The role of myeloid cell type-specific inducible nitric oxide synthase in the development of smoke-induced pulmonary hypertension

Abstract

COPD, comprising chronic bronchitis and emphysema, represents one of the five leading causes of death worldwide. The disease is progressive and currently incurable, with the treatment options limited to those controlling the symptoms. Underlying molecular mechanisms leading to COPD include increased oxidative stress, the imbalance between proteolytic activity and anti-proteolytic defense and influx of inflammatory cells. Moreover, recent findings from preclinical models and COPD patients prompted the hypothesis that pulmonary vascular alterations are an early phenomenon of the COPD pathology and a possible driver of parenchymal destruction. In addition, abnormally high mean pulmonary arterial pressure is present in up to 90% of COPD patients. Although PH is associated with increased risk of exacerbations and decreased survival in COPD patients, efficient pharmacological options are not available. Some of the mechanisms implicated in the pathogenesis of COPD-PH are endothelial dysfunction, hypoxia, vascular pruning and loss of capillary bed. Moreover, activation of inflammatory cells might be a contributing factor to PH development in COPD. The previous work from the group in which the current thesis was done suggested that iNOS may represent a key player in the pathogenesis of smoke-induced PH as well as emphysema. In smoke-exposed mice, iNOS inhibition prevented and reversed parenchymal destruction, PH and pulmonary vascular remodeling. Moreover, experiments with chimeric mice showed that iNOS expression in bone marrow-derived cells is driving the pulmonary vascular alterations, but not emphysema development. However, it remained unclear which bone marrow-derived cell type drives the process and what the respective mechanism is. In this study, I aimed to identify the iNOS-expressing cell type driving smoke-induced PH and to decipher pro-proliferative pathways involved in this process. To address this question I used myeloid cell-specific iNOS knockout mice in chronic smoke exposure to monitor the development of PH and emphysema and co-cultures of macrophages and PASMC to decipher underlying pathways. Interestingly, myeloid cell-specific iNOS knockout mice were protected against smoke-induced PH but not emphysema. Moreover, myeloid cell-specific iNOS deletion ameliorated several smoke-induced changes in lung inflammatory cell composition and phenotype. Specifically, iNOS knockout in myeloid cells prevented the increase in expression of CD206, a marker of M2 polarization, on interstitial macrophages in smoke-exposed lungs. In vitro, smoke-induced pro-proliferative signaling in co-cultures of M2-polarized macrophages and PASMC was abolished by iNOS deletion in phagocytic cells. Importantly, numerous CD206-positive and iNOS-positive macrophages accumulated in the proximity of remodeled vessels in the lungs of COPD patients, as shown by immunohistochemistry. Interestingly, described effects on the composition and activity of lung macrophages were hypoxia-independent and represented the smoke-specific signaling events in the remodeling of the pulmonary vasculature, as myeloid cell-specific knockout mice developed PH after chronic exposure to hypoxia. Regarding the underlying molecular pathways, I found differences in IL-4 signaling between WT and iNOS-deficient M2 macrophages. Moreover, I demonstrated increased phosphorylation of ERK and p38 kinases, previously implicated in the proliferation of vascular cells, in PASMC co-cultured with iNOS-expressing macrophages, smoke-induced WT animals, as well as in remodeled pulmonary vessels in the lung of COPD patients. In summary, my results demonstrate that iNOS deletion in myeloid cells confers protection against PH in smoke-exposed mice and provide evidence for the communication between M2-like macrophages and PASMC in underlying pulmonary vascular remodeling.