1. Immunology and Inflammation
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ENaC-mediated sodium influx exacerbates NLRP3-dependent inflammation in cystic fibrosis

  1. Thomas Scambler
  2. Heledd H Jarosz-Griffiths
  3. Samuel Lara-Reyna
  4. Shelly Pathak
  5. Chi Wong
  6. Jonathan Holbrook
  7. Fabio Martinon
  8. Sinisa Savic
  9. Daniel Peckham
  10. Michael F McDermott  Is a corresponding author
  1. University of Leeds, United Kingdom
  2. University of Lausanne, Switzerland
  3. St James’s University Hospital, United Kingdom
  4. St James’ University Hospital, United Kingdom
Research Article
Cite this article as: eLife 2019;8:e49248 doi: 10.7554/eLife.49248
8 figures, 1 table and 2 additional files

Figures

LPS-induced IL-18 secretion in human bronchial epithelial cells is higher in cells with CF-associated mutations and is NLRP3 inflammasome dependent.

Human bronchial epithelial cell (HBEC) lines (BEAS-2B (WT), IB3-1 (ΔF508/W1282X), CuFi1 (ΔF508/ ΔF508), CuFi4 (ΔF508/G551D) (n = 3 independent experiments) were unstimulated or stimulated with Lipopolysaccharide, from Escherichia coli K12 (LPS Ultrapure), which specifically targets TLR4 (10 ng/mL) for 4 hr before being stimulated for 4 hr with Flagellin (10 ng/mL with Lipofectamine 2000) for NLRC4 inflammasome, TcdB (10 ng/mL) for Pyrin inflammasome or poly(dA:dT) dsDNA (1 μg/mL with Lipofectamine 2000) for AIM2 inflammasome. ELISA assays were used to detect (A) IL-18. To monitor NLRP3 inflammasome activation, HBEC (n = 3 independent experiments) were pre-incubated with MCC950 (15 μM), OxPAPC (30 μg/mL) and YVAD (2 μg/mL) for 1 hr before a stimulation with LPS (10 ng/mL, 4 hr), and ATP (5 mM) for the final 30 min. ELISA assays were used to detect (B) IL-18 and (D) colourimetric assay used to detect caspase-1 activity in protein lysates for LPS/ATP and LPS/ATP/MCC950). (D) Necrosis and pyroptosis are represented as superimposed bar charts. Total necrosis was measured using LDH release assay. For pyroptotic cell death, each sample/condition was repeated in parallel with a caspase-1 inhibitor (YVAD (2 mg/mL, 1 hr)) pre-treatment. The total necrosis level was then taken away from the caspase-1 inhibited sample, or ‘caspase-1 independent’ necrosis, with the remaining LDH level termed ‘caspase-1 dependent necrosis’ or pyroptosis. Cells were then stimulated with LPS (10 ng/mL, 4 hr), and ATP (5 mM) for final 30 min. The assay was performed with HBEC lines (n = 3 independent experiments). (◦) Significance for Total Necrosis (●) Significance for Pyroptosis. A 2-way ANOVA with Tukey’s multiple comparison test was performed (p values * =< 0.05, ** =< 0.01, *** =< 0.001 and **** =< 0.0001).

https://doi.org/10.7554/eLife.49248.002
Figure 2 with 1 supplement
LPS-induced IL-1β/IL-18 secretion in human monocytes is higher in CF and is NLRP3 inflammasome dependent.

