertension (PAH) is a disease characterized by an increase in pulmonary vascular resistance and right ventricular (RV) failure. We aimed to determine the effects of human mesenchymal stem cell (hMSC) therapy in a SU5416/hypoxia (SuH) mice model of PAH. Methods and Results: C57BL/6 mice (20-25 g) were exposure to 4 weeks of hypoxia combined vascular endothelial growth factor receptor antagonism (20 mg/kg SU5416; weekly s.c. injections; PAH mice). Control mice were housed in room air. Following 2 weeks of SuH exposure, we injected 5 × 105 hMSCs cells suspended in 50 µL of vehicle (0.6 U/mL DNaseI in PBS) through intravenous injection in the caudal vein. PAH mice were treated only with vehicle. Ratio between pulmonary artery acceleration time and RV ejection time (PAAT/RVET), measure by echocardiography, was significantly reduced in the PAH mice, compared with controls, and therapy with hMSCs normalized this. Significant muscularization of the PA was observed in the PAH mice and hMSC reduced the number of fully muscularized vessels. RV free wall thickness was higher in PAH animals than in the controls, and a single injection of hMSCs reversed RV hypertrophy. Levels of markers of exacerbated apoptosis, tissue inflammation and damage, cell proliferation and oxidative stress were significantly greater in both lungs and RV tissues from PAH group, compared to controls. hMSC injection in PAH animals normalized the expression of these molecules which are involved with PAH and RV dysfunction development and the state of chronicity. Conclusion: These results indicate that hMSCs therapy represents a novel strategy for the treatment of PAH in the future.

Human adipose tissue mesenchymal stromal cells and their extracellular vesicles act differentially on lung mechanics and inflammation in experimental allergic asthma.

BACKGROUND: Asthma is a chronic inflammatory disease that can be difficult to treat due to its complex pathophysiology. Most current drugs focus on controlling the inflammatory process, but are unable to revert the changes of tissue remodeling. Human mesenchymal stromal cells (MSCs) are effective at reducing inflammation and tissue remodeling; nevertheless, no study has evaluated the therapeutic effects of extracellular vesicles (EVs) obtained from human adipose tissue-derived MSCs (AD-MSC) on established airway remodeling in experimental allergic asthma. METHODS: C57BL/6 female mice were sensitized and challenged with ovalbumin (OVA). Control (CTRL) animals received saline solution using the same protocol. One day after the last challenge, each group received saline, 105 human AD-MSCs, or EVs (released by 105 AD-MSCs). Seven days after treatment, animals were anesthetized for lung function assessment and subsequently euthanized. Bronchoalveolar lavage fluid (BALF), lungs, thymus, and mediastinal lymph nodes were harvested for analysis of inflammation. Collagen fiber content of airways and lung parenchyma were also evaluated. RESULTS: In OVA animals, AD-MSCs and EVs acted differently on static lung elastance and on BALF regulatory T cells, CD3+CD4+ T cells, and pro-inflammatory mediators (interleukin [IL]-4, IL-5, IL-13, and eotaxin), but similarly reduced eosinophils in lung tissue, collagen fiber content in airways and lung parenchyma, levels of transforming growth factor-ß in lung tissue, and CD3+CD4+ T cell counts in the thymus. No significant changes were observed in total cell count or percentage of CD3+CD4+ T cells in the mediastinal lymph nodes. CONCLUSIONS: In this immunocompetent mouse model of allergic asthma, human AD-MSCs and EVs effectively reduced eosinophil counts in lung tissue and BALF and modulated airway remodeling, but their effects on T cells differed in lung and thymus. EVs may hold promise for asthma; however, further studies are required to elucidate the different mechanisms of action of AD-MSCs versus their EVs.

Potential of PEGylated Toll-Like Receptor 7 Ligands for Controlling Inflammation and Functional Changes in Mouse Models of Asthma and Silicosis.

Prior investigations show that signaling activation through pattern recognition receptors can directly impact a number of inflammatory lung diseases. While toll-like receptor (TLR) 7 agonists have raised interest for their ability to inhibit allergen-induced pathological changes in experimental asthma conditions, the putative benefit of this treatment is limited by adverse effects. Our aim was to evaluate the therapeutic potential of two PEGylated purine-like compounds, TMX-302 and TMX-306, characterized by TLR7 partial agonistic activity; therefore, the compounds are expected to induce lower local and systemic adverse reactions. In vitro approaches and translation to murine models of obstructive and restrictive lung diseases were explored. In vitro studies with human PBMCs showed that both TMX-302 and TMX-306 marginally affects cytokine production as compared with equivalent concentrations of the TLR7 full agonist, TMX-202. The PEGylated compounds did not induce monocyte-derived DC maturation or B cell proliferation, differently from what observed after stimulation with TMX-202. Impact of PEGylated ligands on lung function and inflammatory changes was studied in animal models of acute lung injury, asthma, and silicosis following Lipopolysaccharide (LPS), allergen (ovalbumin), and silica inhalation, respectively. Subcutaneous injection of TMX-302 prevented LPS- and allergen-induced airway hyper-reactivity (AHR), leukocyte infiltration, and production of pro-inflammatory cytokines in the lung. However, intranasal instillation of TMX-302 led to neutrophil infiltration and failed to prevent allergen-induced AHR, despite inhibiting leukocyte counts in the BAL. Aerosolized TMX-306 given prophylactically, but not therapeutically, inhibited pivotal asthma features. Interventional treatment with intranasal instillation of TMX-306 significantly reduced the pulmonary fibrogranulomatous response and the number of silica particles in lung interstitial space in silicotic mice. These findings highlight the potential of TMX-306, emphasizing its value in drug development for lung diseases, and particularly silicosis.

Fish oil has beneficial effects on allergen-induced airway inflammation and hyperreactivity in mice.

BACKGROUND: Fish oil (FO) is rich in n-3 polyunsaturated fatty acids (PUFA), which have been suggested to be anti-inflammatory and are associated with improvement of several inflammatory diseases. In this study, we investigated the influence of FO on allergen-induced lung inflammation and airway hyperreactivity in mice. METHODS: Male A/J mice were fed either a standard-chow (SC) or a FO diet (FO) for 8 weeks. After 4 weeks, each group was further randomized for ovalbumin (SC-OVA and FO-OVA) or saline (SC-SAL and FO-SAL) challenge. Resistance and elastance were measured at baseline and after aerosolized methacholine, 24h after the last challenge. Bronchoalveolar lavage (BAL) was performed for leukocyte counts. Lung tissue mucus deposition, peribronchiolar matrix deposition and eosinophil infiltration were quantified. Serum immunoglobulin E (IgE) and IgG1 (ref 2.2), lung IL-4, IL-5, IL-10, IL-13, IL-17, INFγ and eotaxin-1 and 2 were detected by ELISA and nuclear factor kappa B (NFκB), GATA-3 and peroxisome proliferator-activated receptor gamma (PPARγ) expression was measured by Western blot. RESULTS: Levels of serum IgE and IgG1 were significantly higher in OVA sensitized mice. OVA challenge resulted in increased eosinophil infiltration, increased inflammatory cytokine production, peribronchiolar matrix and mucus deposition and airway hyperreactivity to aerosolized methacholine. Elevated lung NFκB and GATA-3 expression was noted in OVA-challenged mice. These changes were attenuated in mice fed with FO diet. Higher PPARγ expression was also detected in the lungs from the FO-fed groups. CONCLUSION: Our results demonstrate that FO intake attenuated classical asthma features by suppressing the systemic sensitization, thus providing evidence that FO might be a prophylactic alternative for asthma prevention.