Carla P. Jones

Differential Mobilization of Subsets of Progenitor Cells from the Bone Marrow

G-CSF stimulates mobilization of hematopoietic progenitor cells (HPCs) from bone marrow by disrupting the CXCR4/SDF-1alpha retention axis. We show here that distinct factors and mechanisms regulate the mobilization of endothelial (EPCs) and stromal progenitor cells (SPCs). Pretreatment of mice with VEGF did not disrupt the CXCR4/SDF-1alpha chemokine axis but stimulated entry of HPCs into the cell cycle via VEGFR1, reducing their migratory capacity in vitro and suppressing their mobilization in vivo. In contrast, VEGF pretreatment enhanced EPC mobilization via VEGFR2 in response to CXCR4 antagonism. Furthermore, SPC mobilization was detected when the CXCR4 antagonist was administered to mice pretreated with VEGF, but not G-CSF. Thus, differential mobilization of progenitor cell subsets is dependent upon the cytokine milieu that regulates cell retention and proliferation. These findings may inform studies investigating mechanisms that regulate progenitor cell recruitment in disease and can be exploited to provide efficacious stem cell therapy for tissue regeneration.

IL-9 Governs Allergen-induced Mast Cell Numbers in the Lung and Chronic Remodeling of the Airways

RATIONALE IL-9 is a pleiotropic cytokine that has multiple effects on structural as well as numerous hematopoietic cells, which are central to the pathogenesis of asthma. OBJECTIVES The contribution of IL-9 to asthma pathogenesis has thus far been unclear, due to conflicting reports in the literature. These earlier studies focused on the role of IL-9 in acute inflammatory models; here we have investigated the effects of IL-9 blockade during chronic allergic inflammation. METHODS Mice were exposed to either prolonged ovalbumin or house dust mite allergen challenge to induce chronic inflammation and airway remodeling. MEASUREMENTS AND MAIN RESULTS We found that IL-9 governs allergen-induced mast cell (MC) numbers in the lung and has pronounced effects on chronic allergic inflammation. Anti-IL-9 antibody-treated mice were protected from airway remodeling with a concomitant reduction in mature MC numbers and activation, in addition to decreased expression of the profibrotic mediators transforming growth factor-β1, vascular endothelial growth factor, and fibroblast growth factor-2 in the lung. Airway remodeling was associated with impaired lung function in the peripheral airways and this was reversed by IL-9 neutralization. In human asthmatic lung tissue, we identified MCs as the main IL-9 receptor expressing population and found them to be sources of vascular endothelial growth factor and fibroblast growth factor-2. CONCLUSIONS Our data suggest an important role for an IL-9-MC axis in the pathology associated with chronic asthma and demonstrate that an impact on this axis could lead to a reduction in chronic inflammation and improved lung function in patients with asthma.

IL-25 drives remodelling in allergic airways disease induced by house dust mite

Background Overexpression of the transforming growth factor β family signalling molecule smad2 in the airway epithelium provokes enhanced allergen-induced airway remodelling in mice, concomitant with elevated levels of interleukin (IL)-25. Objective We investigated whether IL-25 plays an active role in driving this airway remodelling. Methods Anti-IL-25 antibody was given to mice exposed to either inhaled house dust mite (HDM) alone, or in conjunction with an adenoviral smad2 vector which promotes an enhanced remodelling phenotype. Results Blocking IL-25 in allergen-exposed mice resulted in a moderate reduction in pulmonary eosinophilia and levels of T helper type 2 associated cytokines, IL-5 and IL-13. In addition, IL-25 neutralisation abrogated peribronchial collagen deposition, airway smooth muscle hyperplasia and airway hyperreactivity in control mice exposed to HDM and smad2-overexpressing mice. IL-25 was shown to act directly on human fibroblasts to induce collagen secretion. Recruitment of endothelial progenitor cells to the lung and subsequent neovascularisation was also IL-25 dependent, demonstrating a direct role for IL-25 during angiogenesis in vivo. Moreover, the secretion of innate epithelial derived cytokines IL-33 and thymic stromal lymphopoietin (TSLP) was completely ablated. Conclusions In addition to modulating acute inflammation, we now demonstrate a role for IL-25 in orchestrating airway remodelling. IL-25 also drives IL-33 and TSLP production in the lung. These data delineate a wider role for IL-25 in mediating structural changes to the lung following allergen exposure and implicate IL-25 as a novel therapeutic target for the treatment of airway remodelling in asthma.

