(426) Metformin improves HMGB1-induced mucociliary dysfunction in CF airway epithelial cells

Background: Cystic fibrosis-related diabetes mellitus (CFRD) is a major predictor of worse lung function and one of the most common extrapulmonary comorbidities of CF. Hyperglycemia triggers a pro-inflammatory response by inducing expression of advanced glycation end products (AGEs) and damage-associated molecular patterns (DAMPs). High mobility group box protein 1 (HMGB1) functions as a DAMP and promotes inflammation via activation of the receptor for AGEs (RAGE). HMGB1 levels are elevated in sputum and serum of those with CF, particularly at the onset of CFRD, and are associated with worse lung function. However, how elevated levels of HMGB1 impact mucociliary function remains unclear. We therefore sought to investigate the role of HMGB1 in primary CF bronchial epithelial (CFBE) cells and test whether relevant concentrations of metformin, a common anti-diabetic medication with anti-inflammatory properties, could reverse the effects of HMGB1 on mucociliary function.

Methods: F508del CFBE cells were re-differentiated at the air-liquid interface (ALI) and cultured in normal glucose (5.5 mM) media. Cells were treated basolaterally with elexacaftor (1 µM), tezacaftor (5 µM), ivacaftor (1 µM) (ETI) for 24h. Metformin (1 µM) and recombinant HMGB1 (10 ng/mL) were also added basolaterally. Cells were mounted in Ussing chambers and CFTR-dependent short-circuit currents were recorded upon addition of CFTR_inh-172 (10 µM) after ivacaftor (1 µM) and forskolin (10 µM) stimulation in the presence of amiloride (10 µM). ASL volume was estimated by meniscus scanning. Ciliary beat frequency (CBF) was measured using SAVA. HMGB1 expression was measured by ELISA. Gene expression was measured by droplet digital PCR (ddPCR).

Results: F508del CFBE cells exposed to high glucose (12.5 mM) and treated with ETI for 24h showed a significant increase in CFTR conductance but no improvement in ASL volumes compared to controls. The addition of metformin further increased CFTR activity leading to significant improvements in ASL volumes in ETI-treated F508del CFBE cells under high glucose. Metformin also reduced HMGB1 levels in the basolateral media. Treatment of F508del CFBE cells cultured under normal glucose with HMGB1 caused ASL volume depletion, reduced CBF, and increases in mRNA expressions of interleukin-1β (IL-1β), transforming growth factor-β1 (TGF-β1), and tumor necrosis factor-α (TNF-α) after 24h even in the presence of ETI. Metformin reduced the expression of RAGE and rescued the effects of HMGB1 on ASL volumes, CBF, and the expressions of IL-1β, TNF-α, and TGF-β1.

Conclusions: Our data show that clinically relevant doses of metformin can improve F508del-CFTR recovery by ETI in CFBE cells under hyperglycemia in vitro, possibly by reducing levels of HMGB1. Physiological concentrations of HMGB1 can induce inflammation and mucociliary dysfunction even in the presence of ETI. Metformin reverses the effects of HMGB1 on mucociliary dysfunction and inflammation in vitro. These data set the stage for clinical trials to test the efficacy of metformin as an anti-inflammatory agent to improve ion channel and mucociliary function in CFRD.

Acknowledgements: Supported by NIH (R01 HL157942 and R01 HL133240) and CFF (SALATH18I0).

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