Primary monocytes from HC and CF (HC, n = 10; CF, n = 10) were unstimulated or stimulated with LPS which specifically targets TLR4 (10 ng/mL) for 4 hr before being stimulated for 4 hr with Flagellin (10 ng/mL with Lipofectamine 2000) for NLRC4 inflammasome, or TcdB (10 ng/mL) for Pyrin inflammasome or poly(dA:dT) dsDNA (1 μg/mL with Lipofectamine 2000) for AIM2 inflammasome. ELISA assays were used to detect (A) IL-18 and (B) IL-1β cytokine secretion in supernatants. To monitor NLRP3 inflammasome activation, primary monocytes from HC, CF, SAID and NCFB (HC, n = 10; CF, n = 10; SAID, n = 4; NCFB, n = 4) were pre-incubated with MCC950 (15 μM), OxPAPC (30 μg/mL) and YVAD (2 μg/mL) for 1 hr before a stimulation with LPS (10 ng/mL, 4 hr), and ATP (5 mM) for the final 30 min. ELISA assays were used to detect (C) IL-18 and (D) IL-1β cytokine secretion in supernatants and (E) a colourimetric assay was used to detect caspase-1 activity in protein lysates (HC, n = 10; CF, n = 10; SAID, n = 4; NCFB, n = 4). (F) Flow cytometry was used to detect ASC specks in supernatants of primary monocytes from HC, CF, SAID and NCFB (HC, n = 10; CF, n = 10; SAID, n = 6; NCFB, n = 4) for ±LPS/ATP and (HC, n = 5; CF, n = 5) for MCC950 with LPS/ATP. (G) Necrosis and pyroptosis are represented as superimposed bar charts. Total necrosis was measured using LDH release assay. For pyroptotic cell death, each sample/condition was repeated in parallel with a caspase-1 inhibitor (YVAD (2 mg/mL, 1 hr)) pre-treatment. The total necrosis level was taken away from the caspase-1 inhibited sample, or ‘caspase-1 independent’ necrosis, with the remaining LDH level termed ‘caspase-1 dependent necrosis’ or pyroptosis. Cells were then stimulated with LPS (10 ng/mL, 4 hr), and ATP (5 mM) for final 30 min. The assay was performed with primary monocytes from HC, CF, SAID and NCFB (HC, n = 10; CF, n = 10; SAID, n = 4; NCFB, n = 4). (◦) Significance for Total Necrosis (●) Significance for pyroptosis. A 2-way ANOVA statistical test was performed, with Tukey post-hoc correction (p values * =< 0.05, ** =< 0.01, *** =< 0.001 and **** =< 0.0001; error bars ± SEM). Inhibitor treatments in panels a-c were found to significantly reduce cytokine secretion and caspase-1 activity to **p =< 0.01 or less, for CF and SAID groups respectively. Significance values not displayed on the graph.

https://doi.org/10.7554/eLife.49248.003
Figure 2—figure supplement 1
Primary monocytes from HC, CF, SAID and NCFB (HC n = 9, CF n = 9, SAID n = 4, NCFB n = 4) were unstimulated or stimulated with LPS (10 ng/ml, 4 hr) or LPS (10 ng/ml, 4 hr) and ATP (5 mM) for the final 30 min.

ELISAs were used to detect (A) IL-18, and (B) IL-1β cytokine secretion in supernatants. (C) PBMCs were unstimulated or stimulated with LPS (10 ng/ml, 4 hr) or LPS (10 ng/ml, 4 hr) and ATP (5 mM) for the final 30 min. Taqman RT-qPCR was used to measure IFNγ gene expression and (D) Luminex was used to measure IFNγ secretion from peripheral blood mononuclear cells (PBMC) populations from HCs and patients with CF-associated mutations (n = 10).

https://doi.org/10.7554/eLife.49248.004
Figure 3 with 1 supplement
Inflammatory serum cytokine signature in CF.

(A) ELISA assays were used to detect IL-18 (HC, n = 10, CF, n = 30, SAID, n = 10, NCFB, n = 4), (B) IL-1β (HC, n = 10, CF, n = 30, SAID, n = 10, NCFB, n = 4), (C) IL-1Ra (HC, n = 10, CF, n = 30, SAID, n = 7, NCFB, n = 4) in patient sera. Outliers in SAID group for IL-1β and IL-1Ra correspond to HIDS one and A20 deficiency (D) Flow cytometry was used to detect ASC specks (HC, n = 10, CF, n = 15, SAID, n = 10, NCFB, n = 4) in patient sera. (E) A colorimetric assay to detect caspase-1 activity in sera of patients with CF, SAID and NCFB as a percentage of HC (HC, n = 10, CF, n = 15, SAID, n = 4, NCFB, n = 4). Of note, an undetermined amount of detected IL-1Ra is attributed to circulating Anakinra (recombinant IL-1Ra) specifically in the SAID cohort. The Kruskal-Wallis non-parametric test, with Dunn’s multiple comparison test, was performed (p values * =< 0.05, ** =< 0.01, *** =< 0.001 and **** =< 0.0001; error bars ± S.E.M).

