Bioactive Thymosin Alpha-1 Does Not Influence F508del-CFTR Maturation and Activity

Abstract

Deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is the most frequent mutation causing cystic fibrosis (CF). F508del-CFTR is misfolded and prematurely degraded. Recently thymosin a-1 (Tα-1) was proposed as a single molecule-based therapy for CF, improving both F508del-CFTR maturation and function by restoring defective autophagy. However, three independent laboratories failed to reproduce these results. Lack of reproducibility has been ascribed by the authors of the original paper to the use of DMSO and to improper handling. Here, we address these potential issues by demonstrating that Tα-1 changes induced by DMSO are fully reversible and that Tα-1 peptides prepared from different stock solutions have equivalent biological activity. Considering the negative results here reported, six independent laboratories failed to demonstrate F508del-CFTR correction by Tα-1. This study also calls into question the autophagy modulator cysteamine, since no rescue of mutant CFTR function was detected following treatment with cysteamine, while deleterious effects were observed when bronchial epithelia were exposed to cysteamine plus the antioxidant food supplement EGCG. Although these studies do not exclude the possibility of beneficial immunomodulatory effects of thymosin α-1, they do not support its utility as a corrector of F508del-CFTR.

Figure 1

MS, MS/MS and Ion Mobility analysis of thymosin alpha-1 peptide. (A) High-resolution MS spectra of both Abcam (top) and CRIBI (bottom) peptides, measured for charge state +3. The plot shows the measured mass spectrum (blue line) and the overlapped theoretical isotopic profile for the calculated brute formula C₁₂₉H₂₁₅N₃₃O₅₅ with charge state 3. This brute formula differs from the one calculated for native Tα-1 (C₁₂₇H₂₁₃N₃₃O₅₄) for one acetyl group (CH3-CO-). This confirms that both peptides are acetylated. (B) MS spectra for 4.18 μM Tα-1. In panels A and B, peptide was initially dissolved in 1% acetic acid (Panel A) or DMSO (Panel B) and then diluted 1000X in 50% ACN, pH 2 (denaturing conditions). In panels C and D, the peptide was again dissolved in 1% acetic acid (Panel C) or DMSO (Panel D) but then diluted 1000X in 10 mM ammonium acetate, pH 7.4 (native state conditions). Charge states 3+ (1037 m/z) and 4+ (778 m/z) are visible in both native and denaturing conditions, although with different relative intensities. By contrast, charge state 5+ (630.1 m/z) is only visible in denaturing conditions (Panels A and B), thus indicating the loss of native 3D conformation of Tα-1. (C) IMS analysis of Tα-1. Mobilograms (drift time Vs ion intensity plots) for charge state 3+ of Tα-1 a brought to native conditions from 1% acetic acid (red) or DMSO stocks (green) and brought to denaturing conditions from 1% acetic acid (blue) or DMSO stocks (purple).

Figure 2

Evaluation of thymosin α-1 effect on induction of cell apoptosis. (A) Analysis by means of high-content confocal imaging of MCF-7 breast cancer cells following 72-hour treatment with Tα-1 (100 µM) pre-diluted as 200X stock in DMSO or ddH₂O. (B) Dot-plot reporting quantification of apoptotic MCF-7 breast cancer cells treated as in A. (C) High-content confocal image analysis of CFBE41o- bronchial epithelial cells after 72-hour treatment with Tα-1 (100 µM) as in A. (D) Dot-plot showing quantification of the number of apoptotic CFBE41o-cells treated as in A. Mean values ± SD are shown (n = 5). ***P < 0.001 versus respective negative control by ANOVA.

Figure 3

Thymosin α-1 (Tα-1) effect on human bronchial epithelia (HBE) derived from F508del-CFTR homozygous patients. Representative traces of short-circuit current measurements performed on HBE epithelia derived from a homozygous F508del patient were assayed upon a 24-hour treatment with DMSO alone (0.1%), Tα-1 (100 ng/ml + 0.1% DMSO), VX-809 (3 μM), cysteamine (250 μM) or cysteamine plus EGCG (80 μM). The bar graphs show the corresponding monolayer resistance, CFTR-mediated currents and CaCC-mediated currents Mean values ± SD are shown (n = 3–6). ****P < 0.0001 versus negative control by ANOVA.

Figure 4

Evaluation of Tα-1 effect as CFTR modulator on human primary bronchial epithelia (HBE) derived from CF patients and on F508del-CFTR-expressing FRT cells. (A) Representative traces of short-circuit current measurements performed on HBE derived from a homozygous F508del patient (donor code CFBE 13–35) after 24-hour treatment with vehicle alone (0.1% DMSO), Tα-1 (100 ng/ml), or VX-809 (3 μM). (B) Bar graphs reporting CFTR-mediated currents as measured during Ussing chamber recordings of HBE (generated from cultures derived from 3 homozygous F508del patients) treated as described in A. (C) Representative traces obtained during Ussing chamber recordings of FRT epithelia treated as in A. (D) Bar graphs summarizing CFTR-mediated currents derived from experiments as those described in A. Data are means ± SD (n = 3–6). **P < 0.01, *P < 0.05 versus negative control by ANOVA.

Figure 5

Thymosin α1 or cysteamine + EGCG do not improve F508del-CFTR function in primary human bronchial epithelia. (a) Effect of 24 hours treatment with thymosin α1 (100 ng/ml), VX-809 (3 μM), cysteamine (250 μM) + EGCG (80 μM) or combinations on the I_sc of human bronchial epithelia with CFTR^{F508del/F508del} genotype (CF-HBE). Short-circuit currents were recorded in an intact monolayer with equimolar chloride concentrations in both chambers. CFTR-mediated currents were induced by sequential stimulation with forskolin (Fsk, 20 μM) and VX-770 (3 µM) followed by CFTRinh-172 (172, 20 µM) to completely inhibit CFTR (b) Quantification of the CFTR_inh-172 sensitive current in CF-HBE isolated from three homozygous F508del CF patients after single or combination treatment shown as μA/cm² (left axis) or expressed as percentage of the mean WT-CFTR currents measured in WT-HBE, isolated from five donors (right axis). N.s. - not significant, *P < 0.05, **P < 0.01 by paired two-tailed Student’s t-test. Data are means ± SEM.

Figure 6

Evaluation of thymosin α-1 as F508del-CFTR rescue maneuver on human CF primary bronchial epithelia (HBE). (A) Representative traces obtained from Ussing chamber experiments on HBE generated from a homozygous F508del patient (donor code CF-BEX05), after incubation for 24-hour with vehicle, Tα-1 (100 ng/ml, pre-diluted in acqueous buffer), VX-809 (3 μM). (B) Dot plot reporting CFTR-mediated (top) and CaCC-mediated (bottom) currents measured during the experiments depicted in (A). Mean values ± SD are shown (n = 4). **P < 0.01 versus negative control by ANOVA.

Figure 7

Biochemical evaluation of the effect of Tα-1 on mutant CFTR expression pattern in HBE from a F508del homozygous patient. Epithelia were treated with the indicated compounds for 48 hours. CFTR was immunoprecipitated from lysates using rabbit anti-CFTR antibody 155 and then detected by Western blot analysis with antibody CFFT-596. The mature, complex-glycosylated (band C) and the immature, core-glycosylated (band B) forms of CFTR protein are indicated by arrows. Full-length (uncropped) blots/gels are presented in Supplementary File 1.