Bone marrow-derived stromal cells (BMSCs) protect against acute lung injury (ALI). To determine the role of BMSC mitochondria in the protection, we airway-instilled mice first with lipopolysaccharide (LPS), then with mouse BMSCs (mBMSCs). Live optical studies revealed that mBMSCs formed connexin 43 (Cx43)-containing gap junctional channels (GJCs) with the alveolar epithelium, releasing mitochondria-containing microvesicles that the epithelium engulfed. The presence of BMSC mitochondria in the epithelium was evident optically, as also by the presence of human mitochondrial DNA in mouse lungs in which we instilled human BMSCs (hBMSCs). The mitochondrial transfer increased alveolar ATP. LPS-induced ALI, indicated by alveolar leukocytosis and protein leak, inhibition of surfactant secretion and high mortality, was markedly abrogated by wild type mBMSCs, but not by mutant, GJC-incompetent mBMSCs, or by mBMSCs with dysfunctional mitochondria. This is the first evidence that BMSCs protect against ALI by restituting alveolar bioenergetics through Cx43-dependent alveolar attachment and mitochondrial transfer.

The ability to generate lung and airway epithelial cells from human pluripotent stem cells (hPSCs) would have applications in regenerative medicine, drug screening and modeling of lung disease, and studies of human lung development. We established, based on developmental paradigms, a highly efficient method for directed differentiation of hPSCs into lung and airway epithelial cells. Long-term differentiation in vivo and in vitro yielded basal, goblet, Clara, ciliated, type I and type II alveolar epithelial cells. Type II alveolar epithelial cells generated were capable of surfactant protein-B uptake and stimulated surfactant release, providing evidence of specific function. Inhibiting or removing agonists to signaling pathways critical for early lung development in the mouse—retinoic acid, Wnt and BMP—recapitulated defects in corresponding genetic mouse knockouts. The capability of this protocol to generate most cell types of the respiratory system suggests its utility for deriving patient-specific therapeutic cells.

Recapitulation of lung development from human pluripotent stem cells (hPSCs) in three dimensions (3D) would allow deeper insight into human development, as well as the development of innovative strategies for disease modeling, drug discovery and regenerative medicine1. We report here the generation from hPSCs of lung bud organoids (LBOs) that contain mesoderm and pulmonary endoderm and develop into branching airway and early alveolar structures after xenotransplantation and in Matrigel 3D culture. Expression analysis and structural features indicated that the branching structures reached the second trimester of human gestation. Infection in vitro with respiratory syncytial virus, which causes small airway obstruction and bronchiolitis in infants2, led to swelling, detachment and shedding of infected cells into the organoid lumens, similar to what has been observed in human lungs3. Introduction of mutation in HPS1, which causes an early-onset form of intractable pulmonary fibrosis4,5, led to accumulation of extracellular matrix and mesenchymal cells, suggesting the potential use of this model to recapitulate fibrotic lung disease in vitro. LBOs therefore recapitulate lung development and may provide a useful tool to model lung disease.

Tissue-resident macrophages of barrier organs constitute the first line of defense against pathogens at the systemic interface with the ambient environment. In lung, resident alveolar macrophages (AMs) provide sentinel function against inhaled pathogens1. Bacterial constituents ligate toll-like receptors (TLRs) on AMs2, causing AMs to secrete proinflammatory cytokines3 that activate alveolar epithelial receptors4, leading to recruitment of neutrophils that engulf pathogens5,6. However, since the AM-induced immune response could itself cause tissue injury, it is unclear how AMs modulate the response to prevent injury. Here, through real-time alveolar imaging in situ, we show that a subset of AMs attached to the alveolar wall, formed connexin 43 (Cx43)-containing gap junctional channels (GJCs) with the epithelium. During lipopolysaccharide (LPS)-induced inflammation, the AMs remained alveolus-attached and sessile, and they established intercommunication through synchronized Ca2+ waves, using the epithelium as the conducting pathway. The intercommunication was immunosuppressive, involving Ca2+ dependent activation of Akt, since AM-specific knockout of Cx43 enhanced alveolar neutrophil recruitment and secretion of proinflammatory cytokines in the bronchoalveolar lavage (BAL). The picture emerges of a novel immunomodulatory process in which a subset of alveolus-attached AMs intercommunicates immunosuppressive signals to reduce endotoxin-induced lung inflammation.

