SOCS1 and -3 proteins are released by alveolar macrophages into exosomes and microparticles, respectively, which are then taken up by alveolar epithelial cells, resulting in inhibition of STAT signaling. This process was dampened by exposure to cigarette smoke and may thus be important in suppressing airway inflammation.

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Differentiation of fibroblasts into a-smooth muscle actin (SMA)-expressing myofibroblasts represents a critical step in the pathogenesis of fibrotic disorders, and is generally regarded as irreversible. Prostaglandin E 2 (PGE 2 ) has been shown to prevent multiple aspects of fibroblast activation, including the differentiation of fibroblasts to myofibroblasts. Here, we investigated its ability to reverse this differentiated phenotype. Fetal and adult lung fibroblasts were induced to differentiate into myofibroblasts by 24-hour culture with transforming growth factor (TGF)-b1 or endothelin-1. Cells were then treated without or with PGE 2 for various intervals and assessed for a-SMA expression. In the absence of PGE 2 treatment, a-SMA expression induced by TGF-b1 was persistent and stable for up to 8 days. By contrast, PGE 2 treatment effected a dose-dependent decrease in a-SMA and collagen I expression that was observed 2 days after PGE 2 addition, peaked at 3 days, and persisted through 8 days in culture. This effect was not explained by an increase in myofibroblast apoptosis, and indeed, reintroduction of TGF-b1 2 days after addition of PGE 2 prompted dedifferentiated fibroblasts to re-express a-SMA, indicating redifferentiation to myofibroblasts. This effect of PGE 2 was associated with inhibition of focal adhesion kinase signaling, and a focal adhesion kinase inhibitor was also capable of reversing myofibroblast phenotype. These data unambiguously demonstrate reversal of established myofibroblast differentiation. Because many patients have established or even advanced fibrosis by the time they seek medical attention, this capacity of PGE 2 has the potential to be harnessed for therapy of late-stage fibrotic disorders.Keywords: E prostanoid receptor; transforming growth factor-b1; endothelin-1; a-smooth muscle actin; focal adhesion kinase Pathologic scarring or fibrosis results in impaired organ function in diseases such as cirrhosis, diabetes, end-stage renal disease, scleroderma, and pulmonary fibrosis (1, 2). The accumulation of myofibroblasts within pathologic lesions is a pivotal feature of many fibrotic disorders (1, 2). Fibroblasts possess the potential to differentiate into myofibroblasts, which are distinguished from fibroblasts by their expression of contractile proteins, such as a-smooth muscle actin (a-SMA), and their exuberant production of extracellular matrix proteins, such as collagen I. This expression of a-SMA and increased extracellular matrix production endow myofibroblasts with the ability to participate in wound contraction (3). Because the differentiation of fibroblasts to myofibroblasts is generally considered irreversible (4), resolution of normal wound repair is thought to require apoptosis of myofibroblasts (5). By contrast, pathologic fibrosis occurs when myofibroblasts fail to apoptose and instead accumulate and persist within tissues, contributing to progressive scarring. Indeed, idiopathic pulmonary fibrosis (IPF)-the most common and fatal type of lung fibrosis-is characterized b...

Background: PGE 2 inhibits TGF-␤1-induced myofibroblast differentiation, but the mechanism is incompletely understood. Results: PGE 2 inhibits ␣-SMA transcription in human lung fibroblasts by preventing both up-regulation of SRF expression and nuclear translocation of MRTF-A. Conclusion: PGE 2 blocks myofibroblast differentiation by targeting two critical determinants of contractile gene expression. Significance: These actions provide a mechanistic basis for therapeutic targeting of lung fibrosis.

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.

JAK-STAT signaling mediates the actions of numerous cytokines and growth factors, and its endogenous brake is the family of SOCS proteins. Consistent with their intracellular roles, SOCS proteins have never been identified in the extracellular space. Here we report that alveolar macrophages can secrete SOCS1 and -3 in exosomes and microparticles, respectively, for uptake by alveolar epithelial cells and subsequent inhibition of STAT activation. Secretion is tunable and occurs both in vitro and in vivo. SOCS secretion into lung lining fluid was diminished by cigarette smoking in humans and mice. Secretion and transcellular delivery of vesicular SOCS proteins thus represent a new model for the control of inflammatory signaling, which is subject to dysregulation during states of inflammation.

Preservation of gas exchange mandates that the pulmonary alveolar surface restrain unnecessarily harmful inflammatory responses to the many challenges to which it is exposed. These responses reflect the crosstalk between alveolar epithelial cells (AECs) and resident alveolar macrophages (AMs). We recently determined that AMs can secrete suppressors of cytokine signaling (SOCS) proteins within microparticles (MPs). Uptake of these SOCS-containing vesicles by epithelial cells inhibits cytokine-induced STAT activation. However, the ability of epithelial cells to direct AM release of SOCS-containing vesicles in response to inflammatory insults has not been studied. Here we report that SOCS3 protein was elevated in bronchoalveolar lavage fluid (BALF) of both virus- and bacteria-infected mice, as well as in an in vivo LPS model of acute inflammation. In vitro studies revealed that conditioned medium from LPS-stimulated AECs (AEC-CM) enhanced AM SOCS3 secretion above basal levels. Increased amounts of PGE2 were present in AEC-CM after LPS challenge, and both pharmacologic inhibition of PGE2 synthesis in AECs and neutralization of PGE2 in AEC-CM implicated this prostanoid as the major AEC-derived factor mediating enhanced AM SOCS3 secretion. Moreover, pharmacologic blockade of PGE2 synthesis or genetic deletion of a PGE2 synthase similarly attenuated the increase in BALF SOCS3 noted in lungs of mice challenged with LPS in vivo. These results demonstrate a novel tunable form of crosstalk in which AECs utilize PGE2 as a signal to “request” SOCS3 from AMs in order to dampen their endogenous inflammatory responses during infection.

Myofibroblasts are the major cellular source of collagen, and their accumulationvia differentiation from fibroblasts and resistance to apoptosis-is a hallmark of tissue fibrosis. Clearance of myofibroblasts by de-differentiation and restoration of apoptosis sensitivity has the potential to reverse fibrosis. Prostaglandin E2 (PGE2) and mitogens such as FGF2 have each been shown to de-differentiate myofibroblasts, but the resultant cellular phenotypes have neither been comprehensively characterized nor compared. Here we show that PGE2 elicited de-differentiation of human lung myofibroblasts via cAMP/PKA while FGF2 utilized MEK/ERK. The two mediators yielded transitional cells with distinct transcriptomes, with FGF2 promoting but PGE2 inhibiting proliferation and survival. The gene expression pattern in fibroblasts isolated from the lungs of mice undergoing resolution of experimental fibrosis resembled that of myofibroblasts treated with PGE2 in vitro. We conclude that myofibroblast dedifferentiation can proceed via distinct programs exemplified by treatment with PGE2 and FGF2, with that occurring in vivo most closely resembling the former.