Investigating the role of macrophages in pulmonary arterial hypertension using the MacLow mouse model

Macrophages are proposed to play an important regulatory role in the pathogenesis of pulmonary arterial hypertension (PAH), as excessive infiltration is detected around vascular lesions in patients and animal models of the disease. The exact ‘causal’ role for macrophages, and whether their presence or absence is required for the vascular remodelling seen in PAH remains unclear. Using a novel inducible macrophage depletion model (MacLow mouse) I aimed to determine the role of macrophages in the pulmonary arterial remodeling associated with PAH. Macrophage depletion was induced in MacLow mice by administration of doxycycline, where macrophage- specific induction of the cytotoxic diphtheria toxin A chain (DTA) is driven by the CD68 promoter. Male but not female MacLow mice developed a spontaneous PAH phenotype compared to controls following six week doxycycline treatment, this was associated with increased right ventricular hypertrophy and pulmonary vascular remodelling. Immunohistochemical analysis of diseased lungs demonstrated dominancy of the CD206+ macrophages suggesting that M2- like macrophage population may drive this PAH phenotype. Differentiation towards M2 phenotype has been also observed in vitro using primary cells obtained from MacLow mice. Furthermore, by tracing the origin of the effector macrophages responsible for the development of MacLow- induced PAH using different chimeric mice, it seems that the major contributor cells originate within the lung tissue and that male bone marrow cells were not sufficient to induce disease in female recipient. Finally, culturing of bone marrow-derived macrophages and alveolar macrophages showed unique characteristic differences in term of their polarization, expression of DTA and expression of leukotriene B4 following stimulation with doxycycline. The data suggested that alveolar macrophage might be resistant to the doxycycline-induced depletion which may lead to activation of the alveolar macrophages following loss of the interstitial subtype. In conclusion, loss of CD68+ macrophages in MacLow mice led to the development of PAH spontaneously in male and not female mice, and the pathogenesis might involve activation of alveolar macrophages. Also these data further highlight the gender imbalance in PAH and add immune cells to this paradigm.