https://doi.org/10.7554/eLife.49248.005
Figure 3—source data 1

Serum cytokine levels for all patient groups.

https://doi.org/10.7554/eLife.49248.008
Figure 3—figure supplement 1
Inflammatory serum cytokine signature in CF.

ELISA assays were used to detect (A) TNF and (B) IL-6 (HC n = 10, CF n = 30, SAID n = 7, NCFB = 4) in patient serum. The Kruskal-Wallis non-parametric test, with Dunn’s multiple comparison test, was performed (p values * = 0.05, ** = 0.01, *** = 0.001 and **** = 0.0001; error bars ± S.E.M).

https://doi.org/10.7554/eLife.49248.006
Figure 3—figure supplement 1—source data 1

Serum cytokine levels for all patient groups.

https://doi.org/10.7554/eLife.49248.007
Figure 4 with 1 supplement
Dysregulated Na+ and K+ in cells with CF-associated mutations can be modulated with ENaC inhibitors.

Intracellular Na+ was detected using an AM ester of sodium indictor SBFI (S-1263) and (B, D) intracellular K+ was detected using an AM ester of potassium indictor PBFI (P-1266); changes in fluorescence were measured by fluorimeter post-stimulation with 5 mM ATP in (A, B) monocytes (HC = 7, CF = 7) (E, F) HBECs (n = 3 independent experiments). Cells were pre-treated with the following: amiloride (100 μM), S18 derived peptide (25 μM, 4 hr) with LPS (10 ng/mL, 4 hr) and ATP (5 mM) for the final 30 min. A 2-way ANOVA with Tukey’s multiple comparison test was performed (p values * =< 0.05, ** =< 0.01, *** =< 0.001 and **** =< 0.0001) (*) indicate significance when comparing HC with CF-associated mutants. (•) indicate significance between treatments within the same cell line. (C) Endogenous β-ENaC protein expression was detected using western blot in BEAS-2B HBEC, HC and CF monocytes (C) and densitometry analysis of total β-ENaC (bands A, B, C indicated on blot) was quantified in (D) for CF relative to HC (n = 3 independent experiments). (G) BEAS-2B, IB3-1, CuFi-1 and CuFi-4 HBEC lines and densitometry analysis of total β−ENaC (bands A, B, C indicated on blot) was quantified in (H) (n = 3 independent experiments). Band A represents complex N-Glycosylation, 110 kDa β-ENaC (found when associated as ENaC complex); Band B represents Endo-H sensitive N-Glycosylation, 96 kDa β-ENaC; Band C represents immature non-glycosylated, 66 kDa β−ENaC. The Mann-Whitney non-parametric test was performed (p values * =≤ 0.05).

https://doi.org/10.7554/eLife.49248.009
Figure 4—figure supplement 1
Increased sodium influx in cells with CF-associated mutations.

(A, C) Intracellular Na+ was detected using an AM ester of sodium indictor SBFI (S-1263) and (B, D) intracellular K+ was detected using an AM ester of potassium indictor PBFI (P-1266); changes in fluorescence were measured by fluorimeter post-stimulation in (A, B) monocytes (HC = 7, CF = 7) (C, D) HBECs (n = 3 independent experiments). Cells were pre-treated with the following: EIPA (10 mM, 1 hr) and ouabain (100 nM, 24 hr) before a stimulation with LPS (10 ng/mL, 4 hr) and ATP (5 mM) for the final 30 min. A 2-way ANOVA with Tukey’s multiple comparison test was performed (p values * = 0.05, ** = 0.01, *** = 0.001 and **** = 0.0001) (*) indicate significance when comparing HC with CF-associated mutatns. (•) indicate significance between treatments within the same cell line. (E) Gene expression of b-ENaC in HC vs CF (n = 1), represented as DCT. The Mann-Whitney non-parametric test was performed (p values * = 0.05).

https://doi.org/10.7554/eLife.49248.010
Figure 5 with 2 supplements
Inhibition of amiloride-sensitive sodium channels modulates inflammation in cells with CF-associated mutations.

