(436) Characterizing Lactate as a Key Metabolite in the Cystic Fibrosis Lung Environment

Background: Lactate, an end product of glycolysis, is elevated in the sputum of people with cystic fibrosis (pwCF) and correlates with decreased pulmonary function. Lactate can serve a multifunctional role in both immune and epithelial cells. Macrophages, a key component of innate immunity, have previously been shown to undergo changes in polarization and metabolism in response to high levels of lactate in tumor microenvironment (TME) models. In TME, the lactate-enriched environment can also promote epithelial-mesenchymal transition (EMT), leading to loss of tight junctions and changes in the epithelial surface. Taking inspiration from TME models, the purpose of this study is to investigate the effects of lactate in the CF lung environment, specifically in macrophages and epithelial cells.

Methods: Monocyte-derived macrophages (MDMs) were isolated from human donor blood and cultured for 6 days in M-CSF then either stimulated with IFNγ and LPS or left naïve and treated with 10 mM L-lactate. MDM gene expression was analyzed using RT-qPCR. Metabolic analysis was performed on a Seahorse Analyzer XFe96.

CF bronchial epithelial cells (CFBE41o-; CFBE cells) were cultured at air-liquid interface and allowed to differentiate for 14 days. We infected CFBE cells for 6 hrs, with and without lactate pre-treatment (10mM L-lactate), with Pseudomonas aeruginosa and evaluated CFUs. Lactate-treated CFBE cells were also assayed for ATP concentration and cell proliferation.












Results:

Lactate-treated naïve and activated (IFNγ/LPS stimulated) macrophages exhibit decreased TNFα transcription, while stimulated macrophages also exhibit elevated transcription of the lactate signaling receptors, GPR81 and GPR132. Additionally, we found lactate-treated naïve macrophages have increased maximal and spare respiratory capacity, compared to untreated. These preliminary results suggest that elevated lactate, as is found in the CF lung environment, can reduce the expression of pro-inflammatory cytokines in classically activated (M1) macrophages. In addition, naïve recruited macrophages encountering a lactate-rich environment exhibit both a more oxidative metabolic shift and gene expression shift consistent with alternative activation (M2) phenotype. Taken together, our results suggest that elevated lactate levels in the CF lung environment are sufficient to reduce the killing capacity of activated and recruited macrophages during infection, which we are planning to test in future experiments.

In the CF respiratory epithelium, we found no significant change in cell proliferation with lactate treatment, but ATP concentration was significantly increased 4 hours post lactate treatment. CFBE cells pre-treated with lactate exhibited increased biofilm biogenesis. Our results also suggest that while lactate does not appear to be used as fuel source for proliferation, it does induce a metabolic change or signaling event in the short term as demonstrated by increased ATP concentrations. Given the role of lactate in driving EMT in the TME, we are investigating similar mechanisms in the CF airway environment as a potential mechanism for the enhanced biofilm biogenesis phenotype.

Conclusions: Our results suggest that lactate levels encountered in the CF airway may dysregulate cellular functions of the respiratory epithelium and innate immune cells to support the development of chronic infections in CF.

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