1. Human Biology and Medicine
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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
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Cite this article as: eLife 2019;8:e49248 doi: 10.7554/eLife.49248

Abstract

Cystic Fibrosis (CF) is a monogenic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, resulting in defective CFTR-mediated chloride and bicarbonate transport, with dysregulation of epithelial sodium channels (ENaC). These changes alter fluid and electrolyte homeostasis and result in an exaggerated proinflammatory response driven, in part, by infection. We tested the hypothesis that NLRP3 inflammasome activation and ENaC upregulation drives exaggerated innate-immune responses in this multisystem disease. We identify an enhanced proinflammatory signature, as evidenced by increased levels of IL-18, IL-1β, caspase-1 activity and ASC-speck release in monocytes, epithelia and serum with CF-associated mutations; these differences were reversed by pretreatment with NLRP3 inflammasome inhibitors and notably, inhibition of amiloride-sensitive sodium (Na+) channels. Overexpression of β-ENaC, in the absence of CFTR dysfunction, increased NLRP3-mediated inflammation, indicating that dysregulated, ENaC-dependent signalling may drive exaggerated inflammatory responses in CF. These data support a role for sodium in modulating NLRP3 inflammasome activation.

https://doi.org/10.7554/eLife.49248.001

Introduction

Cystic fibrosis (CF) is the most common life-threatening autosomal recessive disease to affect Caucasian populations. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) result in reduced expression and function of the CFTR with the most common mutation (ΔF508/ΔF508) resulting in inadequate processing of the protein and subsequent intracellular trapping in the endoplasmic reticulum (ER) (Elborn, 2016). Clinical manifestations of this debilitating condition include repeated pulmonary infections, innate immune-driven episodes of inflammation and inflammatory arthritis (Elborn, 2016; Whitsett and Alenghat, 2015; Bals et al., 1999; Montgomery et al., 2017). The CFTR protein is widely expressed in a variety of cells and tissues where it functions as an anion channel, conducting chloride (Cl-) and bicarbonate (HCO3-) ions, and as a regulator of a range of epithelial transport proteins, including the epithelial sodium channel (ENaC) (König et al., 2002; Konstas et al., 2003; Kunzelmann, 2003; Berdiev et al., 2009).

The NLRP3 inflammasome is an important inflammatory pathway in CF but there is little indication as to whether this reflects underlying innate autoinflammation or activation in response to chronic bacterial, viral and fungal infections, including pathogens such as Pseudomonas aeruginosa, and Burhkholderia cepacia complex (Iannitti et al., 2016; Rimessi et al., 2015; Montgomery et al., 2017; Fritzsching et al., 2015; Kiedrowski and Bomberger, 2018). We propose that CF exhibits many hallmarks of an autoinflammatory condition (Peckham et al., 2017; McGonagle and McDermott, 2006; McDermott et al., 1999), with infiltration by innate immune cells (macrophages and neutrophils) at target sites, and a lack of autoantibodies or autoreactive T cells (McDermott et al., 1999). The NLRP3 intracellular protein complex is a sensor that detects changes in cellular homeostasis rather than directly sensing common pathogenic or endogenous motifs. Multiple cellular events have been observed to trigger NLRP3 activation, including K+ efflux, Na+ influx, Cl- efflux and Ca2+ signalling (Schorn et al., 2011; Muñoz-Planillo et al., 2013; Katsnelson and George, 2013; Domingo-Fernández et al., 2017; Green et al., 2018; Hafner-Bratkovič and Pelegrín, 2018). All known canonical inflammasomes, including the NLRP3 inflammasome, function as signalling platforms for caspase-1-driven activation of IL-1-type cytokines (IL-1β and IL-18). One of IL-18’s main functions is induction of cell-mediated immunity and IFN-γ secretion by natural killer (NK) and T-cells, whereas IL-1β has a central role in inducing fever with immune cell proliferation, differentiation and apoptosis. Inflammasome activation induces a programmed cell death downstream of the activation of caspases-1, 4, 5, and 11. Gasdermin D (GSDMD) is cleaved by said caspases, oligomerizing and forming pores (13 nm) in the plasma membrane, releasing mature IL-1β and IL-18 and triggering cell lysis, or pyroptosis (Cookson and Brennan, 2001; Platnich and Muruve, 2019).

