The Role of MIF on Eosinophil Biology and Eosinophilic Inflammation.

Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine that participates in innate and adaptive immune responses. MIF contributes to the resistance against infection agents, but also to the cellular and tissue damage in infectious, autoimmune, and allergic diseases. In the past years, several studies demonstrated a critical role for MIF in the pathogenesis of type-2-mediated inflammation, including allergy and helminth infection. Atopic patients have increased MIF amounts in affected tissues, mainly produced by immune cells such as macrophages, Th2 cells, and eosinophils. Increased MIF mRNA and protein are found in activated Th2 cells, while eosinophils stock pre-formed MIF protein and secrete high amounts of MIF upon stimulation. In mouse models of allergic asthma, the lack of MIF causes an almost complete abrogation of the cardinal signs of the disease including mucus secretion, eosinophilic inflammation, and airway hyper-responsiveness. Additionally, blocking the expression of MIF in animal models leads to significant reduction of pathological signs of eosinophilic inflammation such as rhinitis, atopic dermatitis, eosinophilic esophagitis and helminth infection. A number of studies indicate that MIF is important in the effector phase of type-2 immune responses, while its contribution to Th2 differentiation and IgE production is not consensual. MIF has been found to intervene in different aspects of eosinophil physiology including differentiation, survival, activation, and migration. CD4+ T cells and eosinophils express CD74 and CXCR4, receptors able to signal upon MIF binding. Blockage of these receptors with neutralizing antibodies or small molecule antagonists also succeeds in reducing the signals of inflammation in experimental allergic models. Together, these studies demonstrate an important contribution of MIF on eosinophil biology and in the pathogenesis of allergic diseases and helminth infection.

Multiple doses of adipose tissue-derived mesenchymal stromal cells induce immunosuppression in experimental asthma.

In experimental house dust mite (HDM)-induced allergic asthma, therapeutic administration of a single dose of adipose tissue-derived mesenchymal stromal cells (MSCs) ameliorates lung inflammation but is unable to reverse remodeling. We hypothesized that multiple doses of MSCs might exert better therapeutic effects by reducing lung inflammation and remodeling but might also result in immunosuppressive effects in experimental asthma. HDM was administered intranasally in C57BL/6 mice. After the last HDM challenge, mice received two or three doses of MSCs (105 cells per day) or saline intravenously. An additional cohort of mice received dexamethasone as a positive control for immunosuppression. Two and three doses of MSCs reduced lung inflammation, levels of interleukin (IL)-4, IL-13, and eotaxin; total leukocyte, CD4+ T-cell, and eosinophil counts in bronchoalveolar lavage fluid; and total leukocyte counts in bone marrow, spleen, and mediastinal lymph nodes. Two and three doses of MSCs also reduced collagen fiber content and transforming growth factor-ß levels in lung tissue; however, the three-dose regimen was more effective, and reduced these parameters to control levels, while also decreasing α-actin content in lung tissue. Two and three doses of MSCs improved lung mechanics. Dexamethasone, two and three doses of MSCs similarly increased galectin levels, but only the three-dose regimen increased CD39 levels in the thymus. Dexamethasone and the three-dose, but not the two-dose regimen, also increased levels of programmed death receptor-1 and IL-10, while reducing CD4+ CD8low cell percentage in the thymus. In conclusion, multiple doses of MSCs reduced lung inflammation and remodeling while causing immunosuppression in HDM-induced allergic asthma.

Serum from Asthmatic Mice Potentiates the Therapeutic Effects of Mesenchymal Stromal Cells in Experimental Allergic Asthma.

Asthma is a chronic inflammatory disease characterized by airway inflammation and remodeling, which can lead to progressive decline of lung function. Although mesenchymal stromal cells (MSCs) have shown beneficial immunomodulatory properties in preclinical models of allergic asthma, effects on airway remodeling have been limited. Mounting evidence suggests that prior exposure of MSCs to specific inflammatory stimuli or environments can enhance their immunomodulatory properties. Therefore, we investigated whether stimulating MSCs with bronchoalveolar lavage fluid (BALF) or serum from asthmatic mice could potentiate their therapeutic properties in experimental asthma. In a house dust mite (HDM) extract asthma model in mice, unstimulated, asthmatic BALF-stimulated, or asthmatic serum-stimulated MSCs were administered intratracheally 24 hours after the final HDM challenge. Lung mechanics and histology; BALF protein, cellularity, and biomarker levels; and lymph-node and bone marrow cellularity were assessed. Compared with unstimulated or BALF-stimulated MSCs, serum-stimulated MSCs further reduced BALF levels of interleukin (IL)-4, IL-13, and eotaxin, total and differential cellularity in BALF, bone marrow and lymph nodes, and collagen fiber content, while increasing BALF IL-10 levels and improving lung function. Serum stimulation led to higher MSC apoptosis, expression of various mediators (transforming growth factor-ß, interferon-γ, IL-10, tumor necrosis factor-α-stimulated gene 6 protein, indoleamine 2,3-dioxygenase-1, and IL-1 receptor antagonist), and polarization of macrophages to M2 phenotype. In conclusion, asthmatic serum may be a novel strategy to potentiate therapeutic effects of MSCs in experimental asthma, leading to further reductions in both inflammation and remodeling than can be achieved with unstimulated MSCs. Stem Cells Translational Medicine 2019;8:301&312.

