Bartman, Colleen M

Recognizing that antioxidant systems are impaired in the airways of premature infants, we used human fetal airway cells to explore the impact of oxygen on iron regulation and iron-mediated cell death (ferroptosis). We find moderate hyperoxia impairs antioxidant systems that prevent ferroptosis and dysregulates iron metabolism, while severe hyperoxia has a negative effect on mechanisms driving the early onset of ferroptosis. Inhibitors of ferroptosis decrease iron and lipid peroxidation, demonstrating links between oxygen and iron regulation in developing airways.

The studies demonstrate the differential effects of the nicotinic acetylcholine receptors (nAChRs) regulating airway hyperreactivity (AHR) and remodeling in a mixed allergen model of asthma. We compared wild-type (WT) versus global α7nAChR knockout (Glα7KO) versus smooth muscle-specific α7nAChR KO (smα7KO) mice. We found that smα7KO improved lung function and AHR in mice challenged with MA and in the presence of nicotine, whereas MA-challenged Glα7KO in the presence of nicotine showed worsened AHR.

AbstractSupplemental O2 (hyperoxia) is a critical intervention for premature infants (<34 weeks) but consequently is associated with development of bronchial airway hyperreactivity (AHR) and asthma. Clinical practice shifted toward the use of moderate hyperoxia (<60% O2), but risk for subsequent airway disease remains. In mouse models of moderate hyperoxia, neonatal mice have increased AHR with effects on airway smooth muscle (ASM), a cell type involved in airway tone, bronchodilation, and remodeling. Understanding mechanisms by which moderate O2 during the perinatal period initiates sustained airway changes is critical to drive therapeutic advancements toward treating airway diseases. We propose that cellular clock factor BMAL1 is functionally important in developing mouse airways. In adult mice, cellular clocks target pathways highly relevant to asthma pathophysiology and Bmal1 deletion increases inflammatory response, worsens lung function, and impacts survival outcomes. Our understanding of BMAL1 in the developing lung is limited, but our previous findings show functional relevance of clocks in human fetal ASM exposed to O2. Here, we characterize Bmal1 in our established mouse neonatal hyperoxia model. Our data show that Bmal1 KO deleteriously impacts the developing lung in the context of O2 and these data highlight the importance of neonatal sex in understanding airway disease.

With continued smoking of tobacco products and expanded use of nicotine delivery devices worldwide, understanding the impact of smoking and vaping on respiratory health remains a major global unmet need. Although multiple studies have shown a strong association between smoking and asthma, there is a relative paucity of mechanistic understanding of how elements in cigarette smoke impact the airway. Recognizing that nicotine is a major component in both smoking and vaping products, it is critical to understand the mechanisms by which nicotine impacts airways and promotes lung diseases such as asthma. There is now increasing evidence that α7 nicotinic acetylcholine receptors (α7nAChRs) are critical players in nicotine effects on airways, but the mechanisms by which α7nAChR influences different airway cell types have not been widely explored. In this review, we highlight and integrate the current state of knowledge regarding nicotine and α7nAChR in the context of asthma and identify potential approaches to alleviate the impact of smoking and vaping on the lungs.

Chronic airway disease is a short- and long-term consequence of premature birth. Understanding effects of O2 exposure during the perinatal period is key to identify targetable mechanisms that initiate and sustain adverse airway changes. Our findings show a beneficial effect of exogenous H2S on developing mouse airway structure and function with notable sex differences. H2S donors alleviate effects of O2 on airway hyperreactivity, contractility, airway smooth muscle thickness, and extracellular matrix deposition.

Asthma is a major healthcare burden, which is further exacerbated by smoking. Recognizing the smoking risk of asthma, understanding the effects of nicotine on asthmatic airways becomes critical. Surprisingly, the mechanisms of nicotine action, even in normal and especially asthmatic airways, are understudied. Accordingly, the goal of this research is to investigate how nicotine influences asthmatic airways in terms of mitochondrial structure and function, via the a7nAChR.

Cellular senescence is emerging as a driver of idiopathic pulmonary fibrosis (IPF), a progressive and fatal disease with limited effective therapies. The senescence-associated secretory phenotype (SASP), involving the release of inflammatory cytokines and profibrotic growth factors by senescent cells, is thought to be a product of multiple cell types in IPF, including lung fibroblasts. NF-κB is a master regulator of the SASP, and its activity depends on the phosphorylation of p65/RelA. The purpose of this study was to assess the role of Pim-1 kinase as a driver of NF-κB-induced production of inflammatory cytokines from low-passage IPF fibroblast cultures displaying markers of senescence. Our results demonstrate that Pim-1 kinase phosphorylates p65/RelA, activating NF-κB activity and enhancing IL-6 production, which in turn amplifies the expression of PIM1, generating a positive feedback loop. In addition, targeting Pim-1 kinase with a small molecule inhibitor dramatically inhibited the expression of a broad array of cytokines and chemokines in IPF-derived fibroblasts. Furthermore, we provide evidence that Pim-1 overexpression in low-passage human lung fibroblasts is sufficient to drive premature senescence, in vitro. These findings highlight the therapeutic potential of targeting Pim-1 kinase to reprogram the secretome of senescent fibroblasts and halt IPF progression.

