Scott H. Wettlaufer

Lung Resident Mesenchymal Stem Cells Isolated From Human Lung Allografts Inhibit T Cell Proliferation via a Soluble Mediator

Development of allograft rejection continues to be the major determinant of morbidity and mortality postlung transplantation. We have recently demonstrated that a population of donor-derived mesenchymal stem cells is present in human lung allografts and can be isolated and expanded ex vivo. In this study, we investigated the impact of lung resident mesenchymal stem cells (LR-MSCs), derived from allografts of human lung transplant recipients, on T cell activation in vitro. Similar to bone marrow-derived MSCs, LR-MSCs did not express MHC II or the costimulatory molecules CD80 or CD86. In vitro, LR-MSCs profoundly suppressed the proliferative capacity of T cells in response to a mitogenic or an allogeneic stimulus. The immunosuppressive function of LR-MSCs was also noted in the absence of direct cell contact, indicating that LR-MSCs mediated their effect predominantly via a soluble mediator. LR-MSCs isolated from lung transplant recipients demonstrated PGE2 secretion at baseline (385 ± 375 pg/ml), which increased in response to IL-1β (1149 ± 1081 pg/ml). The addition of PG synthesis inhibitors (indomethacin and NS-398) substantially abrogated LR-MSC-mediated immunosuppression, indicating that PGE2 may be one of the major soluble mediators impacting T cell activity. This is the first report to demonstrate that human tissue-derived MSCs isolated from an allogeneic environment have the potential to mediate immunological responses in vitro.

Prostaglandin E2 induces fibroblast apoptosis by modulating multiple survival pathways

Although the lipid mediator prostaglandin E2 (PGE2) exerts antifibrotic effects by inhibiting multiple fibroblast functions, its ability to regulate fibroblast survival is unknown. Here, we examined the effects of this prostanoid on apoptosis and apoptosis pathways in normal and fibrotic lung fibroblasts. As compared to medium alone, 24 h of treatment with PGE2 increased apoptosis of normal lung fibroblasts in a dose‐dependent manner (EC50~50 nM), as measured by annexin V staining, caspase 3 activity, cleavage of poly‐ADP‐ribose polymerase, and single‐stranded DNA levels. PGE2 also potentiated apoptosis elicited by Fas ligand plus cycloheximide. These proapoptotic actions were dependent on signaling through the EP2/EP4 receptors and by downstream activation of both caspases 8 and 9. Silencing and gene deletion of PTEN demonstrated that the effects of PGE2 involved decreased activity of the prosurvival molecule Akt. PGE2 also down‐regulated expression of survivin, an inhibitor of apoptosis, and increased expression of Fas. Fibroblasts from patients with pulmonary fibrosis exhibited resistance to the apoptotic effects of PGE2. These findings show for the first time that, in contrast to its effects on many other cell types, PGE2 promotes apoptosis in lung fibroblasts through diverse pathways. They provide another dimension by which PGE2 may inhibit, and perhaps even reverse, fibrogenesis in patients with interstitial lung disease.—Huang, S. K., White, E. S., Wettlaufer, S. H., Grifka, H., Hogaboam, C. M., Thannickal, V. J., Horowitz, J. C., Peters‐Golden, M. Prostaglandin E2 induces fibroblast apoptosis by modulating multiple survival pathways. FASEB J. 23, 4317–4326 (2009). www.fasebj.org

The antifibrotic effects of plasminogen activation occur via prostaglandin E2 synthesis in humans and mice