ELISA assays were used to detect IL-18 (A) and (B) IL-1β in monocytes from HC (n = 9 amiloride, n = 10 S18), patients with CF (n = 10), SAID (n = 4) and NCFB (n = 4) and IL-18 (E) HBEC (n = 3, amiloride independent experiments) (F) HBEC (n = 3, S18 independent experiments). (C) Colourimetric assay was used to detect caspase-1 activity in protein lysates (HC n = 11, CF n = 11) and (D) flow cytometry was used to detect ASC specks in supernatant of primary monocytes (HC n = 5, CF n = 5). Cell stimulation was as follows: Amiloride (100 μM or 10 μM, 1 hr) or S18 derived peptide (25 μM, 4 hr) were used as a pre-treatment before a stimulation with LPS (10 ng/mL, 4 hr) and ATP (5 mM) for the final 30 min. (E, F) SCNN1B over-expression in BEAS-2B cells increases pro-inflammatory cytokine secretion. (E) BEAS-2B cells were transiently transfected with 10µg SCNN1B cDNA (+) or a pcDNA3.1 vector only control (-) for 48 hr then stimulated with LPS (10 ng/mL, 4 hr) and ATP (5 mM) for the final 30 min (n = 3 independent experiments). Cells were lysed and immunoblotted for β-ENaC and β-actin. (F) ELISA assays were used to detect IL-18 in the supernatant fraction. A 2-way ANOVA with Tukey’s multiple comparison test was performed (p values * =≤ 0.05, ** =≤ 0.01, *** =≤ 0.001 and **** =≤ 0.0001) (*) indicate significance, when comparing HC with CF. (•) indicate significance between treatments within the same cell line.

https://doi.org/10.7554/eLife.49248.011
Figure 5—figure supplement 1
Inhibition of amiloride-sensitive sodium channels modulates inflammation in cells with CF-associated mutations.

(A) ELISA assays were used to detect IL-18 in HBECs (n = 3 independent experiments) and (C) IL-18 and (D) IL-1β and (F) TNF in monocytes from HC (n = 10), patients with CF (n = 10), SAID (n = 4) and NCFB (n = 4). Cell stimulation was as follows: (F) Amiloride (100 mM or 10 mM, 1 hr) (A, C, D, E), EIPA (10 mM, 1 hr), were used as a pre-treatment before a stimulation with LPS (10 ng/mL, 4 hr) and ATP (5 mM) for the final 30 min. (H) All the above stimulations were used to detect caspase-1 activity using a colorimetric assay in HBECs and for EIPA in monocytes (G). A 2-way ANOVA with Tukey’s multiple comparison test was performed (p values * = 0.05, ** = 0.01, *** = 0.001 and **** = 0.0001) (*) indicate significance when comparing HC with CF-associated mutants. (•) indicate significance between treatments within the same cell line.

https://doi.org/10.7554/eLife.49248.012
Figure 5—figure supplement 2
Inhibition of amiloride-sensitive sodium channels modulates inflammation in cells with CF-associated mutations.

(A, D, G) ELISA assays were used to detect IL-18 and (B, E, H) IL-1β in monocytes from HC (n = 10), patients with CF (n = 10), SAID (n = 4) and NCFB (n = 4) and (C, F) IL-18 in HBECs (n = 3 independent experiments). Cell stimulation was as follows: (A–C) Amiloride (100 mM or 10 mM, 1 hr), (D–F) EIPA (10 mM, 1 hr), (G–I) S18 derived peptide (25 mM, 4 hr) were used as a pre-treatment before a stimulation with LPS (10 ng/mL, 4 hr) and Nigericin (1µM) for the final 30 min. A 2-way ANOVA with Tukey’s multiple comparison test was performed (p values * = 0.05, ** = 0.01, *** = 0.001 and **** = 0.0001) (*) indicate significance when comparing HC with CF-associated mutants. (•) indicate significance between treatments within the same cell line.

https://doi.org/10.7554/eLife.49248.013
SCNN1B over-expression in BEAS-2B cells increases pro-inflammatory cytokine secretion.

