Selective Inhibition of Bromodomain-Containing Protein 4 Reduces Myofibroblast Transdifferentiation and Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis is a lethal disease driven by myofibroblast expansion. Currently no therapies exist that target the epigenetic mechanisms controlling myofibroblast transdifferentiation, which is responsible for unregulated extracellular matrix (ECM) production. We have recently shown that bromodomain-containing protein 4 (BRD4), an epigenetic regulator that forms a scaffold for nuclear activators and transcription factors, is essential for TGFß-induced myofibroblast transdifferentiation. However, its role in the development and progression of pulmonary fibrosis in vivo has not been established. Here, we evaluate the hypothesis that BRD4 bromodomain interactions mediate myofibroblast expansion and fibrosing disease in vivo. C57BL/6J mice challenged with intratracheal bleomycin were systemically treated with a selective allosteric inhibitor of the BRD4 bromodomain 1 (BD1), ZL0591 (10 mg/kg), during the established fibrotic phase (14 days post-bleomycin) in a rigorous therapeutic paradigm. Eleven days after initiation of ZL0591 treatment (25 days post-bleomycin), we detected a significant improvement in blood O2 saturation compared to bleomycin/vehicle control. Twenty-eight days post-bleomycin, we observed a reduction in the volumetric Hounsfield Unit (HU) density by micro computed tomography (µCT) in the ZL0591-treated group, as well as a reduction in collagen deposition (hydroxyproline content) and severity of injury (Ashcroft scoring). Myofibroblast transdifferentiation was measured by smooth muscle α-actin (αSMA) staining, indicating a loss of this cell population in the ZL0591-treated group, and corresponded to reduced transcript levels of myofibroblast-associated extracellular matrix genes, tenascin-C and collagen 1α1. We conclude that BRD4 BD1 interactions are critical for myofibroblast transdifferentiation and fibrotic progression in a mouse model of pulmonary fibrosis.

Interleukin-1α Is a Critical Mediator of the Response of Human Bronchial Fibroblasts to Eosinophilic Inflammation.

Eosinophils contribute to allergic inflammation in asthma in part via elaboration of a complex milieu of soluble mediators. Human bronchial fibroblasts (HBF) respond to stimulation by these mediators by acquiring a pro-inflammatory profile including induction of interleukin 6 (IL6) and IL8. This study sought to determine key component(s) of eosinophil soluble factors that mediate IL6 and IL8 induction in HBF. HBF treated with eosinophil-derived soluble mediators were analyzed for gene expression, intracellular signaling, and IL6 and IL8 secretion following inhibition of inflammatory signaling. Segmental allergen bronchoprovocation (SBP-Ag) was performed in mild asthmatics and bronchoalveolar lavage fluid was analyzed for eosinophils and cytokines. We found that signaling via the IL1α/IL1 receptor is an essential component of the response of HBF to eosinophil-derived soluble factors. IL1α-dependent activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) signaling is required to induce IL6 secretion. However, NFκB signaling is dispensable for the induction of IL8, whereas Src is required. IL1α is associated with eosinophilic inflammation in human airways after SBP-Ag. Conclusions: IL1α appears to be a critical component of the soluble eosinophil-derived milieu that drives pro-inflammatory bronchial fibroblast responses and associates with eosinophilic inflammation following SBP-Ag. Disruption of IL1α-signaling could modify the downstream effects of eosinophilic inflammation on airway remodeling.

Eosinophil cytolysis on Immunoglobulin G is associated with microtubule formation and suppression of rho-associated protein kinase signalling.

BACKGROUND: The presence of eosinophils in the airway is associated with asthma severity and risk of exacerbations. Cell-free eosinophil granules are found in tissues in eosinophilic diseases, including asthma. This suggests that eosinophils have lysed and released cellular content, likely harming tissues. OBJECTIVE: The present study explores the mechanism of CD32- and αMß2 integrin-dependent eosinophil cytolysis of IL3-primed blood eosinophils seeded on heat-aggregated immunoglobulin G (HA-IgG). METHODS: Cytoskeletal events and signalling pathways potentially involved in cytolysis were assessed using inhibitors. The level of activation of the identified events and pathways involved in cytolysis was measured. In addition, the links between these identified pathways and changes in degranulation (exocytosis) and adhesion were analysed. RESULTS: Cytolysis of IL3-primed eosinophils was dependent on the production of reactive oxygen species (ROS) and downstream phosphorylation of p-38 MAPK. In addition, formation of microtubule (MT) arrays was necessary for cytolysis and was accompanied by changes in MT dynamics as measured by phosphorylation status of stathmin and microtubule-associated protein 4 (MAP4), the latter of which was regulated by ROS production. Reduced ROCK signalling preceded cytolysis, which was associated with eosinophil adhesion and reduced migration. CONCLUSION AND CLINICAL RELEVANCE: In this CD32- and αMß2 integrin-dependent adhesion model, lysing eosinophils exhibit reduced migration and ROCK signalling, as well as both MT dynamic changes and p-38 phosphorylation downstream of ROS production. We propose that interfering with these pathways would modulate eosinophil cytolysis and subsequent eosinophil-driven tissue damage.