In healthy lungs, ENaC helps maintain normal volume and composition of airway surface liquid (ASL). An absence or reduction in functional CFTR leads to defective CFTR-mediated anion transport and upregulation of ENaC. These changes in normal homeostasis result in fluid hyperabsorption, abnormally thick viscous mucus and defective mucociliary clearance (Althaus, 2013; Boucher, 2019). While there is no literature to support a direct link between ENaC and inflammation in CF, there is indirect evidence to suggest that aberrant sodium (Na+) transport influences the disease process. Overexpression of β-ENaC in mice, results in CF-like lung disease, with ASL dehydration, inflammation and mucous obstruction of bronchial airways (Mall et al., 2004; Zhou et al., 2011; Zhou et al., 2008). In humans, genetic variants in the β- and γ- ENaC chains, leading to functional abnormalities in ENaC, have been associated with bronchiectasis and CF-like symptoms (Fajac et al., 2008). By contrast, rare mutations associated with hypomorphic ENaC activity, can slow disease progression in patients homozygous for the CFTR ∆F508 mutation (Mall et al., 2004; Zhou et al., 2011; Donaldson and Boucher, 2007). These studies highlight the essential role of ENaC in regulating normal airway homeostasis and show that inhibition of ENaC may modify disease progression, either by altering ASL composition or modifying other processes, such as inflammation. Several trials are ongoing to assess the safety and effectiveness of new topical ENaC inhibitors to restoring airway surface liquid and mucociliary clearance in CF (Cystic Fibrosis Foundation, 2017).

The aims of this study were to characterise NLRP3 inflammasome activation in CF and to investigate the role of the epithelial Na+ channel, ENaC, in driving this inflammation through alterations in ionic homeostasis, a known NLRP3 activating event.

In order to fulfil these aims, monocytes and epithelial cells with characterised CF-associated mutations are directly compared to cohorts of NCFB and SAID.The NCFB cohort comprises of individuals with primary ciliary dyskinesia (PCD), a rare, ciliopathic, autosomal recessive genetic disorder affects the movement of cilia in the lining of the respiratory tract. Individuals with PCD suffer from reduced mucus clearance from the lungs, and susceptibility to chronic recurrent respiratory infections, as is the case with CF. By comparing monocytic- and epithelial-driven inflammation in CF and PCD, one is able to distinguish between inflammation due to recurrent infection, as is the case with both CF and NCFB, and inflammation that is downstream of CFTR/ENaC-mediated ionic disturbances, specific to CF.

The SAID patient cohort is composed of an array of systemic autoinflammatory diseases that are defined by an innate immune driven inflammation. The variety of autoinflammatory disorders described in this manuscript demonstrates the broad range of pathophysiology within this rare inflammatory disease spectrum. Here we demonstrate that the intrinsic ionic defect in cells and individuals with CF-associated mutations predisposes hyperactivation of the NLRP3 inflammasome, leading to inappropriate and destructive innate immune driven inflammation, as found in autoinflammation.

Results

Increased NLRP3-dependent IL-18 secretion in human bronchial epithelial cells with CF-associated mutations

In CF, airway epithelial cells have been shown to produce exaggerated levels of proinflammatory cytokines (IL-8 and TNF) characteristic of a hyper-inflammatory phenotype (Venkatakrishnan et al., 2000). However, IL-1β secretion is barely detectable in bronchial/airway epithelial cells and does not greatly increase following stimulus with NLRP3-inflammasome activators, despite being highly responsive to the effects of the IL-1β cytokine itself (Tang et al., 2012; Peeters et al., 2013; Gillette et al., 2013). We explored the effects of the constitutively expressed IL-18 cytokine in HBECs (BEAS-2B (WT) control, IB3-1 (ΔF508/W1282X), CuFi-1 (ΔF508/ΔF508) and CuFi-4 (ΔF508/G551D)) following treatment with activators for the four main caspase-1 driven inflammasomes, NLRC4, pyrin, AIM2 and NLRP3 (Figure 1).