Impact of one versus two doses of mesenchymal stromal cells on lung and cardiovascular repair in experimental emphysema.

BACKGROUND: A single administration of mesenchymal stromal cells (MSCs) has been shown to reduce lung inflammation in experimental elastase-induced emphysema; however, effects were limited in terms of lung-tissue repair and cardiac function improvement. We hypothesized that two doses of MSCs could induce further lung and cardiovascular repair by mitigating inflammation and remodeling in a model of emphysema induced by multiple elastase instillations. We aimed to comparatively investigate the effects of one versus two doses of MSCs, administered 1 week apart, in a murine model of elastase-induced emphysema. METHODS: C57BL/6 mice were randomly divided into control (CTRL) and emphysema (E) groups. Mice in the E group received porcine pancreatic elastase (0.2 IU, 50 µL) intratracheally once weekly for four consecutive weeks; the CTRL animals received sterile saline (50 µL) using the same protocol. Three hours after the last instillation, the E group was further randomized to receive either saline (SAL) or murine MSCs (105 cells) intratracheally, in one or two doses (1 week apart). Fourteen days later, mice were euthanized, and all data analyzed. RESULTS: Both one and two doses of MSCs improved lung mechanics, reducing keratinocyte-derived chemokine and transforming growth factor-ß levels in lung homogenates, total cell and macrophage counts in bronchoalveolar lavage fluid (BALF), and collagen fiber content in airways and blood vessels, as well as increasing vascular endothelial growth factor in lung homogenates and elastic fiber content in lung parenchyma. However, only the two-dose group exhibited reductions in tumor necrosis factor-α in lung tissue, BALF neutrophil and lymphocyte count, thymus weight, and total cellularity, as well as CD8+ cell counts and cervical lymph node CD4+ and CD8+ T cell counts, as well as further increased elastic fiber content in the lung parenchyma and reduced severity of pulmonary arterial hypertension. CONCLUSIONS: Two doses of MSCs enhanced lung repair and improvement in cardiac function, while inducing T cell immunosuppression, mainly of CD8+ cells, in elastase-induced emphysema.

Bosutinib Therapy Ameliorates Lung Inflammation and Fibrosis in Experimental Silicosis.

Silicosis is an occupational lung disease for which no effective therapy exists. We hypothesized that bosutinib, a tyrosine kinase inhibitor, might ameliorate inflammatory responses, attenuate pulmonary fibrosis, and thus improve lung function in experimental silicosis. For this purpose, we investigated the potential efficacy of bosutinib in the treatment of experimental silicosis induced in C57BL/6 mice by intratracheal administration of silica particles. After 15 days, once disease was established, animals were randomly assigned to receive DMSO or bosutinib (1 mg/kg/dose in 0.1 mL 1% DMSO) by oral gavage, twice daily for 14 days. On day 30, lung mechanics and morphometry, total and differential cell count in alveolar septa and granuloma, levels of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-4, transforming growth factor (TGF)-ß, and vascular endothelial growth factor in lung homogenate, M1 and M2 macrophages, total leukocytes, and T cells in BALF, lymph nodes, and thymus, and collagen fiber content in alveolar septa and granuloma were analyzed. In a separate in vitro experiment, RAW264.7 macrophages were exposed to silica particles in the presence or absence of bosutinib. After 24 h, gene expressions of arginase-1, IL-10, IL-12, inducible nitric oxide synthase (iNOS), metalloproteinase (MMP)-9, tissue inhibitor of metalloproteinase (TIMP)-1, and caspase-3 were evaluated. In vivo, in silicotic animals, bosutinib, compared to DMSO, decreased: (1) fraction area of collapsed alveoli, (2) size and number of granulomas, and mononuclear cell granuloma infiltration; (3) IL-1ß, TNF-α, IFN-γ, and TGF-ß levels in lung homogenates, (4) collagen fiber content in lung parenchyma, and (5) viscoelastic pressure and static lung elastance. Bosutinib also reduced M1 cell counts while increasing M2 macrophage population in both lung parenchyma and granulomas. Total leukocyte, regulatory T, CD4+, and CD8+ cell counts in the lung-draining lymph nodes also decreased with bosutinib therapy without affecting thymus cellularity. In vitro, bosutinib led to a decrease in IL-12 and iNOS and increase in IL-10, arginase-1, MMP-9, and TIMP-1. In conclusion, in the current model of silicosis, bosutinib therapy yielded beneficial effects on lung inflammation and remodeling, therefore resulting in lung mechanics improvement. Bosutinib may hold promise for silicosis; however, further studies are required.

