Anti-inflammatory effects of elexacaftor/tezacaftor/ivacaftor in adults with cystic fibrosis heterozygous for F508del

Abstract

Inflammation is a key driver in the pathogenesis of cystic fibrosis (CF). We assessed the effectiveness of elexacaftor/tezacaftor/ivacaftor (ETI) therapy on downregulating systemic and immune cell-derived inflammatory cytokines. We also monitored the impact of ETI therapy on clinical outcome. Adults with CF, heterozygous for F508del (n = 19), were assessed at baseline, one month and three months following ETI therapy, and clinical outcomes were measured, including sweat chloride, lung function, weight, neutrophil count and C-reactive protein (CRP). Cytokine quantifications were measured in serum and following stimulation of peripheral blood mononuclear cells (PBMCs) with lipopolysaccharide (LPS) and adenosine triphosphate and analysed using LEGEND plex™ Human Inflammation Panel 1 by flow cytometry (n = 19). ASC specks were measured in serum and caspase-1 activity and mRNA levels determined from stimulated PBMCs were determined. Patients remained stable over the study period. ETI therapy resulted in decreased sweat chloride concentrations (p < 0.0001), CRP (p = 0.0112) and neutrophil count (p = 0.0216) and increased percent predicted forced expiratory volume (ppFEV1) (p = 0.0399) from baseline to three months, alongside a trend increase in weight. Three months of ETI significantly decreased IL-18 (p< 0.0011, p < 0.0001), IL-1β (p<0.0013, p = 0.0476), IL-6 (p = 0.0109, p = 0.0216) and TNF (p = 0.0028, p = 0.0033) levels in CF serum and following PBMCs stimulation respectively. The corresponding mRNA levels were also found to be reduced in stimulated PBMCs, as well as reduced ASC specks and caspase-1 levels, indicative of NLRP3-mediated production of pro-inflammatory cytokines, IL-1β and IL-18. While ETI therapy is highly effective at reducing sweat chloride and improving lung function, it also displays potent anti-inflammatory properties, which are likely to contribute to improved long-term clinical outcomes.

Fig 1. Serum cytokine levels in patients with CF (heterozygous, F508del, other), following treatment with ETI.

Sera were collected at baseline, one month and three months of treatment from patients heterozygous for F508del, and a second minimal function CFTR mutations receiving ETI therapy (n = 19). LegendPlex assays were used to detect levels of A, IL-18; B, IL-1β; C, TNF; D, IL-6, E, IL-10, F IL-8, G IL-23 in sera. H, Flow cytometry was used to detect NLRP3-positive ASC-specks. A one-way ANOVA statistical test, with Tukey’s multiple comparison test was performed. P values for baseline to one month (a) and baseline to three months (b) are shown on each graph. The upper 95% confidence interval for average HC (solid blue line) across 3 months, with block colour shading to lower 5% confidence interval, is displayed for each cytokine.

Fig 2. Cytokine secretion in NLRP3-stimulated CF immune cells isolated from patients with CF (heterozygous, F508del, other), following treatment with ETI.

PBMCs isolated at baseline, one month and three months from patients heterozygous, F508del, other CFTR mutations receiving ETI therapy (n = 19). Following isolation, PBMCs were immediately stimulated with LPS (10 ng/mL, 4 hr), and ATP (5 mM) for the final 30 min. Legend Plex assays were used to detect levels of A, IL-1β; B, IL-18; C, TNF; D, IL-6 and E, IL-10, F IL-8, G, IL-23 secretion from PBMCs. H, caspase-1 activity was detected in stimulated PBMCs at each time point. A two-way ANOVA statistical test with Tukey’s multiple comparison test was performed. P values for baseline to one month (a) and baseline to three months (b) are shown on each graph. The upper 95% confidence interval for average HC (solid blue line) across 3 months, with block colour shading to lower 5% confidence interval is displayed for each cytokine.

Fig 3. mRNA expression in NLRP3-stimulated CF immune cells isolated from patients with CF (heterozygous, F508del, other), following treatment with ETI.

PBMCs isolated at baseline, one month and three months from patients heterozygous, F508del, other CFTR mutations receiving ETI therapy (n = 19). Following isolation, PBMCs were immediately stimulated with LPS (10 ng/mL, 4 hr), and ATP (5 mM) for the final 30 min. RNA was isolated, and mRNA gene expression levels measured A, IL-1β; B, IL-18; C, TNF; D, IL-6 and E, IL-10, F NLRP3, G, TXNIP. A two-way ANOVA statistical test with Tukey’s multiple comparison was performed. P values for baseline to one month (a) and baseline to three months (b) are shown on each graph. The upper 95% confidence interval for average HC (solid blue line) across 3 months, with block colour shading to lower 5% confidence interval is displayed for each cytokine.