Luis A. Ortiz

MESENCHYMAL STEM CELL ENGRAFTMENT IN LUNG IS ENHANCED IN RESPONSE TO BLEOMYCIN EXPOSURE AND AMELIORATES ITS FIBROTIC EFFECTS

Previously we described a reliable method based on immunodepletion for isolating mesenchymal stem cells (MSCs) from murine bone marrow and showed that, after intracranial transplantation, the cells migrated throughout forebrain and cerebellum and adopted neural cell fates. Here we systemically administered MSCs purified by immunodepletion from male bleomycin (BLM)-resistant BALB/c mice into female BLM-sensitive C57BL/6 recipients and quantified engraftment levels in lung by real-time PCR. Male DNA accounted for 2.21 × 10-5% of the total lung DNA in control-treated mice but was increased 23-fold (P = 0.05) in animals exposed to BLM before MSC transplantation. Fluorescence in situ hybridization revealed that engrafted male cells were localized to areas of BLM-induced injury and exhibited an epithelium-like morphology. Moreover, purification of type II epithelial cells from the lungs of transplant recipients resulted in a 3-fold enrichment of male, donor-derived cells as compared with whole lung tissue. MSC administration immediately after exposure to BLM also significantly reduced the degree of BLM-induced inflammation and collagen deposition within lung tissue. Collectively, these studies demonstrate that murine MSCs home to lung in response to injury, adopt an epithelium-like phenotype, and reduce inflammation and collagen deposition in lung tissue of mice challenged with BLM.

Interleukin 1 receptor antagonist mediates the antiinflammatory and antifibrotic effect of mesenchymal stem cells during lung injury

Mesenchymal stem cells (MSCs) have been exploited as cellular vectors to treat a wide array of diseases but the mechanisms responsible for their therapeutic effect remain indeterminate. Previously, we reported that MSCs inhibit bleomycin (BLM)-induced inflammation and fibrosis within the lungs of mice. Interrogation of the MSC transcriptome identified interleukin 1 receptor antagonist (IL1RN) as a potential mediator of this effect. Fractionation studies indicated that MSCs are the principal source of IL1RN in murine bone marrow and that its expression is restricted to a unique subpopulation of cells. Moreover, MSC-conditioned media was shown to block proliferation of an IL-1α-dependent T cell line and inhibit production of TNF-α by activated macrophages in vitro. Studies conducted in mice revealed that MSC administration was more effective than recombinant IL1RN delivered via adenoviral infection or osmotic pumps in inhibiting BLM-induced increases in TNF-α, IL-1α, and IL1RN mRNA in lung, IL1RN protein in bronchoalveolar lavage (BAL) fluid, and trafficking of lymphocytes and neutrophils into the lung. Therefore, MSCs protect lung tissue from BLM-induced injury by blocking TNF-α and IL-1, two fundamental proinflammatory cytokines in lung. Identification of IL1RN-expressing human MSC subpopulations may provide a novel cellular vector for treating chronic inflammatory diseases in humans.

Stem Cells and Cell Therapies in Lung Biology and Lung Diseases

The Alpha-1 Foundation, American Thoracic Society, Pulmonary Fibrosis Foundation, University of Vermont College of Medicine, University of Vermont Department of Medicine, and the Vermont Lung Center provided financial support for the conference. The conference was also supported in part by R13HL088903 from the NHLBI (D.J.W.). This article has an online supplement, which is accessible from this issues table of contents at www.atsjournals.org Conflict of Interest Statement: D.J.W. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.K.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. L.A.O. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.P.-M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. D.J.P has several patent applications submitted and two U. S. patents issued on mesenchymal stem cells; he has received royalties of less than

Loss of Fibroblast Thy-1 Expression Correlates with Lung Fibrogenesis

40,000 a year on several of these through Thomas Jefferson University and Drexel University. Organizing committee participants are as follows: Zea Borok, M.D., University of Southern California; Diane Krause, M.D., Ph.D., Yale University; Luis Ortiz, M.D., University of Pittsburgh; Darwin J. Prockop, M.D., Ph.D., Tulane University; Jeffrey Spees, Ph.D., University of Vermont; Benjamin T. Suratt, M.D., University of Vermont; Daniel J. Weiss, M.D., Ph.D., University of Vermont. A list of conference participants, executive summaries of speaker presentations, and poster abstracts are included in the online supplement.

Mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle microRNAs

Fibroblasts consist of heterogeneous subpopulations that have distinct roles in fibrotic responses. Previously we reported enhanced proliferation in response to fibrogenic growth factors and selective activation of latent transforming growth factor (TGF)-beta in fibroblasts lacking cell surface expression of Thy-1 glycoprotein, suggesting that Thy-1 modulates the fibrogenic potential of fibroblasts. Here we report that compared to controls Thy-1-/- C57BL/6 mice displayed more severe histopathological lung fibrosis, greater accumulation of lung collagen, and increased TGF-beta activation in the lungs 14 days after intratracheal bleomycin. The majority of cells demonstrating TGF-beta activation and myofibroblast differentiation in bleomycin-induced lesions were Thy-1-negative. Histological sections from patients with idiopathic pulmonary fibrosis demonstrated absent Thy-1 staining within fibroblastic foci. Normal lung fibroblasts, in both mice and humans, were predominantly Thy-1-positive. The fibrogenic cytokines interleukin-1 and tumor necrosis factor-alpha induced loss of fibroblast Thy-1 surface expression in vitro, which was associated with Thy-1 shedding, Smad phosphorylation, and myofibroblast differentiation. These results suggest that fibrogenic injury promotes loss of lung fibroblast Thy-1 expression, resulting in enhanced fibrogenesis.

Future Directions in Idiopathic Pulmonary Fibrosis Research. An NHLBI Workshop Report

Mesenchymal stem cells (MSCs) and macrophages are fundamental components of the stem cell niche and function coordinately to regulate haematopoietic stem cell self-renewal and mobilization. Recent studies indicate that mitophagy and healthy mitochondrial function are critical to the survival of stem cells, but how these processes are regulated in MSCs is unknown. Here we show that MSCs manage intracellular oxidative stress by targeting depolarized mitochondria to the plasma membrane via arrestin domain-containing protein 1-mediated microvesicles. The vesicles are then engulfed and re-utilized via a process involving fusion by macrophages, resulting in enhanced bioenergetics. Furthermore, we show that MSCs simultaneously shed micro RNA-containing exosomes that inhibit macrophage activation by suppressing Toll-like receptor signalling, thereby de-sensitizing macrophages to the ingested mitochondria. Collectively, these studies mechanistically link mitophagy and MSC survival with macrophage function, thereby providing a physiologically relevant context for the innate immunomodulatory activity of MSCs.

Atmospheric oxygen inhibits growth and differentiation of marrow-derived mouse mesenchymal stem cells via a p53-dependent mechanism: implications for long-term culture expansion.

The median survival of patients with idiopathic pulmonary fibrosis (IPF) continues to be approximately 3 years from the time of diagnosis, underscoring the lack of effective medical therapies for this disease. In the United States alone, approximately 40,000 patients die of this disease annually. In November 2012, the NHLBI held a workshop aimed at coordinating research efforts and accelerating the development of IPF therapies. Basic, translational, and clinical researchers gathered with representatives from the NHLBI, patient advocacy groups, pharmaceutical companies, and the U.S. Food and Drug Administration to review the current state of IPF research and identify priority areas, opportunities for collaborations, and directions for future research. The workshop was organized into groups that were tasked with assessing and making recommendations to promote progress in one of the following six critical areas of research: (1) biology of alveolar epithelial injury and aberrant repair; (2) role of extracellular matrix; (3) preclinical modeling; (4) role of inflammation and immunity; (5) genetic, epigenetic, and environmental determinants; (6) translation of discoveries into diagnostics and therapeutics. The workshop recommendations provide a basis for directing future research and strategic planning by scientific, professional, and patient communities and the NHLBI.

Tumor Necrosis Factor-α Accelerates the Resolution of Established Pulmonary Fibrosis in Mice by Targeting Profibrotic Lung Macrophages

Large scale expansion of human mesenchymal stem cells (MSCs) is routinely performed for clinical therapy. In contrast, developing protocols for large scale expansion of primary mouse MSCs has been more difficult due to unique aspects of rodent biology. Currently, established methods to isolate mouse MSCs select for rapidly dividing subpopulations that emerge from bone marrow cultures following long‐term (months) expansion in atmospheric oxygen. Herein, we demonstrate that exposure to atmospheric oxygen rapidly induced p53, TOP2A, and BCL2‐associated X protein (BAX) expression and mitochondrial reactive oxygen species (ROS) generation in primary mouse MSCs resulting in oxidative stress, reduced cell viability, and inhibition of cell proliferation. Alternatively, procurement and culture in 5% oxygen supported more prolific expansion of the CD45−ve/CD44+ve cell fraction in marrow, produced increased MSC yields following immunodepletion, and supported sustained MSC growth resulting in a 2,300‐fold increase in cumulative cell yield by fourth passage. MSCs cultured in 5% oxygen also exhibited enhanced trilineage differentiation. The oxygen‐induced stress response was dependent upon p53 since siRNA‐mediated knockdown of p53 in wild‐type cells or exposure of p53−/− MSCs to atmospheric oxygen failed to induce ROS generation, reduce viability, or arrest cell growth. These data indicate that long‐term culture expansion of mouse MSCs in atmospheric oxygen selects for clones with absent or impaired p53 function, which allows cells to escape oxygen‐induced growth inhibition. In contrast, expansion in 5% oxygen generates large numbers of primary mouse MSCs that retain sensitivity to atmospheric oxygen, and therefore a functional p53 protein, even after long‐term expansion in vitro. STEM CELLS 2012;30:975–987