Activin A and TGF-β promote TH9 cell-mediated pulmonary allergic pathology

BACKGROUND IL-9-secreting (T(H)9) T cells are thought to represent a distinct T-cell subset. However, evidence for their functionality in disease is uncertain. OBJECTIVE To define a functional phenotype for T(H)9-driven pathology in vivo. METHODS We used fluorescence-activated cell sorting to identify circulating T(H)9 cells in atopic and nonatopic subjects. In mice we utilized a model of allergic airways disease induced by house dust mite to determine T(H)9 cell function in vivo and the role of activin A in T(H)9 generation. RESULTS Allergic patients have elevated T(H)9 cell numbers in comparison to nonatopic donors, which correlates with elevated IgE levels. In a murine model, allergen challenge with house dust mite leads to rapid T(H)9 differentiation and proliferation, with much faster kinetics than for T(H)2 cell differentiation, resulting in the specific recruitment and activation of mast cells. The TGF-β superfamily member activin A replicates the function of TGF-β1 in driving the in vitro generation of T(H)9 cells. Importantly, the in vivo inhibition of T(H)9 differentiation induced by allergen was achieved only when activin A and TGF-β were blocked in conjunction but not alone, resulting in reduced airway hyperreactivity and collagen deposition. Conversely, adoptive transfer of T(H)9 cells results in enhanced pathology. CONCLUSION Our data identify a distinct functional role for T(H)9 cells and outline a novel pathway for their generation in vitro and in vivo. Functionally, T(H)9 cells promote allergic responses resulting in enhanced pathology mediated by the specific recruitment and activation of mast cells in the lungs.

Overexpression of Smad2 Drives House Dust Mite–mediated Airway Remodeling and Airway Hyperresponsiveness via Activin and IL-25

RATIONALE Airway hyperreactivity and remodeling are characteristic features of asthma. Interactions between the airway epithelium and environmental allergens are believed to be important in driving development of pathology, particularly because altered epithelial gene expression is common in individuals with asthma. OBJECTIVES To investigate the interactions between a modified airway epithelium and a common aeroallergen in vivo. METHODS We used an adenoviral vector to generate mice overexpressing the transforming growth factor-beta signaling molecule, Smad2, in the airway epithelium and exposed them to house dust mite (HDM) extract intranasally. MEASUREMENTS AND MAIN RESULTS Smad2 overexpression resulted in enhanced airway hyperreactivity after allergen challenge concomitant with changes in airway remodeling. Subepithelial collagen deposition was increased and smooth muscle hyperplasia was evident resulting in thickening of the airway smooth muscle layer. However, there was no increase in airway inflammation in mice given the Smad2 vector compared with the control vector. Enhanced airway hyperreactivity and remodeling did not correlate with elevated levels of Th2 cytokines, such as IL-13 or IL-4. However, mice overexpressing Smad2 in the airway epithelium showed significantly enhanced levels of IL-25 and activin A after HDM exposure. Blocking activin A with a neutralizing antibody prevented the increase in lung IL-25 and inhibited subsequent collagen deposition and also the enhanced airway hyperreactivity observed in the Smad2 overexpressing HDM-exposed mice. CONCLUSIONS Epithelial overexpression of Smad2 can specifically alter airway hyperreactivity and remodeling in response to an aeroallergen. Moreover, we have identified novel roles for IL-25 and activin A in driving airway hyperreactivity and remodeling.

