L‐Citrulline Attenuates Inflammation Induced Phenotypic Changes in Neonatal Rat Alveolar Type 1 Cells

Abstract

IntroductionBronchopulmonary dysplasia (BPD) is a chronic lung disease initiated by inflammation and oxidative stress during the early stages of lung development, disrupting alveolar growth. BPD damages alveolar Type 1 (AT1) epithelial cells, causing decreased alveolarization and impaired gas exchange. Recently, it was shown that AT1 cells undergo cell reprogramming into surfactant producing alveolar Type 2 cells (AT2) in hyperoxic and hypoxic lung injury models, causing significant structural damage in the neonatal lung. While current treatments have improved neonatal care, they cause significant side effects. L‐citrulline, a naturally occurring amino acid, protects the newborn rat lung from injury, preserving alveolar growth in various neonatal BPD models. Due to the significance of inflammation in BPD, we sought to investigate the effect of inflammatory stimuli, lipopolysaccharide (LPS) and tumour necrosis factor α (TNF‐α), and L‐citrulline treatment on AT1 cell programming in vitro. We hypothesize that L‐citrulline treatment will prevent inflammation‐induced AT1 cell reprogramming in vitro.MethodsAT1 cells were isolated from newborn rats on postnatal day 4 using fluorescence activated cell sorting. AT1 cells were pre‐treated with L‐citrulline (4mM/mL or 10mM/mL) for 1hr prior to LPS (10µg/mL), TNF‐α (5ng/mL), or a combination of LPS and TNF‐α for 24hrs in vitro. Whole cell lysates were prepared for immunoblotting. AT1 cells were also assayed for the level of injury‐induced cell viability using the MTT assay.ResultsAT1 cell lysates from the LPS+TNF‐α group had significantly decreased RTI‐40 protein expression compared to the control group (p<0.04 LPS+TNF‐α vs. control, n=6). In the presence of L‐citrulline, RTI‐40 protein expression of cells exposed to LPS+ TNF‐α was maintained (p<0.02 L‐citrulline+LPS+TNF‐α vs. LPS+TNF‐α, n=4) (Figure 1). AT1 cell viability in LPS, TNF‐α, and the combined exposure group was comparable to the control group (n=8) (Figure 2).ConclusionOur preliminary data strongly suggests that L‐citrulline treatment mitigates inflammation‐induced cell reprogramming in neonatal AT1 cells. Our findings show that the injury‐induced decrease in RTI‐40 expression is not due to reduced AT1 cell viability, rather due to phenotypic changes. Given the importance of AT1 cells in alveolarization, gas exchange, and BPD pathogenesis, characterizing the cell specific effects of L‐citrulline on AT1 cell reprograming is considerably critical and highly relevant to identifying a safe and effective treatment for premature infants that can mitigate the effects of inflammation on the developing lung.