Alveolar macrophage apoptosis and TNF-α, but not p53, expression correlate with murine response to bleomycin

Idiopathic pulmonary fibrosis (IPF) is a relentless, fibrotic parenchymal lung disease in which alternatively programmed macrophages produce profibrotic molecules that promote myofibroblast survival and collagen synthesis. Effective therapies to treat patients with IPF are lacking, and conventional therapy may be harmful. We tested the hypothesis that therapeutic lung delivery of the proinflammatory cytokine tumor necrosis factor (TNF)-α into wild-type fibrotic mice would reduce the profibrotic milieu and accelerate the resolution of established pulmonary fibrosis. Fibrosis was assessed in bleomycin-instilled wild-type and TNF-α(-/-) mice by measuring hydroxyproline levels, static compliance, and Massons trichrome staining. Macrophage infiltration and programming status was assessed by flow cytometry of enzymatically digested lung and in situ immunostaining. Pulmonary delivery of TNF-α to wild-type mice with established pulmonary fibrosis was found to reduce their fibrotic burden, to improve lung function and architecture, and to reduce the number and programming status of profibrotic alternatively programmed macrophages. In contrast, fibrosis and alternative macrophage programming were prolonged in bleomycin-instilled TNF-α(-/-) mice. To address the role of the reduced numbers of alternatively programmed macrophages in the TNF-α-induced resolution of established pulmonary fibrosis, we conditionally depleted macrophages in MAFIA (MAcrophage Fas-Induced Apoptosis) mice. Conditional macrophage depletion phenocopied the resolution of established pulmonary fibrosis observed after therapeutic TNF-α delivery. Taken together, our results show for the first time that TNF-α is involved in the resolution of established pulmonary fibrosis via a mechanism involving reduced numbers and programming status of profibrotic macrophages. We speculate that pulmonary delivery of TNF-α or augmenting its signaling pathway represent a novel therapeutic strategy to resolve established pulmonary fibrosis.

Systemic Inhibition of NF-κB Activation Protects from Silicosis

Apoptosis is considered to be a protective mechanism that limits lung injury. However, apoptosis might contribute to the inflammatory burden present in the injured lung. The exposure of mice to bleomycin (BLM) is a well-established model for the study of lung injury. BLM exposure induces DNA damage and enhances tumor necrosis factor (TNF)-alpha expression in the lung. To evaluate the importance of alveolar macrophage (AM) apoptosis in the pathogenesis of lung injury, we exposed BLM-sensitive (C57BL/6) and BLM-resistant (BALB/c) mice to BLM (120 mg/kg) and studied the induction of apoptosis [by light-microscopy changes (2, 8, 12, 24, 48, and 72 h) and annexin V uptake by flow cytometry (24 h)], the secretion of TNF-alpha (measured by ELISA), and the expression of p53 (by immunoblotting) in AM retrieved from these mice. BLM, but not vehicle, induced apoptosis in AM from both murine strains. The numbers of apoptotic AM were significantly greater (P < 0.001) in C57BL/6 mice (52.9%) compared with BALB/c mice (40.8%) as demonstrated by annexin V uptake. BLM induction of apoptosis in AM was preceded by an increased secretion of TNF-alpha in C57BL/6 but not in BALB/c mice. Furthermore, double TNF-alpha receptor-deficient mice, developed on a C57BL/6 background, demonstrated significantly (P < 0.001) lower numbers of apoptotic AM compared with C57BL/6 and BALB/c mice. BLM also enhanced p53 expression in AM from both murine strains. However, p53-deficient mice developed BLM-induced lung injury, exhibited similar lung cell proliferation (measured as proliferating cell nuclear antigen immunostaining), and accumulated similar amounts of lung hydroxyproline (65 +/- 6.9 microgram/lung) as did C57BL/6 (62 +/- 6.5 microgram/lung) mice. Therefore, AM apoptosis is occurring during BLM-induced lung injury in a manner that correlates with murine strain sensitivity to BLM. Furthermore, TNF-alpha secretion rather than p53 expression contributes to the difference in murine strain response to BLM.tumor necrosis factor; strain susceptibility