Mesenchymal stem cells (MSCs), which potentially transdifferentiate into multiple cell types, are increasingly reported to be beneficial in models of organ system injury. However, the molecular mechanisms underlying interactions between MSCs and host cells, in particular endothelial cells (ECs), remain unclear. We show here in a matrigel angiogenesis assay that MSCs are capable of inhibiting capillary growth. After addition of MSCs to EC-derived capillaries in matrigel at EC: MSC ratio of 1:1, MSCs migrated toward the capillaries, intercalated between ECs, established Cx43-based intercellular gap junctional communication (GJC) with ECs, and increased production of reactive oxygen species (ROS). These events led to EC apoptosis and capillary degeneration. In an in vivo tumor model, direct MSC inoculation into subcutaneous melanomas induced apoptosis and abrogated tumor growth. Thus, our findings show for the first time that at high numbers, MSCs are potentially cytotoxic and that when injected locally in tumor tissue they might be effective antiangiogenesis agents suitable for cancer therapy. IntroductionIntense interest in the therapeutic application of bone marrowderived mesenchymal stem cells (MSCs) arises from the possibility that MSCs promote vascular repair. In animal models, intravenous injections of MSCs protected against heart failure by enhancing cardiac myocyte survival 1 and blocked lipopolysaccharide-induced acute lung injury by reducing total cell and proinflammatory cytokines in the lung. 2 In a collagen gel model, MSCs promoted survival of capillaries grown from human umbilical vein endothelial cells (HUVECs). 3 Despite these findings, the lack of conclusive evidence supporting a beneficial effect of MSCs in the clinical setting 4 indicates that mechanisms underlying MSC-endothelial cell (EC) interactions require better understanding.Several reports indicate that these interactions result from direct contact between MSCs and host cells. The MSC-induced responses include induction of gene transcription in ECs, 3 mitochondrial transfer in A549 cells, 5 and interleukin-10 (IL-10) secretion in alveolar macrophages. 6 In the context of tumor growth, MSCs recruit ECs to induce angiogenesis in stable tissue 7 as well as in tumors, 8 raising the possibility that MSCs might promote tumor growth. By contrast, intravenously injected MSCs are capable of abrogating growth of the Kaposi sarcoma, 9 suggesting that MSCs potentially possess cytotoxic properties. However, the mechanisms by which MSCs engage ECs are not understood and might involve gap junctional communication (GJC), as proposed for MSCcardiomycyte interactions. 10 Here, we addressed MSC-EC interactions in a capillary culture with the expectation that MSCs would enhance angiogenesis. However, surprisingly, addition of MSCs caused dose-dependent EC cytotoxicity that was attributable to the formation of MSC-EC GJC and the production of MSC-derived reactive oxygen species (ROS). The combined effect of these responses was capillary destruction. Further...

SUMMARY Regulatory T cell (Treg cell) responses and apoptotic cell clearance (efferocytosis) represent critical arms of the inflammation resolution response. We sought to determine whether these processes may be linked through Treg cell-mediated enhancement of efferocytosis. In zymosan-induced peritonitis and LPS-induced lung injury, Treg cells increased early in resolution, and Treg cell depletion decreased efferocytosis. In advanced atherosclerosis, where defective efferocytosis drives disease progression, Treg cell expansion improved efferocytosis. Mechanistic studies revealed the following sequence: (i) Treg cells secreted interleukin (IL)-13, which stimulated IL-10 production in macrophages; (ii) autocrine signaling by IL-10 induced Vav1 in macrophages; and (iii) Vav1 activated Rac1 to promote apoptotic cell engulfment. In summary, Treg cells promote macrophage efferocytosis during inflammation resolution via a transcellular signaling pathway that enhances apoptotic cell internalization. These findings suggest an expanded role of Treg cells in inflammation resolution and provide a mechanistic basis for Treg cell-enhancement strategies for non-resolving inflammatory diseases.

Endothelium forms a physical barrier that separates blood from tissue. Communication between blood and tissue occurs through the delivery of molecules and circulating substances across the endothelial barrier by directed transport either through or between cells. Inflammation promotes macromolecular transport by decreasing cell-cell and cell-matrix adhesion and increasing centripetally directed tension, resulting in the formation of intercellular gaps. Inflammation may also increase the selected transport of macromolecules through cells. Significant progress has been made in understanding the molecular and cellular mechanisms that account for constitutive endothelial cell barrier function and also the mechanisms activated during inflammation that reduce barrier function. Current concepts of mechanisms regulating endothelial cell barrier function were presented in a symposium at the 2000 Experimental Biology Conference and are reviewed here.

Considerable progress has been made in understanding the basic mechanisms that regulate fluid and protein exchange across the endothelial and epithelial barriers of the lung under both normal and pathological conditions. Clinically relevant lung injury occurs most commonly from severe viral and bacterial infections, aspiration syndromes, and severe shock. The mechanisms of lung injury have been identified in both experimental and clinical studies. Recovery from lung injury requires the reestablishment of an intact endothelial barrier and a functional alveolar epithelial barrier capable of secreting surfactant and removing alveolar edema fluid. Repair mechanisms include the participation of endogenous progenitor cells in strategically located niches in the lung. Novel treatment strategies include the possibility of cell-based therapy that may reduce the severity of lung injury and enhance lung repair.