Plasminogen activation to plasmin protects from lung fibrosis, but the mechanism underlying this antifibrotic effect remains unclear. We found that mice lacking plasminogen activation inhibitor-1 (PAI-1), which are protected from bleomycin-induced pulmonary fibrosis, exhibit lung overproduction of the antifibrotic lipid mediator prostaglandin E2 (PGE2). Plasminogen activation upregulated PGE2 synthesis in alveolar epithelial cells, lung fibroblasts, and lung fibrocytes from saline- and bleomycin-treated mice, as well as in normal fetal and adult primary human lung fibroblasts. This response was exaggerated in cells from Pai1-/- mice. Although enhanced PGE2 formation required the generation of plasmin, it was independent of proteinase-activated receptor 1 (PAR-1) and instead reflected proteolytic activation and release of HGF with subsequent induction of COX-2. That the HGF/COX-2/PGE2 axis mediates in vivo protection from fibrosis in Pai1-/- mice was demonstrated by experiments showing that a selective inhibitor of the HGF receptor c-Met increased lung collagen to WT levels while reducing COX-2 protein and PGE2 levels. Of clinical interest, fibroblasts from patients with idiopathic pulmonary fibrosis were found to be defective in their ability to induce COX-2 and, therefore, unable to upregulate PGE2 synthesis in response to plasmin or HGF. These studies demonstrate crosstalk between plasminogen activation and PGE2 generation in the lung and provide a mechanism for the well-known antifibrotic actions of the fibrinolytic pathway.

Resident Tissue-Specific Mesenchymal Progenitor Cells Contribute to Fibrogenesis in Human Lung Allografts

Fibrotic obliteration of the small airways leading to progressive airflow obstruction, termed bronchiolitis obliterans syndrome (BOS), is the major cause of poor outcomes after lung transplantation. We recently demonstrated that a donor-derived population of multipotent mesenchymal stem cells (MSCs) can be isolated from the bronchoalveolar lavage (BAL) fluid of human lung transplant recipients. Herein, we study the organ specificity of these cells and investigate the role of local mesenchymal progenitors in fibrogenesis after lung transplantation. We demonstrate that human lung allograft-derived MSCs uniquely express embryonic lung mesenchyme-associated transcription factors with a 35,000-fold higher expression of forkhead/winged helix transcription factor forkhead box (FOXF1) noted in lung compared with bone marrow MSCs. Fibrotic differentiation of MSCs isolated from normal lung allografts was noted in the presence of profibrotic mediators associated with BOS, including transforming growth factor-β and IL-13. MSCs isolated from patients with BOS demonstrated increased expression of α-SMA and collagen I when compared with non-BOS controls, consistent with a stable in vivo fibrotic phenotype. FOXF1 mRNA expression in the BAL cell pellet correlated with the number of MSCs in the BAL fluid, and myofibroblasts present in the fibrotic lesions expressed FOXF1 by in situ hybridization. These data suggest a key role for local tissue-specific, organ-resident, mesenchymal precursors in the fibrogenic processes in human adult lungs.

Airway remodeling in murine asthma correlates with a defect in PGE2 synthesis by lung fibroblasts.

Asthma is a chronic lung disease characterized by local inflammation that can result in structural alterations termed airway remodeling. One component of airway remodeling involves fibroblast accumulation and activation, resulting in deposition of collagen I around small bronchi. Prostaglandin E(2) (PGE(2)) is the main eicosanoid lipid mediator produced by lung fibroblasts, and it exerts diverse anti-fibrotic actions. Dysregulation of the PGE(2) synthesis/response axis has been identified in human pulmonary fibrotic diseases and implicated in the pathogenesis of animal models of lung parenchymal fibrosis. Here we investigated the relationship between the fibroblast PGE(2) axis and airway fibrosis in an animal model of chronic allergic asthma. Airway fibrosis increased progressively as the number of airway challenges with antigen increased from 3 to 7 to 12. Compared with cells from control lungs, fibroblasts grown from the lungs of asthmatic animals, regardless of challenge number, exhibited no defect in the ability of PGE(2) or its analogs to inhibit cellular proliferation and collagen I expression. This correlated with intact expression of the EP(2) receptor, which is pivotal for PGE(2) responsiveness. However, cytokine-induced upregulation of PGE(2) biosynthesis as well as expression of cyclooxygenase-2 (COX-2) and microsomal PGE synthase-1 declined with increasing numbers of antigen challenges. In addition, treatment with the COX-2-selective inhibitor nimesulide potentiated the degree of airway fibrosis following repeated allergen challenge. Because endogenous COX-2-derived PGE(2) acts as a brake on airway fibrosis, the inability of fibroblasts to upregulate PGE(2) generation in the inflammatory milieu presented by repeated allergen exposure could contribute to the airway remodeling and fibrosis observed in chronic asthma.

