In this proposal, we will address the often debated role of the neonatal alveolar macrophage (AM) as an important contributor to impaired immune responses in the developing cystic fibrosis (CF) lung. AM are important in the initial defenses against bacteria and viruses, including respiratory syncytial virus (RSV), an important viral pathogen which causes significant morbidity in the CF lung. As one of few laboratories focused on the effects of oxidative stress on the function of AM in the newborn lung, we have begun to evaluate the effects of cftr mutation on neonatal AM using a mouse cftr knockout gut corrected model (cftr KO). While cftr KO mice do not spontaneously develop characteristic pulmonary pathologies of human CF lung per se, they serve as excellent models to evaluate the intrinsic cftr alterations in neonatal AM, in the absence of pulmonary infection. In cftr KO neonatal mice, oxidative stress was increased in the bronchoalveolar lavage fluid (BALF), even in the absence of infection. Cftr KO AM had dramatic increases in TGF?and the M2 marker arginase 1 (Arg1), and depressed phagocytosis. This suggests that the cftr mutation shifts the neonatal AM's activation state away from pathogen clearance (classically activated,M1) towards immunosuppression (alternative activation,M2). Interestingly, control neonatal AM treated with the BALF obtained from young children with CF also mimic the effects of the cftr KO AM with increased oxidant stress, TGF?and M2 polarization. A central role for CF-related decreases in the antioxidant glutathione (GSH) was demonstrated by the ability of extracellular GSH to attenuate the increased TGF?and Arg1 and improve phagocytosis in cftr KO AM and CFBALF-exposed AM. This suggested that oxidant-induced up- regulation of TGF?modulated the neonatal cftr KO AM's inflammatory state; but increasing extracellular GSH blunted CF-induced TGF?signaling and its downstream effects on AM polarity and function. Our central hypothesis is that decreased GSH pools due to cftr mutation increase AM oxidant stress and impair phagocytosis by increasing TGF? thereby shifting neonatal AM towards M2 activation and diminishing the neonatal lung's defenses against RSV.
In Aim 1, we will determine the effects of cftr mutations on baseline and RSV-stimulated state of manipulated cells lines, and primary neonatal AM isolated from cftr KO, ?08 and G551D mutated neonatal mice.
In Aim 2, we will examine the mechanisms by which maintaining GSH pools modulates the immune functions of neonatal CF AM. Alterations in AM activation state and subsequent changes in inflammatory signaling may compromise RSV clearance. This impaired response by AM may then be the signal that promotes sustained neutrophil recruitment into the airspace. With early identification of the CF newborn, the development of potential early therapeutic interventions could modify pulmonary inflammation and infection before respiratory symptoms occur. This proposal will begin to address whether the neonatal AM is an appropriate target for the development of such potential therapeutic strategies.
In this proposal, we will address the often debated role of the neonatal alveolar macrophage (AM) as an important contributor to impaired immune responses in the developing cystic fibrosis (CF) lung using cell lines and mice models incorporating the cftr mutation or deletion. With early identification of infants with CF, the development of potential early therapeutic interventions could modify pulmonary inflammation and infection from viruses such as respiratory syncytial virus before inflammatory respiratory symptoms occur. This proposal will begin to address whether the neonatal AM is an appropriate target for the development of such potential therapies.
| Gauthier, Theresa W; Grunwell, Jocelyn R; Ping, Xiao-Du et al. (2017) Impaired defenses of neonatal mouse alveolar macrophage withcftrdeletion are modulated by glutathione and TGF?1. Physiol Rep 5: |