BEAS-2B cells were transiently transfected with 10µg SCNN1B cDNA (+) or a pcDNA3.1 vector only control (-) for 48 hr then stimulated with LPS (10 ng/mL, 4 hr) and ATP (5 mM) for the final 30 min (n = 3 independent experiments). Cells were lysed and immunoblotted for β-ENaC and β-actin a). ELISA assays were used to detect IL-18 in the supernatant b) fraction. A 2-way ANOVA with Tukey’s multiple comparison test was performed (p values * =≤ 0.05, ** =≤ 0.01, *** =≤ 0.001 and **** =≤ 0.0001).

https://doi.org/10.7554/eLife.49248.014
A schematic diagram of the proposed excessive NLRP3 inflammasome activation observed in individuals and cells with CF-associated mutations.

Without functional CFTR, inhibition of ENaC currents is diminished leading to increased intracellular Na+ levels. Dysregulation of ENaC-dependent Na2+ influx leads to increased K+ efflux (via unknown mechanism) and NLRP3 inflammasome activation, with subsequent release of IL-1β and IL-18. In the CF airway, K+ efflux is exacerbated upon K+ channel stimulation by endotoxins, DAMPs or PAMPs, leading to aberrant NLRP3 inflammasome activation and excessive IL-1β and IL-18 secretion. Blocking ENaC currents with S18 peptide restores Na+ and K+ levels which reduces NLRP3-mediated production of IL-1β and IL-18.