Prostaglandin E2 As an Inhibitory Modulator of Fibrogenesis in Human Lung Allografts

RATIONALE Donor mesenchymal stromal/stem cell (MSC) expansion and fibrotic differentiation is associated with development of bronchiolitis obliterans syndrome (BOS) in human lung allografts. However, the regulators of fibrotic differentiation of these resident mesenchymal cells are not well understood. OBJECTIVES This study examines the role of endogenous and exogenous prostaglandin (PG)E2 as a modulator of fibrotic differentiation of human lung allograft-derived MSCs. METHODS Effect of PGE2 on proliferation, collagen secretion, and α-smooth muscle actin (α-SMA) expression was assessed in lung-resident MSCs (LR-MSCs) derived from patients with and without BOS. The response pathway involved was elucidated by use of specific agonists and antagonists. MEASUREMENT AND MAIN RESULTS PGE2 treatment of LR-MSCs derived from normal lung allografts significantly inhibited their proliferation, collagen secretion, and α-SMA expression. On the basis of pharmacologic and small-interfering RNA approaches, a PGE2/E prostanoid (EP)2/adenylate cyclase pathway was implicated in these suppressive effects. Stimulation of endogenous PGE2 secretion by IL-1β was associated with amelioration of their myofibroblast differentiation in vitro, whereas its inhibition by indomethacin augmented α-SMA expression. LR-MSCs from patients with BOS secreted significantly less PGE2 than non-BOS LR-MSCs. Furthermore, BOS LR-MSCs were found to be defective in their ability to induce cyclooxygenase-2, and therefore unable to up-regulate PGE2 synthesis in response to IL-1β. BOS LR-MSCs also demonstrated resistance to the inhibitory actions of PGE2 in association with a reduction in the EP2/EP1 ratio. CONCLUSIONS These data identify the PGE2 axis as an important autocrine-paracrine brake on fibrotic differentiation of LR-MSCs, a failure of which is associated with BOS.

Differential regulation by leukotrienes and calcium of Fcγ receptor-induced phagocytosis and Syk activation in dendritic cells versus macrophages

Macrophage (MØ) phagocytosis via the Fc receptor for immunoglobulin G (FcγR) requires the spleen tyrosine kinase (Syk) and serves an important antimicrobial function. We have reported previously that FcγR‐mediated ingestion and Syk activation in MØ are amplified by and depend on the proinflammatory lipid mediator leukotriene B4 (LTB4). Although FcγR‐mediated ingestion is also important for antigen uptake, there is no information about LTB4 regulation of these processes in dendritic cells (DCs). In this study, we compared murine bone marrow (BM)‐derived DCs to MØ from BM, peritoneum, and the pulmonary alveolar space. Neither phagocytosis nor Syk activation in DCs was influenced by exogenous LTB4. Unlike the various MØ populations, Syk activation in DCs was likewise unaffected by pharmacologic or genetic strategies to inhibit endogenous LTB4 synthesis or to block the high‐affinity LTB4 receptor BLT1. DCs were refractory to regulation by LTB4 despite the fact that they expressed BLT1 and mobilized intracellular calcium in response to its ligation. This resistance to LTB4 in DCs instead reflected the fact that in contrast to MØ, Syk activation in DCs was itself entirely independent of calcium. These results identify a fundamental difference in FcγR signaling between DCs and MØ, which may relate to the divergent, functional consequences of target ingestion in the two cell types.

Reversal of the Transcriptome by Prostaglandin E2 during Myofibroblast Dedifferentiation