CXCR2 Mediates the Recruitment of Endothelial Progenitor Cells During Allergic Airways Remodeling

Airway remodeling is a central feature of asthma and includes the formation of new peribronchial blood vessels, which is termed angiogenesis. In a number of disease models, bone marrow‐derived endothelial progenitor cells (EPCs) have been shown to contribute to the angiogenic response. In this study we set out to determine whether EPCs were recruited into the lungs in a model of allergic airways disease and to identify the factors regulating EPC trafficking in this model. We observed a significant increase in the number of peribronchial blood vessels at day 24, during the acute inflammatory phase of the model. This angiogenic response was associated with an increase in the quantity of EPCs recoverable from the lung. These EPCs formed colonies after 21 days in culture and were shown to express CD31, von Willebrand factor, and vascular endothelial growth factor (VEGF) receptor 2, but were negative for CD45 and CD14. The influx in EPCs was associated with a significant increase in the proangiogenic factors VEGF‐A and the CXCR2 ligands, CXCL1 and CXCL2. However, we show directly that, while the CXCL1 and CXCL2 chemokines can recruit EPCs into the lungs of allergen‐sensitized mice, VEGF‐A was ineffective in this respect. Further, the blockade of CXCR2 significantly reduced EPC numbers in the lungs after allergen exposure and led to a decrease in the numbers of peribronchial blood vessels after allergen challenge with no effect on inflammation. The data presented here provide in vivo evidence that CXCR2 is critical for both EPC recruitment and the angiogenic response in this model of allergic inflammation of the airways. STEM CELLS 2009;27:3074–3081

Annexin A1 regulates neutrophil clearance by macrophages in the mouse bone marrow

Under homeostatic conditions, a proportion of senescent CXCR4hi neutrophils home from the circulation back to the bone marrow, where they are phagocytosed by bone marrow macrophages. In this study, we have identified an unexpected role for the anti‐inflammatory molecule annexin A1 (AnxA1) as a critical regulator of this process. We first observed that AnxA1−/− mice have significantly increased neutrophil numbers in their bone marrow while having normal levels of GM and G colony‐forming units, monocytes, and macrophages. Although AnxA1−/− mice have more neutrophils in the bone marrow, a greater proportion of these cells are senescent, as determined by their higher levels of CXCR4 expression and annexin V binding. Consequently, bone marrow neutrophils from AnxA1−/− mice exhibit a reduced migratory capacity in vitro. Studies conducted in vitro also show that expression of AnxA1 is required for bone marrow macrophages, but not peritoneal macrophages, to phagocytose apoptotic neutrophils. Moreover, in vivo experiments indicate a defect in clearance of wild‐type neutrophils in the bone marrow of AnxA1−/− mice. Thus, we conclude that expression of AnxA1 by resident macrophages is a critical determinant for neutrophil clearance in the bone marrow.—Dalli, J., Jones, C. P., Cavalcanti, D. M., Farsky, S. H., Perretti, M., Rankin, S. M. Annexin A1 regulates neutrophil clearance by macrophages in the mouse bone marrow. FASEB J. 26, 387–396 (2012). www.fasebj.org

Bone Marrow-Derived Stem Cells and Respiratory Disease

Adult bone marrow contains a number of discrete populations of progenitor cells, including endothelial, mesenchymal, and epithelial progenitor cells and fibrocytes. In the context of a range of diseases, endothelial progenitor cells have been reported to promote angiogenesis, mesenchymal stem cells are potent immunosuppressors but can also contribute directly to tissue regeneration, and fibrocytes have been shown to induce tissue fibrosis. This article provides an overview of the basic biology of these different subsets of progenitor cells, reporting their distinct phenotypes and functional activities. The differences in their secretomes are highlighted, and the relative role of cellular differentiation vs paracrine effects of progenitor cells is considered. The article reviews the literature examining the contribution of progenitor cells to the pathogenesis of respiratory disease, and discusses recent studies using bone marrow progenitor cells as stem cell therapies in the context of pulmonary hypertension, COPD, and asthma.