Bone Marrow, Adipose, and Lung Tissue-Derived Murine Mesenchymal Stromal Cells Release Different Mediators and Differentially Affect Airway and Lung Parenchyma in Experimental Asthma.

Mesenchymal stromal cells (MSCs) from different sources have differential effects on lung injury. To compare the effects of murine MSCs from bone marrow (BM), adipose tissue (AD), and lung tissue (LUNG) on inflammatory and remodeling processes in experimental allergic asthma, female C57BL/6 mice were sensitized and challenged with ovalbumin (OVA) or saline (C). Twenty-four hours after the last challenge, mice received either saline (50 µl, SAL), BM-MSCs, AD-MSCs, or LUNG-MSCs (105 cells per mouse in 50 µl total volume) intratracheally. At 1 week, BM-MSCs produced significantly greater reductions in resistive and viscoelastic pressures, bronchoconstriction index, collagen fiber content in lung parenchyma (but not airways), eosinophil infiltration, and levels of interleukin (IL)-4, IL-13, transforming growth factor (TGF)-ß, and vascular endothelial growth factor (VEGF) in lung homogenates compared to AD-MSCs and LUNG-MSCs. Only BM-MSCs increased IL-10 and interferon (IFN)-γ in lung tissue. In parallel in vitro experiments, BM-MSCs increased M2 macrophage polarization, whereas AD-MSCs and LUNG-MSCs had higher baseline levels of IL-4, insulin-like growth factor (IGF), and VEGF secretion. Exposure of MSCs to serum specimens obtained from asthmatic mice promoted reductions in secretion of these mediators, particularly in BM-MSCs. Intratracheally administered BM-MSCs, AD-MSCs, and LUNG-MSCs were differentially effective at reducing airway inflammation and remodeling and improving lung function in the current model of allergic asthma. In conclusion, intratracheal administration of MSCs from BM, AD, and LUNG were differentially effective at reducing airway inflammation and remodeling and improving lung function comparably reduced inflammation and fibrogenesis in this asthma model. However, altered lung mechanics and lung remodeling responded better to BM-MSCs than to AD-MSCs or LUNG-MSCs. Moreover, each type of MSC was differentially affected in a surrogate in vitro model of the in vivo lung environment. Stem Cells Translational Medicine 2017;6:1557-1567.

Effects of different mesenchymal stromal cell sources and delivery routes in experimental emphysema.

We sought to assess whether the effects of mesenchymal stromal cells (MSC) on lung inflammation and remodeling in experimental emphysema would differ according to MSC source and administration route. Emphysema was induced in C57BL/6 mice by intratracheal (IT) administration of porcine pancreatic elastase (0.1 UI) weekly for 1 month. After the last elastase instillation, saline or MSCs (1×105), isolated from either mouse bone marrow (BM), adipose tissue (AD) or lung tissue (L), were administered intravenously (IV) or IT. After 1 week, mice were euthanized. Regardless of administration route, MSCs from each source yielded: 1) decreased mean linear intercept, neutrophil infiltration, and cell apoptosis; 2) increased elastic fiber content; 3) reduced alveolar epithelial and endothelial cell damage; and 4) decreased keratinocyte-derived chemokine (KC, a mouse analog of interleukin-8) and transforming growth factor-ß levels in lung tissue. In contrast with IV, IT MSC administration further reduced alveolar hyperinflation (BM-MSC) and collagen fiber content (BM-MSC and L-MSC). Intravenous administration of BM- and AD-MSCs reduced the number of M1 macrophages and pulmonary hypertension on echocardiography, while increasing vascular endothelial growth factor. Only BM-MSCs (IV > IT) increased the number of M2 macrophages. In conclusion, different MSC sources and administration routes variably reduced elastase-induced lung damage, but IV administration of BM-MSCs resulted in better cardiovascular function and change of the macrophage phenotype from M1 to M2.

Effects of bone marrow mononuclear cells from healthy or ovalbumin-induced lung inflammation donors on recipient allergic asthma mice.