Myofibroblasts, the major effector cells in pathologic fibrosis, derive from the differentiation of fibroblasts driven by mediators such as transforming growth factor-β1 (TGF-β1) and biomechanical signals. Although the myofibroblast has traditionally been considered a terminally differentiated cell, the lipid mediator prostaglandin E2 (PGE2) has been shown to not only prevent but also reverse myofibroblast differentiation, as characterized by the ability of PGE2 to diminish expression of collagen I and α-smooth muscle actin in established myofibroblasts. Here, we use microarrays to examine the extent of transcriptomic changes that occur during TGF-β1-induced differentiation and PGE2-induced dedifferentiation of myofibroblasts. Normal primary human adult lung fibroblasts were cultured for 24 hours with or without TGF-β1 and treated for 48 hours with PGE2. Gene expression levels were assessed from total RNA on the Affymetrix U219 microarray. TGF-β1 up-regulated 588 genes and down-regulated 689 genes compared with control cells. PGE2 reversed the expression of 363 (62%) of the TGF-β1-up-regulated genes and 345 (50%) of the TGF-β1-down-regulated genes. Genes up-regulated by TGF-β1 and reversed by PGE2 were enriched in annotations for Cell Adhesion, Contractile Fiber, and Actin Binding, whereas genes down-regulated by TGF-β1 but subsequently reversed by PGE2 were enriched in annotations for Glycoprotein, Polysaccharide Binding, and Regulation of Cell Migration. Surprisingly, the genes whose expression was affected by PGE2 differed between TGF-β1-induced myofibroblasts and undifferentiated fibroblasts. These data demonstrate the capacity of PGE2 to effect marked global alterations in the transcriptomic program of differentiated myofibroblasts and emphasize the considerable plasticity of these cells.

Mechanisms and modulation of microvesicle uptake in a model of alveolar cell communication

Extracellular vesicles, including exosomes and shed microvesicles (MVs), can be internalized by recipient cells to modulate function. Although the mechanism by which extracellular vesicles are internalized is incompletely characterized, it is generally considered to involve endocytosis and an initial surface-binding event. Furthermore, modulation of uptake by microenvironmental factors is largely unstudied. Here, we used flow cytometry, confocal microscopy, and pharmacologic and molecular targeting to address these gaps in knowledge in a model of pulmonary alveolar cell-cell communication. Alveolar macrophage–derived MVs were fully internalized by alveolar epithelial cells in a time-, dose-, and temperature-dependent manner. Uptake was dependent on dynamin and actin polymerization. However, it was neither saturable nor dependent on clathrin or receptor binding. Internalization was enhanced by extracellular proteins but was inhibited by cigarette smoke extract via oxidative disruption of actin polymerization. We conclude that MV internalization occurs via a pathway more consistent with fluid-phase than receptor-dependent endocytosis and is subject to bidirectional modulation by relevant pathologic perturbations.

Distinct PKA regulatory subunits mediate PGE2 inhibition of TGFβ-1-stimulated collagen I translation and myofibroblast differentiation

Prostaglandin E2 (PGE2), via cAMP signaling, inhibits a variety of fibroblast functions relevant to fibrogenesis. Among these are their translation of collagen I protein and their differentiation to myofibroblasts. PKA is central to these actions, with cAMP binding to regulatory (R) subunits leading to the release of catalytic subunits. Here we examined the role of specific PKAR subunit isoforms in these inhibitory actions in transforming growth factor β-1 (TGFβ-1)-stimulated human lung fibroblasts (HLFs). HLFs expressed all four R subunit isoforms. siRNA-mediated knockdown of subunits PKARIα and PKARIIα had no effect on PGE2 inhibition of either process. However, knockdown of PKARIβ selectively attenuated PGE2 inhibition of collagen I protein expression, whereas knockdown of PKARIIβ selectively attenuated PGE2 inhibition of expression of the myofibroblast differentiation marker, α-smooth muscle actin (α-SMA). cAMP analogs that selectively activate either PKARIβ or PKARIIβ exclusively inhibited collagen I synthesis or differentiation, respectively. In parallel, the PKARIβ agonist (but not a PKARIIβ agonist) reduced phosphorylation of two proteins involved in protein translation, protein kinase B (AKT) and mammalian target of rapamycin (mTOR). By contrast, the PKARIIβ agonist (but not a PKARIβ agonist) reduced levels of the differentiation-associated phosphorylated focal adhesion kinase (p-FAK) as well as the relative mRNA and protein expression of serum response factor (SRF), a transcription factor necessary for myofibroblast differentiation. Our results demonstrate that cAMP inhibition of collagen I translation and myofibroblast differentiation reflects the actions of distinct PKAR subunits.