Troubleshooting: Quantification of mobilization of progenitor cell subsets from bone marrow in vivo.

INTRODUCTION The molecular mechanisms that control the mobilization of specific stem cell subsets from the bone marrow are currently being intensely investigated. It is anticipated that boosting the mobilization of these stem cells via pharmacological intervention will not only produce more effective strategies for bone marrow transplant patients, but also provide novel therapeutic approaches for tissue regeneration. METHODS Measurement of stem cell mobilization by sampling peripheral blood is problematic because it is technically difficult to accurately determine absolute numbers of rare progenitor cells by blood sampling. Furthermore a rise in progenitors may be caused by release of stem cells from tissues other than the bone marrow (e.g. spleen and adipose), or indeed an inhibition of stem cell homing back to the bone marrow or other tissues. Finally it is not possible to distinguish whether the pharmacological agent is acting directly at the level of the bone marrow or mobilizing progenitors by a distinct indirect mechanism. To resolve these problems, we have developed a technique that allows perfusion of the vasculature of the hind limb bone marrow in situ in mice. In this system, the femoral artery and vein are cannulated in situ such that the femur and tibia bone marrow are perfused in isolation under anaesthesia. As such, pharmacological agents can be administered directly into the bone marrow vasculature. Mobilized cells are then collected via the femoral vein and colony assays performed in defined growth media to allow identification of haematopoietic, endothelial, and mesenchymal progenitor cells. We have used this system to determine the ability of a CXCR4 antagonist to mobilize these distinct types of progenitor cells from the bone marrow of mice pre-conditioned with either G-CSF or VEGF. RESULTS AND CONCLUSION This isolated hind limb perfusion system has allowed comparisons to be made between cytokines (G-CSF and VEGF) that act chronically, either alone or in combination with agents that act acutely on the bone marrow (CXCR4 antagonist) on their ability to directly mobilize specific populations of stem cells. Data obtained therefore gives a more accurate understanding of the efficacy of different mobilizing strategies compared to peripheral blood analysis.

Lung Macrophages Contribute to House Dust Mite Driven Airway Remodeling via HIF-1α

HIF-1α is a transcription factor that is activated during hypoxia and inflammation and is a key regulator of angiogenesis in vivo. During the development of asthma, peribronchial angiogenesis is induced in response to aeroallergens and is thought to be an important feature of sustained chronic allergic inflammation. Recently, elevated HIF-1α levels have been demonstrated in both the lung tissue and bronchoalveolar lavage of allergic patients, respectively. Therefore, we investigated the role of HIF-1α on the development of angiogenesis and inflammation following acute and chronic allergen exposure. Our data shows that intranasal exposure to house dust mite (HDM) increases the expression of HIF-1α in the lung, whilst reducing the expression of the HIF-1α negative regulators, PHD1 and PHD3. Blockade of HIF-1α in vivo, significantly decreased allergic inflammation and eosinophilia induced by allergen, due to a reduction in the levels of IL-5 and Eotaxin-2. Importantly, HIF-1α blockade significantly decreased levels of VEGF-A and CXCL1 in the lungs, which in turn led to a profound decrease in the recruitment of endothelial progenitor cells and a reduction of peribronchial angiogenesis. Furthermore, HDM or IL-4 treatment of primary lung macrophages resulted in significant production of both VEGF-A and CXCL1; inhibition of HIF-1α activity abrogated the production of these factors via an up-regulation of PHD1 and PHD3. These findings suggest that novel strategies to reduce the expression and activation of HIF-1α in lung macrophages may be used to attenuate allergen-induced airway inflammation and angiogenesis through the modulation of VEGF-A and CXCL1 expression. Clinical Relevance This study provides new insights into the role of HIF-1α in the development of peribronchial angiogenesis and inflammation in a murine model of allergic airway disease. These findings indicate that strategies to reduce activation of macrophage derived HIF-1α may be used as a target to improve asthma pathology.