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

Under basal conditions, IL-18 levels in the HBECs were undetectable in the absence of a stimulus. All inflammasomes were primed with lipopolysaccharide from Escherichia coli K12 (LPS), which specifically targets TLR4 and is used to promote pro-IL-18/IL-1β expression; this was followed by stimulation with established activators of the inflammasomes, NLRC4 (flagellin), pyrin (TcdB) and AIM-2 (dsDNA). There were no differences between cells with or without CF-associated mutations for activation of these particular inflammasomes (Figure 1A). However, when HBECs were stimulated with LPS, followed by ATP, a specific NLRP3 inflammasome activating signal that nucleates NLRP3 assembly with ASC, pyrin and caspase-1, IL-18 secretion was upregulated in CF-associated mutant cell lines, IB3-1 (p<0.0001) and CuFi-1 (p<0.0001) relative to the BEAS-2B control. When these cells were pre-treated with small molecule inhibitors of the NLRP3 inflammasome signalling pathway (MCC950; NLRP3, OxPAPC;TLR4, YVAD;caspases), IL-18 secretion was reduced (Figure 1B) in the CF-associated mutant HBEC lines thereby confirming NLRP3 inflammasome as the source of the elevated IL-18 inflammatory cytokine. Consistent with increased IL-18 cytokine levels, caspase-1 activation was also elevated in the CF-associated mutant HBEC lines relative to control post-LPS and ATP stimulation in vitro, and was depleted by MCC950 pre-treatment (Figure 1C).

It is well established that NLRP3 inflammasome triggers sterile inflammatory responses and pyroptosis, which is a proinflammatory form of programmed cell death initiated by the activation of inflammatory caspases (Bergsbaken et al., 2009). To examine this, we monitored cell death whereby pyroptosis was distinguished from necrosis by pretreating cells with a caspase inhibitor and using lactose dehydrogenase (LDH) as a measure of necrosis. Elevated pyroptosis was present after LPS and ATP stimulation in the HBEC line IB3-1 (Figure 1D), consistent with an NLRP3-mediated hyper-inflammatory phenotype.

Increased NLRP3-dependent IL-1β/IL-18 secretion in human monocytes with CF-associated mutations

We next explored NLRP3 inflammasome activation in primary monocytes (main producers of IL-18 and IL-1β, along with neutrophils) derived from HC, CF, SAID, and NCFB (Figure 2). Under basal conditions primary monocytes, isolated from HC and CF, showed no significant difference in the secretion of IL-18 and IL-1β cytokines (Figure 2A,B) or when monocytes were stimulated with LPS alone across all patient groups (Figure 2—figure supplement 1A,B). As with the HBECs, there was also no statistical difference between HC and CF in LPS mediated activation of inflammasomes, NLRC4, pyrin and AIM-2.

Figure 2 with 1 supplement see all
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

In primary monocytes stimulated with LPS, followed by ATP to activate the NLRP3 inflammasome, we observed hyper-responsiveness in IL-18 (p<0.0001) and IL-1β (p=0.0009) secretion in CF monocytes relative to HC. When these cells were pretreated with NLRP3 inflammasome pathway inhibitors (MCC950; NLRP3, OxPAPC;TLR4, YVAD;caspase-1), their secretions were significantly abrogated across all patient groups (HC, CF, SAID and NCFB) (Figure 2C,D). Downstream, IL-18 acts on NK and T-cells to express and secrete IFN-γ (Kim et al., 2015); we monitored IFN-γ gene expression and secretion and found they were increased, post-NLRP3-inflammasome activation, in PBMCs from patients with CF compared to HC (Figure 2—figure supplement 1C,D).

We next examined cell death in HC, CF, SAID and NCFB monocytes (Figure 2G). Elevated pyroptosis was present after LPS and ATP stimulation in monocytes from patients with CF, and also in those diagnosed with SAID. Notably, caspase-1-independent necrosis was also elevated in CF and SAID monocytes (Figure 2E). To understand the relationship between elevated pyroptosis and downstream inflammation, the presence of ASC protein aggregates (specks), key inflammasome components, were measured in the cell supernatants, post-NLRP3 inflammasome activation. ASC-specks were elevated in stimulated monocytes from patients with CF-associated mutations, and also in those diagnosed with SAID (Figure 2F), and were reduced with MCC950 treatment. Similar to HBECs, caspase-1 activation was also elevated in CF and SAID monocytes, post-LPS and ATP stimulation in vitro, and were depleted by MCC950 pretreatment in the monocytes (Figure 2E).