Premature infants are frequently and intermittently administered supplemental oxygen during hypoxic episodes, resulting in cycles of intermittent hypoxia and hyperoxia. The relatively hypoxic in utero environment is important for lung development while hyperoxia during the neonatal period is recognized as detrimental towards the development of diseases such as bronchopulmonary dysplasia and bronchial asthma. Understanding early mechanisms that link hypoxic, hyperoxic, and intermittent hypoxic-hyperoxic exposures to altered airway structure and function are key to developing advanced therapeutic approaches in the clinic. Changes in oxygen availability can be detrimental to cellular function and contribute to oxidative damage. Here, we sought to determine the effect of oxygen on mitochondria in human fetal airway smooth muscle cells exposed to either 5% O2, 21% O2, 40% O2, or cycles of 5% and 40% O2 (intermittent hypoxia-hyperoxia). Reactive oxygen species production, altered mitochondrial morphology, and changes in mitochondrial respiration were assessed in the context of the antioxidant N-acetylcysteine. Our findings show developing airway smooth muscle is differentially responsive to hypoxic, hyperoxic, or intermittent hypoxic-hyperoxic exposure in terms of mitochondrial structure and function. Cycling O2 decreased mitochondrial branching and branch length similar to hypoxia and hyperoxia in the presence of antioxidants. Additionally, hypoxia decreased overall mitochondrial respiration while the addition of antioxidants increased respiration in normoxic and O2-cycling conditions. These studies show the necessity of balancing oxidative damage and antioxidant defense systems in the developing airway.

Differentiation of human bronchial epithelial cells (HBEs) in air-liquid interface (ALI) cultures recapitulates organotypic modeling of the in vivo environment. Although ALI cultures are invaluable for studying the respiratory epithelial barrier, loss-of-function studies are limited by potentially cytotoxic reagents in classical transfection methods, the length of the differentiation protocol, and the number of primary epithelial cell passages. Here, we present the efficacy and use of a simple method for small interfering RNA (siRNA) transfection of normal HBEs (NHBEs) in ALI cultures that does not require potentially cytotoxic transfection reagents and does not detrimentally alter the physiology or morphology of NHBEs during the differentiation process. This transfection protocol introduces a reproducible and efficient method for loss-of-function studies in HBE ALI cultures that can be leveraged for modeling the respiratory system and airway diseases.

Supplemental O2 (hyperoxia) is necessary for preterm infant survival but is associated with development of bronchial airway hyperreactivity and childhood asthma. Understanding early mechanisms that link hyperoxia to altered airway structure and function are key to developing advanced therapies. We previously showed that even moderate hyperoxia (50% O2) enhances intracellular calcium ([Ca2+]i) and proliferation of human fetal airway smooth muscle (fASM), thereby facilitating bronchoconstriction and remodeling. Here, we introduce cellular clock biology as a novel mechanism linking early oxygen exposure to airway biology. Peripheral, intracellular clocks are a network of transcription-translation feedback loops that produce circadian oscillations with downstream targets highly relevant to airway function and asthma. Premature infants suffer circadian disruption whereas entrainment strategies improve outcomes, highlighting the need to understand relationships between clocks and developing airways. We hypothesized that hyperoxia impacts clock function in fASM and that the clock can be leveraged to attenuate deleterious effects of O2 on the developing airway. We report that human fASM express core clock machinery ( PER1, PER2, CRY1, ARNTL/BMAL1 , CLOCK) that is responsive to dexamethasone (Dex) and altered by O2. Disruption of the clock via siRNA-mediated PER1 or ARNTL knockdown alters store-operated calcium entry (SOCE) and [Ca2+]i response to histamine in hyperoxia. Effects of O2 on [Ca2+]i are rescued by driving expression of clock proteins, via effects on the Ca2+ channels IP3R and Orai1. These data reveal a functional fASM clock that modulates [Ca2+]i regulation, particularly in hyperoxia. Harnessing clock biology may be a novel therapeutic consideration for neonatal airway diseases following prematurity.