RESUMO
BACKGROUND: Chronic lung allograft dysfunction (CLAD) is the leading cause of mortality in lung transplant recipients. CLAD is characterized by respiratory failure owing to the accumulation of fibrotic cells in small airways and alveoli, inducing tissue contraction and architectural destruction. However, the source of the fibroblastic cells and the mechanism(s) underlying the accumulation and activation remain unexplained. Mesenchymal stromal cells (MSCs) are multipotent progenitors that are normally located in the lung tissue but can be isolated from the alveolar space in lung transplant recipients, where they have a profibrotic phenotype. Our objective was to identify the mediator(s) inducing migration and contractile differentiation of lung tissue MSCs. METHODS: Bronchoalveolar lavage (BAL) (7 healthy controls and 21 lung transplant recipients), CCL2, HGF, TGFB, EGF, and PDGF-BB and autotaxin were measured by enzyme-linked immunosorbent assay. BAL (7 healthy controls and 31 lung transplant recipients) lysophosphatidic acid (LPA) (16:0, 18:0, 18:1, 22:4) was measured by liquid chromatography with tandem mass spectrometry. The effect of inhibition of candidate mediators on BAL-mediated chemoattraction of MSCs and contraction of MSC-spiked collagen gel assays was assessed. BAL cells from a lung transplant recipient with CLAD were analyzed by single-cell RNA sequencing. RESULTS: We first demonstrate that BAL fluid from lung transplant recipients and particularly those with CLAD is potently chemoattractive to human lung tissueâderived MSCs and induces a contractile phenotype. After excluding several candidate mediators, we show that LPA blockade completely abrogated transplant recipient BALâmediated chemoattraction of MSCs and contraction of MSC-spiked collagen gels. Furthermore, LPA levels were enriched in transplant recipient BAL, and LPA replicated the observed in vitro profibrotic effects of transplant recipient BAL. Finally, we identify BAL monocyteâderived macrophages with autotaxin (ENPP2) and fibrotic transcriptional signature. CONCLUSIONS: Autotaxin-expressing alveolar macrophages are present in CLAD BAL. These cells potentially provide a local source of autotaxin/LPA that drives MSC recruitment and tissue contraction in CLAD. These cells are analogous to an aberrant macrophage population recently identified in idiopathic pulmonary fibrosis, suggesting an overlap in pathogenesis between CLAD and other forms of lung fibrosis.
Assuntos
Líquido da Lavagem Broncoalveolar/citologia , Transplante de Pulmão , Pulmão/metabolismo , Lisofosfolipídeos/metabolismo , Células-Tronco Mesenquimais/citologia , Fibrose Pulmonar/metabolismo , Transplantados , Adulto , Idoso , Biomarcadores/metabolismo , Movimento Celular , Colágeno/metabolismo , Feminino , Seguimentos , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Fibrose Pulmonar/patologiaRESUMO
BACKGROUND: Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are capable of repairing wounded lung epithelial cells by donating cytoplasmic material and mitochondria. Recently, we characterized two populations of human lung-derived mesenchymal stromal cells isolated from digested parenchymal lung tissue (LT-MSCs) from healthy individuals or from lung transplant recipients' bronchoalveolar lavage fluid (BAL-MSCs). The aim of this study was to determine whether LT-MSCs and BAL-MSCs are also capable of donating cytoplasmic content and mitochondria to lung epithelial cells. METHODS: Cytoplasmic and mitochondrial transfer was assessed by co-culturing BEAS2B epithelial cells with Calcein AM or Mitotracker Green FM-labelled MSCs. Transfer was then measured by flow cytometry and validated by fluorescent microscopy. Molecular inhibitors were used to determine the contribution of microtubules/tunnelling nanotubes (TNTs, cytochalasin D), gap junctions (carbenoxolone), connexin-43 (gap26) and microvesicles (dynasore). RESULTS: F-actin microtubules/TNTs extending from BM-MSCs, LT-MSCs and BAL-MSCs to bronchial epithelial cells formed within 45 minutes of co-culturing cells. Each MSC population transferred a similar volume of cytoplasmic content to epithelial cells. Inhibiting microtubule/TNTs, gap junction formation and microvesicle endocytosis abrogated the transfer of cytoplasmic material from BM-MSCs, LT-MSCs and BAL-MSCs to epithelial cells. In contrast, blocking connexin-43 gap junction formation had no effect on cytoplasmic transfer. All MSC populations donated mitochondria to bronchial epithelial cells with similar efficiency. Mitochondrial transfer was reduced in all co-cultures after microtubule/TNT or endocytosis inhibition. Gap junction formation inhibition reduced mitochondrial transfer in BM-MSC and BAL-MSC co-cultures but had no effect on transfer in LT-MSC co-cultures. Connexin-43 inhibition did not impact mitochondrial transfer. Finally, bronchial epithelial cells were incapable of donating cytoplasmic content or mitochondria to any MSC population. CONCLUSION: Similar to their bone marrow counterparts, LT-MSCs and BAL-MSCs can donate cytoplasmic content and mitochondria to bronchial epithelial cells via multiple mechanisms. Given that BM-MSCs utilize these mechanisms to mediate the repair of damaged bronchial epithelial cells, both LT-MSCs and BAL-MSCs will probably function similarly.
Assuntos
Células da Medula Óssea/metabolismo , Células Epiteliais/metabolismo , Pulmão/metabolismo , Células-Tronco Mesenquimais/metabolismo , Mitocôndrias/metabolismo , Adulto , Transporte Biológico , Células da Medula Óssea/ultraestrutura , Líquido da Lavagem Broncoalveolar/citologia , Comunicação Celular , Linhagem Celular , Técnicas de Cocultura , Conexina 43/genética , Conexina 43/metabolismo , Células Epiteliais/ultraestrutura , Feminino , Junções Comunicantes/metabolismo , Junções Comunicantes/ultraestrutura , Expressão Gênica , Humanos , Pulmão/ultraestrutura , Masculino , Células-Tronco Mesenquimais/ultraestrutura , Microscopia de Fluorescência , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Pessoa de Meia-Idade , Mitocôndrias/ultraestrutura , Cultura Primária de CélulasRESUMO
Immunosuppressive strategies continue to evolve, with a number of new formulations having been developed in recent years. Although acute rejection rates may have diminished, current protocols of immunosuppression for chronic organ rejection are clearly inadequate. This complication remains the primary cause of graft loss months to years after solid organ transplant. In summary, the overall goal of achieving immune tolerance remains elusive. This chapter will focus on the pharmacological manipulation of the rejection response, reviewing historical and current recommended protocols. A brief outline of potential future pathways of targeted immunosuppression is described.