Proinflammatory cytokines and ASC specks are elevated in CF Sera, and are comparable to patients diagnosed with systemic autoinflammatory disease (SAID)

To understand the extent of systemic inflammation in CF, serum cytokine levels were measured in patients with CF, SAID, NCFB and HC. Serum IL-18 (p=0.0064), IL-1β (p<0.0001) and IL-1Ra (p<0.0001) levels were all significantly elevated in CF samples, with levels comparable to SAIDs (Figure 3A–C). However, in contrast to IL-1-type cytokines, the inflammasome-independent cytokines, TNF and IL-6, were not significantly elevated in patients with CF whereas the levels of these two cytokines were significantly elevated in samples from patients with SAID (Figure 3—figure supplement 1). All SAID patients were on active recombinant IL-1Ra (anakinra) therapy, which will have reduced serum cytokine levels. However, levels of IL-18 and endogenous IL-1Ra were raised in patients with SAID, and are comparable to the proinflammatory IL-1 cytokines found in CF-serum.

Figure 3 with 1 supplement see all
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

We next sought to confirm that serum IL-1-type cytokines were associated with NLRP3 inflammasome activation by detecting the presence of ASC specks, in sera. ASC-specks were significantly elevated in CF (p=0.0007) and SAID sera compared to HC (Figure 3D), reflecting inflammasome-mediated inflammation and pyroptosis (Franklin et al., 2014). The activity of caspase-1, the rate-limiting factor in the activation of all inflammasomes (Mariathasan et al., 2006), was significantly elevated in CF and SAID serum samples compared to NCFB and HC (Figure 3E).

These data suggest that systemic serum cytokine levels from patients with CF are comparable to patients diagnosed with SAID and can be characterised by release of proinflammatory IL-1-type cytokine family members (IL-1β and IL-18), associated with NLRP3 inflammasome activation.

Dysregulated na+ and K+ in cells with CF-associated mutations can be modulated with ENaC inhibitors

As K+ efflux and Na+ influx are thought to occur upstream of NLRP3 inflammasome activation and ENaC overactivation is a recognised event in cells with CF-associated mutations, we monitored intracellular concentrations of K+ and Na+ to determine if they were dysregulated in CF. The concentration gradient of Na+ and K+ is essential for cellular homeostasis, including resting potential, nutrient transport, cell volume and signal transduction. Here we tested the hypothesis that dysregulated ENaC-mediated Na+ transport alters the Na+/K+ gradient, enhancing K+ efflux and downstream NLRP3 inflammasome activation. We found that intracellular Na+ levels were significantly higher in CF cells [monocytes (p<0.0001), HBEC (p<0.0001)], at the peak of Na+ influx, which was recorded immediately following stimulus with ATP. This corresponded with a superior reduction in intracellular K+ [monocytes (p<0.0001), HBEC (p<0.0001)], suggestive of greater K+ efflux in these cells, upon ATP stimulation (Figure 4A,B,E,F, Figure 4—figure supplement 1). The magnitude of the observed Na+ and K+ fluxes was significantly reduced by ENaC inhibition (using both amiloride and the SPLUNC1-derived peptide, S18, a highly stable and specific small molecule inhibitor of ENaC channels). Pretreatment with 5-(N-ethyl-N-isopropyl)-amiloride, EIPA, a broad-spectrum Na+ channel inhibitor with lower potency for ENaC, also reduced intracellular Na+ and increased intracellular K+ in CF monocytes and HBEC lines, but not to the same extent as amiloride or S18 (Figure 4A,B,E,F, Figure 4—figure supplement 1). To corroborate our findings, we used ouabain, a Na+/K+-ATPase inhibitor, which was associated with a higher intracellular Na+ in CF cells compared to HC. Collectively, these data suggest that higher intracellular Na+ levels in CF, mainly driven by ENaC, predispose cells to a greater K+ efflux upon stimulation with ATP. Na+ influx has been described as a modulator of NLRP3 activation, dependent on K+ efflux (Schorn et al., 2011; Muñoz-Planillo et al., 2013; Katsnelson and George, 2013).

Figure 4 with 1 supplement see all
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

As inhibition of ENaC activity (by amiloride and S18) modulated elevated intracellular Na+ levels in cells with CF-associated mutations, we measured β-ENaC protein expression in HBEC lines and found significantly increased expression, in CuFi-1 and CuFi-4 cells relative to BEAS-2B control (p=<0.05) (Figure 4G,H). Furthermore, the β-ENaC gene was expressed in monocytes with a significant increase in β-ENaC protein levels noted in CF monocytes compared to HC (p=<0.05) (Figure 4—figure supplement 1, Figure 4C,D).