https://doi.org/10.7554/eLife.49248.015
Author response image 1

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
AntibodyRabbit polyclonal anti-SCNN1BAvia Systems Biology, San DiegoCat# ARP72375_P050;
RRID: AB_2811256
WB
1:500
AntibodyGoat polyclonal anti-Rabbit IgG (H+L) Poly-HRP Secondary AntibodyThermoFisher ScientificCat# 32260; RRID: AB_1965959WB 1:4000
AntibodyRabbit polyclonal anti-actin-βGeneTexCat# GTX109639, RRID: AB_1949572WB
1:20:000
AntibodyMouse monoclonal Phycoerythrin anti-ASC (TMS-1)BiolegendCat# 653903,
RRID: AB_2564507
5 µL/ ml
Cell line (Homo-sapiens)BEAS-2B cell lineATCCATCC CRL-9609
Cell line (Homo-sapiens)IB3-1ATCCATCC CRL-2777
Cell line (Homo-sapiens)CuFi-1 cell lineATCCATCC CRL-4013
Cell line (Homo-sapiens)CuFi-4 cell lineATCCATCC CRL-4015
Commercial assay or kitMycoAlertTMLonzaCat# LT07-118
Biological samples (Homo-sapiens)Human Blood SamplesSt James's University HospitalHealth Research Authority REC reference 17/YH/0084
Chemical compound, drugLymphoprepAxis ShieldCat# 1114544
Chemical compound, drugPan Monocyte Isolation Kit, humanMiltenyi BiotecCat# 130-096-537
Chemical compound, drugLipopolysacchride Ultrapure EKInvivoGenCat# tlrl-eklps10ng/ml
Chemical compound, drugMCC950Cayman ChemicalCat# CAY17510-115 nM, 1 hr
Chemical compound, drugYVADInvivoGenCat# inh-yvad2 μg/ mL,
1 hr
Chemical compound, drugOxPAPCInvivoGenCat# tlrl-oxp130 μg/ mL, 1 hr
Chemical compound, drugAmiloride (hydrochloride)Cayman ChemicalCat# 2629510 μM, 100 μM,
1 hr
Chemical compound, drugSPLUNC1-derived peptide, S18Gift from Spyryx Biosciences, Inc25 μM, 4 hr
Chemical compound, drug5-(N-ethyl-N-isopropyl)-Amiloride (EIPA)Cayman ChemicalCat# 1154-25-210 μM, 1 hr
Chemical compound, drugOuabainTorcis BioscienceCat# 630-60-4100 nM,
24 hr
Chemical compound, drugATPInvivoGenCat# tlrl-atpl5 mM,
30 min
Chemical compound, drugpoly(dA:dT) dsDNAInvivoGenCat# tlrl-patn1 μg/ mL,
1 hr
Chemical compound, drugTcdBCayman ChemicalCat# CAY19665-5010 ng/mL,
1 hr
Chemical compound, drugFlagellinInvivoGenCat# tlrl-pbsfla10 ng/mL,
1 hr
Commercial assay or kitPierce BCA Protein Assay KitThermoFisher ScientificCat# 23225
Chemical compound, drugPhosSTOPMerckCat# 4906845001
Chemical compound, drugPierce Protease Inhibitor Mini TabletsThermoFisher ScientificCat# A32955
Chemical compound, drugImmobilon Western Chemiluminescent HRP SubstrateMerckCat# WBKLS0500
Commercial assay or kitIL-1 beta Human Matched Antibody PairThermoFisher ScientificCat# CHC1213Assay sensitivity < 31.2 pg/mL
Commercial assay or kitIL-18 Human Matched Antibody PairThermoFisher ScientificCat# BMS267/2MSTAssay sensitivity 78 pg/mL
Commercial assay or kitIL-6 Human Matched Antibody PairThermoFisher ScientificCat# CHC1263Assay sensitivity 15.6 pg/mL
Commercial assay or kitTNF alpha Human Matched Antibody PairThermoFisher ScientificCat# CHC1753Assay sensitivity < 15.6 pg/mL
Commercial assay or kitIL1RA Human Matched Antibody PairThermoFisher ScientificCat# CHC1183Assay sensitivity < 31.2 pg/mL
Chemical compound, drug(TMB) substrate solutionSigmaCat# T0440
Commercial assay or kitCaspase-1 Colorimetrix AssayR and D SystemsCat# BF15100
Commercial assay or kitHigh-Capacity cDNA Reverse Transcription KitThermoFisher ScientificCat# 4368814
Recombinant DNA reagentSCNN1B cDNA plasmidAddgeneCat# 83429
Recombinant DNA reagentpcDNA3.1 cDNA plasmidGift from N.M Hooper, Manchester
Chemical compound, drugsodium-sensitive molecule SBFIThermoFisher ScientificCat# S-126310 mM,
100 min
Chemical compound, drugpotassium-sensitive molecule PBFIThermoFisher ScientificCat# P-126610 mM,
100 min
Chemical compound, drugPluronic F-127SigmaCat# P2443
Software, algorithmGraphPad Prism7Graphpad software

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

Additional files

Supplementary file 1

Donor demographics.

Patients with Cystic Fibrosis (CF), systemic autoinflammatory diseases (SAID), non-CF bronchiectasis (NCFB) and healthy controls (HC). All CF donors were F508del/F508del homozygous (n = 30) with no sign of infection. Three of the patients with NCFB had primary ciliary dyskinesia (PCD) and one patient with NCFB had an unknown genotype. All patients with a SAID had characterised mutations in a known disease-causing gene (Tumor Necrosis Factor Receptor Associated Periodic Syndrome (TRAPS) n = 2, Muckle-Wells n = 2, A20 haploinsufficiency n = 1, Pyrin-Associated Autoinflammation with Neutrophilic Dermatosis (PAAND) n = 1, Familial Mediterranean Fever (FMF) n = 2, Hyper IgD Syndrome (HIDS) n = 2 and Schnitzler syndrome n = 1). BMI: Body Mass Index; FEV: Forced expiratory volume; CRP: C-reactive protein.

https://doi.org/10.7554/eLife.49248.016
Transparent reporting form
https://doi.org/10.7554/eLife.49248.017

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