INTRODUCTION: Asthma is characterized by a chronic inflammatory process which may lead to several changes in bone marrow cell composition. We hypothesized that bone marrow mononuclear cells (BMMCs) obtained from ovalbumin (OVA)-induced lung inflammation mice may promote different effects compared to BMMCs from healthy donors in a model of allergic asthma. METHODS: C57BL/6 mice were randomly assigned to two groups. In the OVA group, mice were sensitized and challenged with ovalbumin, while healthy animals (control group) received saline using the same protocol. BMMCs were analyzed by flow cytometry 24 hours after the last challenge. After BMMC characterization, another group of OVA mice were further randomized into three subgroups to receive intratracheal saline (BMMC-SAL), BMMCs from control or BMMCs from OVA mice (BMMC-Control and BMMC-OVA, respectively; 2x106 cells/mouse), 24 hours after the last challenge. RESULTS: BMMC-OVA exhibited an increased percentage of eosinophils, monocytes and hematopoietic precursors, while mesenchymal stem cells decreased, as compared with BMMC-Control. BMMCs from both donor groups reduced airway resistance, alveolar collapse, bronchoconstriction index, eosinophil infiltration, collagen fiber content in alveolar septa and levels of interleukin (IL)-4, IL-5, IL-13, interferon-γ, transforming growth factor-ß, and vascular endothelial growth factor in lung homogenates. However, the benefits of BMMCs were significantly more pronounced when cells were obtained from control donors. CONCLUSION: Both BMMC-Control and BMMC-OVA reduced the inflammatory and remodeling processes; nevertheless, BMMC-Control led to a greater improvement in lung morphofunction, which may be due to different BMMC composition and/or properties.

IL-13 immunotoxin accelerates resolution of lung pathological changes triggered by silica particles in mice.

Instillation of silica into the lungs of rodents results in pathological changes that strongly mimic human silicosis, an occupational lung disease marked by restrictive airway obstruction, inflammation, and fibrosis. Because IL-13 is a pivotal proinflammatory and fibrogenic cytokine, we examined whether a recombinant immunotoxin comprised of human IL-13 and a mutated form of Pseudomonas exotoxin (IL-13-PE) might affect pathological features of experimental silicosis. Mice received a single intranasal instillation of silica particles and were treated with intranasal IL-13-PE every other day from days 21 to 27 postsilica. The sensitivity of putative cell targets to IL-13-PE was also assessed in in vitro settings. Upregulation of IL-13, its receptor subunits IL-13Rα1 and IL-13Rα2, and shared receptor IL-4Rα were associated with development of granulomatous lung inflammation triggered by silica. IL-13-PE inhibited silica-induced granuloma and fibrotic responses noted at 24 h and 15 d after the last treatment. Upregulation of TNF-α, TGF-ß, and chemokines, as well as increased collagen deposition and airway hyperreactivity to methacholine were all clearly sensitive to IL-13-PE. In addition, IL-13-PE inhibited both IL-13-induced proliferation of cultured lung fibroblasts from silicotic mice and silica-induced IL-8 generation from A549 cells. In conclusion, our findings show that therapeutic treatment with IL-13-PE can reverse important pathological features caused by inhalation of silica particles, suggesting that this recombinant immunotoxin is a promising molecular template in drug discovery for the treatment of silicosis.

Two for one: cyclic AMP mediates the anti-inflammatory and anti-spasmodic properties of the non-anesthetic lidocaine analog JMF2-1.

Inhalation of JMF2-1, an analog of lidocaine with reduced anesthetic activity, prevents airway contraction and lung inflammation in experimental asthma models. We sought to test if the JMF2-1 effects are a consequence of increased intracellular cAMP levels in asthma cell targets, such as smooth muscle cells and T cells. Functional effect of JMF2-1 on carbachol-induced contraction of intact or epithelial-denuded rat trachea was assessed in conventional organ baths. cAMP was quantified by radioimmunoassay in cultured guinea pig tracheal smooth muscle cells, as well as lymph node cells from BALB/c mice, exposed to JMF2-1. We found that JMF2-1 (0.1-1mM) concentration-dependently inhibited epithelium-intact tracheal ring contraction induced by carbachol challenge. The antispasmodic effect remained unaltered following epithelium removal or pretreatment with NG-nitro-L-arginine methyl ester (100µM), but it was clearly sensitive to 9-(tetrahydro-2-furyl) adenine (SQ22,536, 100µM), an adenylate cyclase inhibitor. JMF2-1 (300 and 600µM) also dose-dependently increased cAMP intracellular levels of both cultured airway smooth muscle cells and T lymphocytes. This effect was consistently abrogated by SQ22,536 and reproduced by forskolin in both systems. JMF2-1 induced apoptosis of anti-CD3 activated T cells in a mechanism sensitive to zIETD, indicating that JMF2-1 mediates caspase-8-dependent apoptosis. Furthermore, forskolin also inhibited anti-CD3 induced T cell proliferation and survival. Our results suggest that JMF2-1 inhibits respiratory smooth muscle contraction as well as T cell proliferation and survival through enhancement of intracellular cAMP levels. These findings may help to explain the anti-inflammatory and antispasmodic effects of JMF2-1 observed in previous studies.