Inhibition of amiloride-sensitive sodium channels modulates inflammation in CF

We next sought to determine the extent to which dysregulated Na+ levels contribute to the observed NLRP3 inflammasome activation in cells with CF-associated mutations. We utilised small molecule inhibitors, for potent inhibition of ENaC, for in vitro NLRP3 inflammasome activation assays. Notably, amiloride alleviated the augmented cytokine secretion, as well as caspase-1 activity in both primary CF monocytes (IL-18 p=0.0001; IL-1β p=0.0272) and HBEC lines (Figure 5A,B,E) (Montgomery et al., 2017; Fritzsching et al., 2015; Mall et al., 2004). S18 potently inhibited cytokine secretion (IL-18 p=0.0052; IL-1β p=0.0100) and caspase-1 activity exclusively in cells with CF-associated mutations (Figure 5C, Figure 5—figure supplement 1), whereas EIPA had no effect (Figure 5—figure supplement 1A,C,D,E). These data were replicated by nigericin activation of NLRP3, used as a control for ATP, due to ATP’s ability to modulate other ionic channels (Figure 5—figure supplement 2). To corroborate these findings, both amiloride (p<0.0001) and S18 (p=0003) inhibited ASC-speck formation in CF monocytes (Figure 5D), and finally, inhibition of amiloride-sensitive channels did not modulate TNF levels (Figure 5—figure supplement 1F), suggestive of a specific ENaC-NLRP3 axis.

Figure 5 with 2 supplements see all
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

β-ENaC overexpression in BEAS-2B cells increases proinflammatory cytokine secretion

In order to recapitulate the ENaC-NLRP3 axis, as revealed in this study, we overexpressed the β-ENaC chain in the WT BEAS-2B line. This approach has been used previously in a β-ENaC Tg-mouse model of CF, which recreated a CF-like lung disease state, with mucous plugging and excessive inflammation (Mall et al., 2004; Zhou et al., 2011). Overexpression of β-ENaC induced elevated IL-18 secretion at baseline and after LPS and ATP stimulation (p=0.0003) (Figure 6B). These data support the hypothesis that excessive ENaC-mediated Na+ influx intrinsically drives NLRP3 inflammasome activation in CF.

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

Discussion

Here we have described our findings that IL-1-type cytokines were elevated in both monocytes and serum of patients with CF, but two of the major inflammasome-independent cytokines, TNF and IL-6, were not significantly elevated in these patients. In contrast to patients with SAID, these data suggest that the proinflammatory cytokine response in CF has a predominant NLRP3 inflammasome constitution. Furthermore, on NLRP3 inflammasome activation, IL-18 secretion was upregulated in the CF-associated mutant cell lines, IB3-1 (p<0.0001) and CuFi-1 (p<0.0001) relative to the BEAS-2B control, and these levels were reduced by treatment with small molecule inhibition of NLRP3 inflammasome signalling, thereby confirming the NLRP3 inflammasome as a major source of the elevated IL-18 inflammatory cytokine in these cells. Perhaps the most notable feature is the uncovering of a molecular link between enhanced ENaC-dependent Na+ influx, observed in cells with CF-associated mutations, and the exacerbated NLRP3 inflammasome activation. By pretreating these cells with CF-associated mutations with small molecule inhibitors of ENaC, the exaggerated IL-1-type cytokine response in vitro was diminished (Figure 7).

