RESUMEN
Myofibroblasts play key roles in wound healing and pathological fibrosis. Here, we used an RNAi screen to characterize myofibroblast regulatory genes, using a high-content imaging approach to quantify α-smooth muscle actin stress fibers in cultured human fibroblasts. Screen hits were validated on physiological compliance hydrogels, and selected hits tested in primary fibroblasts from patients with idiopathic pulmonary fibrosis. Our RNAi screen led to the identification of STAT3 as an essential mediator of myofibroblast activation and function. Strikingly, we found that STAT3 phosphorylation, while responsive to exogenous ligands on both soft and stiff matrices, is innately active on a stiff matrix in a ligand/receptor-independent, but ROCK- and JAK2-dependent fashion. These results demonstrate how a cytokine-inducible signal can become persistently activated by pathological matrix stiffening. Consistent with a pivotal role for this pathway in driving persistent fibrosis, a STAT3 inhibitor attenuated murine pulmonary fibrosis when administered in a therapeutic fashion after bleomycin injury. Our results identify novel genes essential for the myofibroblast phenotype, and point to STAT3 as an important target in pulmonary fibrosis and other fibrotic diseases.
Asunto(s)
Janus Quinasa 2/metabolismo , Miofibroblastos/metabolismo , Fibrosis Pulmonar/genética , Interferencia de ARN , Factor de Transcripción STAT3/metabolismo , Quinasas Asociadas a rho/metabolismo , Animales , Femenino , Fibroblastos/metabolismo , Humanos , Janus Quinasa 2/genética , Ratones , Ratones Endogámicos C57BL , Fosforilación , Fibrosis Pulmonar/metabolismo , Factor de Transcripción STAT3/genética , Transducción de Señal , Quinasas Asociadas a rho/genéticaRESUMEN
B lymphocytes play an essential regulatory role in the adaptive immune response through Ab production during infection. A less known function of B lymphocytes is their ability to respond directly to infectious Ags through stimulation of pattern recognition receptors expressed on their surfaces. ß-Glucans are carbohydrates present in the cell wall of many pathogenic fungi that can be detected in the peripheral blood of patients during infection. They have been shown to participate in the innate inflammatory response, as they can directly activate peripheral macrophages and dendritic cells. However, their effect as direct stimulators of B lymphocytes has not been yet fully elucidated. The aim of this study was to examine the molecular mechanisms and cytokine profiles generated following ß-glucan stimulation of B lymphocytes, compared with the well-established TLR-9 agonist CpG oligodeoxynucleotide (CpG), and study the participation of ß-glucan-stimulated B cells in the innate immune response. In this article, we demonstrate that ß-glucan-activated B lymphocytes upregulate proinflammatory cytokines (TNF-α, IL-6, and IL-8). Of interest, ß-glucan, unlike CpG, had no effect on B lymphocyte proliferation or IgM production. When compared with CpG (TLR9 agonist), ß-glucan-activated cells secreted significantly higher levels of IL-8. Furthermore, IL-8 secretion was partially mediated by Dectin-1 and required SYK, MAPKs, and the transcription factors NF-κB and AP-1. Moreover, we observed that conditioned media from ß-glucan-stimulated B lymphocytes elicited neutrophil chemotaxis. These studies suggest that ß-glucan-activated B lymphocytes have an important and novel role in fungal innate immune responses.
Asunto(s)
Linfocitos B/inmunología , Quimiotaxis de Leucocito/inmunología , Inmunidad Innata/inmunología , Neutrófilos/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis , Proliferación Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Humanos , Inmunoglobulina M/biosíntesis , Interleucina-6/biosíntesis , Interleucina-8/biosíntesis , Interleucina-8/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lectinas Tipo C/metabolismo , Activación de Linfocitos/inmunología , FN-kappa B/metabolismo , Oligodesoxirribonucleótidos/farmacología , Proteínas Tirosina Quinasas/metabolismo , Quinasa Syk , Linfocitos T/inmunología , Receptor Toll-Like 9/agonistas , Factor de Transcripción AP-1/metabolismo , beta-Glucanos/inmunologíaRESUMEN
BACKGROUND: Abnormal immune responses are believed to be highly relevant in the pathogenesis of chronic obstructive pulmonary disease (COPD). Dendritic cells provide a critical checkpoint for immunity by their capacity to both induce and suppress immunity. Although evident that cigarette smoke, the primary cause of COPD, significantly influences dendritic cell functions, little is known about the roles of dendritic cells in the pathogenesis of COPD. METHODS: The extent of dendritic cell infiltration in COPD tissue specimens was determined using immunohistochemical localization of CD83+ cells (marker of matured myeloid dendritic cells), and CD1a+ cells (Langerhans cells). The extent of tissue infiltration with Langerhans cells was also determined by the relative expression of the CD207 gene in COPD versus control tissues. To determine mechanisms by which dendritic cells accumulate in COPD, complimentary studies were conducted using monocyte-derived human dendritic cells exposed to cigarette smoke extract (CSE), and dendritic cells extracted from mice chronically exposed to cigarette smoke. RESULTS: In human COPD lung tissue, we detected a significant increase in the total number of CD83+ cells, and significantly higher amounts of CD207 mRNA when compared with control tissue. Human monocyte-derived dendritic cells exposed to CSE (0.1-2%) exhibited enhanced survival in vitro when compared with control dendritic cells. Murine dendritic cells extracted from mice exposed to cigarette smoke for 4 weeks, also demonstrated enhanced survival compared to dendritic cells extracted from control mice. Acute exposure of human dendritic cells to CSE induced the cellular pro-survival proteins heme-oxygenase-1 (HO-1), and B cell lymphoma leukemia-x(L) (Bcl-xL), predominantly through oxidative stress. Although activated human dendritic cells conditioned with CSE expressed diminished migratory CCR7 expression, their migration towards the CCR7 ligand CCL21 was not impaired. CONCLUSIONS: These data indicate that COPD is associated with increased numbers of cells bearing markers associated with Langerhans cells and mature dendritic cells, and that cigarette smoke promotes survival signals and augments survival of dendritic cells. Although CSE suppressed dendritic cell CCR7 expression, migration towards a CCR7 ligand was not diminished, suggesting that reduced CCR7-dependent migration is unlikely to be an important mechanism for dendritic cell retention in the lungs of smokers with COPD.