Cystic fibrosis transmembrane conductance regulator dysfunction in platelets drives lung hyperinflammation

Abstract

Cystic fibrosis (CF) lung disease is characterized by an inflammatory response that can lead to terminal respiratory failure. The cystic fibrosis transmembrane conductance regulator (CFTR) is mutated in CF, and we hypothesized that dysfunctional CFTR in platelets, which are key participants in immune responses, is a central determinant of CF inflammation. We found that deletion of CFTR in platelets produced exaggerated acute lung inflammation and platelet activation after intratracheal LPS or Pseudomonas aeruginosa challenge. CFTR loss of function in mouse or human platelets resulted in agonist-induced hyperactivation and increased calcium entry into platelets. Inhibition of the transient receptor potential cation channel 6 (TRPC6) reduced platelet activation and calcium flux, and reduced lung injury in CF mice after intratracheal LPS or Pseudomonas aeruginosa challenge. CF subjects receiving CFTR modulator therapy showed partial restoration of CFTR function in platelets, which may be a convenient approach to monitoring biological responses to CFTR modulators. We conclude that CFTR dysfunction in platelets produces aberrant TRPC6-dependent platelet activation, which is a major driver of CF lung inflammation and impaired bacterial clearance. Platelets and TRPC6 are what we believe to be novel therapeutic targets in the treatment of CF lung disease.

Keywords: Inflammation; Neutrophils; Platelets; Pulmonology.

Figure 1. Lung injury and bacterial lung colony measurements in CF mice after intratracheal LPS or PAO1.

(A and G) BAL WBCs, (B and H) BAL neutrophils, (C and I) BAL total protein, (D and J) BAL thromboxane B2, and BAL NETs as measured by (E and K) NE-DNA and (F and L) citH3-DNA in WT, CFTR^+/–, and CFTR^–/– mice at 48 hours after intratracheal LPS or PAO1. (M) Bacterial lung colony counts in WT, CFTR^+/–, and CFTR^–/– mice after intratracheal PAO1. Data are mean ± SEM of 5 to 8 animals per group. Data were analyzed by 1-way ANOVA. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Figure 2. Lung injury and leukocyte-platelet aggregates in mice with conditional CFTR deletion after intratracheal LPS or PAO1.

(A) BAL WBCs, (B) neutrophils, (C) total protein in CF^fl/fl, CF-LysM, CF-MRP8, and CF-PF4 mice at 48 hours after intratracheal LPS. NPAs in BAL (D) and blood (E), and MPAs in blood (F) at 24 hours after intratracheal LPS. Data are mean ± SEM of 4 to 9 animals per group. Data were analyzed by 1-way ANOVA. (G–N) Lung injury and leukocyte-platelet aggregates in CF^fl/fl and CF-PF4 mice after intratracheal PAO1. (G) BAL WBC, (H) neutrophils, (I) total protein, (J) NPAs, (K) CD62P, (L) blood NPAs, (M) blood MPAs, and (N) lung colonies in CF-PF4 and CF^fl/fl mice after challenge with PAO1. Standard lung injury measurements were performed at 48 hours, and NPAs and MPAs at 24 hours after intratracheal PAO1. Data are mean ± SEM of 5 to 8 animals per group. Data were analyzed by Student’s t test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Figure 3. Effects of CFTR inhibition or deletion on thrombin-induced platelet activation.

(A) Representative gating scheme of platelet activation (CD41⁺ events) quantifying CD62P expression with or without thrombin stimulation in WT or CFTR^–/– platelets with or without CFTRinh-172 (CF172). (B) CD62P expression in platelets from WT, CFTR^–/–, CF^fl/fl, and CF-PF4 mice with or without thrombin stimulation plus vehicle or CFTRinh-172 (CF172). Data are mean ± SEM of 5 to 8 mice. Data were analyzed by 2-way ANOVA. *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001.

Figure 4. Characterization of TRPC6 in platelets.

(A, B) mRNA expression of TRPC isoforms TRPC1 and TRPC6 in platelets from WT, CFTR^–/–, CF^fl/fl, CF-LysM, and CF-PF4 mice. TRPC isoforms 2, 3, 4, 5, and 7 were undetectable (not shown). (C) Immunofluorescence staining and (D) flow cytometry analysis of CD41 (red) and TRPC6 (blue) in platelets from WT and TRPC6^–/– mice (representative of 3 independent experiments). Scale bar: 2.5 μm. (E and F) CD62P expression on platelets from (E) WT, CFTR^–/–, TRPC6^–/–, and CFTR^–/– × TRPC6^–/– mice, and (F) CF^fl/fl and CF-PF4 mice after thrombin challenge with or without incubation with vehicles, CF172, or CF172 plus SKF-96365 (SK). Data are mean ± SEM of 5 to 11 animals per group. Data were analyzed by 2-way ANOVA. *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001.

Figure 5. Calcium entry measured by the ratiometric Indo-1 assay in thrombin-stimulated platelets from mice and humans.

(A) Kinetic tracings of Indo-1 Violet/Blue MFI measured in platelets isolated from CFTR^–/– (blue), TRPC6^–/– (green), and WT (red) mice with 0.125 IU thrombin introduced at 60-second intervals starting at 30 seconds (black arrows). (B) Peak MFI in WT, CFTR^–/–, and TRPC6^–/– platelets incubated with vehicles, CF172, or CF172 plus SK. Data are mean ± SEM of 7 to 11 animals per group. (C) Peak MFI measured in platelets isolated from healthy human and CF subjects not on modulators incubated with vehicles, CF172, or CF172 plus SK. (D) CD62P and (E) PAC-1 expression in platelets from human controls, CF platelets plus modulators (lumacaftor/ivacaftor), and CF platelets not treated with modulators (no modulators). Data are presented as minimum-to-maximum whiskers and box plots showing the median and interquartile ranges. (C–E) n = 6–12 subjects per group. Data in B–E were analyzed by 2-way ANOVA. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Figure 6. Lung injury measurements in CFTR and TRPC6 mutant mice after intratracheal LPS or PAO1.

(A) BAL WBCs, (B) neutrophils, (C) total protein, (D) thromboxane B_2, (E) NETs (NE-DNA ELISA), and (F) NETs (citH3-DNA ELISA) in CFTR × TRPC6 mutant mice (genotypes indicated in x axis label). Data are mean ± SEM of 5 to 6 animals per group. Data were analyzed by 2-way ANOVA. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. (G–J) Lung injury and bacterial counts after intratracheal PAO1. (G) BAL WBCs, (H) neutrophils, (I) total protein, and (J) lung colonies in CFTR and TRPC6 mutant mice. Data are mean ± SEM of 5 to 6 animals per group. Data were analyzed by 2-way ANOVA. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Figure 7. Graphical abstract indicating key events in platelet-induced neutrophilic inflammation in CF.