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

Bronchial epithelial cell (BEC) lines can produce significant quantities of IL-18 on stimulation but secrete only negligible amounts of basal or stimulated IL-1β compared to hematopoietic cells (Tang et al., 2012; Peeters et al., 2013; Gillette et al., 2013). In human BEC models, rhinovirus has been associated with IL-1β release, a process accentuated by dual priming with both ATP and polyinosine polycytidylic acid (Poly I:C, which interacts with TLR3) (Piper et al., 2013; Shi et al., 2012). In general, IL-18 is the predominant cytokine secreted by non-myeloid cells, which are also capable of having activated inflammasomes (Okazawa et al., 2004). This contrasts with PBMCs, which produce large amounts of IL-1β, from both patients with CF and healthy controls (Tang et al., 2012). The differential secretion of IL-18 and IL-1β, observed in this study is noteworthy, with IL-1β secretion being undetectable in HBEC lines, under the conditions used. This disparity may reflect the function of these two inflammasome-processed zymogens; IL-18 is a cytokine that induces recruitment of neutrophils and Th17 differentiation, as well as IFNγ secretion, whereas IL-1β is an intrinsically more destructive cytokine, acting systemically to induce fever, proliferation, differentiation, apoptosis and sensitivity to pain. IL-1β secretion is tightly regulated by a highly controlled process, involving its own gene expression as well as inflammasome priming and assembly, whereas IL-18 is constitutively expressed and only depends on the two signals for inflammasome priming and assembly. In addition, IL-1β is rapidly sequestered, or secretion, by IL-1Ra and soluble IL-1RI and –RII, once secreted, which makes IL-1β particularly difficult to assay and detect. The biological property of IL-1β underlies the severe phenotype of the rare autoinflammatory disease, deficiency of the interleukin-1–receptor antagonist (DIRA), caused by homozygous mutations in the IL1RN gene, which encoding the IL-1Ra protein (Aksentijevich et al., 2009).

The primary consequence of mutations in the CFTR gene is defective CFTR anion transport and upregulation of Na+ transport through dysregulation of ENaC (Muraglia et al., 2019; Peckham et al., 1997). Under in vivo conditions, patients with CF have a more negative baseline airway and nasal potential difference compared to HC and shows an enhanced depolarising response to amiloride, reflecting the inhibition of excessive ENaC mediated Na+ influx (Solomon et al., 2018). In airway epithelial cells, increased basolateral Na/K-ATPase activity has also been reported (Peckham et al., 1997). We hypothesised that dysregulation of Na+ transport might influence NLRP3 inflammasome activation by increasing K+ efflux, a principal trigger for NLRP3 activation (Schorn et al., 2011; Muñoz-Planillo et al., 2013; Katsnelson and George, 2013; Katsnelson et al., 2015; Di et al., 2018; Qu et al., 2017; Zhu et al., 2017; Rivers-Auty and Brough, 2015). We confirmed a dysregulation of Na+ and K+ transport by examining primary innate immune cells and HBEC lines with CF-associated mutations in vitro and discovered that excessive ENaC-mediated Na+ flux, measured indirectly by dependent changes of fluorescence signal, correlate with an increased K+ efflux, an activating signal of the NLRP3 inflammasome and downstream IL-18 and IL-1β secretion. We also show that the systemic serum cytokine signature from patients with CF is comparable to patients diagnosed with SAID, characterised by release of proinflammatory IL-1β and IL-18, which are associated with inflammasome activation. On exploration of the inflammation involved, we found a LPS-induced hyper-responsive NLRP3 inflammasome, exclusively in cells with CF-associated mutations, comparable to monocytes from patients with SAID. It should be noted that the SAID patient cohort is comprised of a variety of autoinflammatory diseases that have differing molecular pathophysiology and varying degrees of innate driven inflammation. This NLRP3 inflammasome activation extended beyond IL-18 and IL-1β secretion, with increased propensity for pyroptotic cell death and associated release of NLRP3 inflammasome components, such as ASC, and induction of IFN-γ secretion in the PBMC population. This type of inflammation has similarities to autoinflammation, particularly the IL-1/IL-18 inflammasome signature observed in serum and also present in vitro. The significantly elevated levels of ASC specks in CF sera (Figure 3D), in addition to the proinflammatory IL-1-type cytokine signature (Figure 3A–C), suggests the presence of a NLRP3 inflammasome agonist. A wide range of possible candidates for such an agonist(s) include infectious pathogens (either CF or non-CF related), the patients’ metabolic milieu or, indeed, other unknown agonists. Further work will be necessary to decipher the complex molecular mechanisms involved. This study does not suggest that inflammation is independent of infections in individuals with CF but that the response to said infections is intrinsically dysregulated and predisposed to excessive and inappropriate degrees of inflammation.

Cystic fibrosis is a monogenic disease, yet consists of varying degrees of pathology depending on the precise CFTR mutation and the extent to which the encoded CFTR protein is subsequently transcribed, translated, folded within the ER, expressed on the plasma membrane, its stability on the surface and its ability to conduct chloride and bicarbonate. There are well documented examples of different classes of CFTR mutations that fail at each one of the above stages of CFTR expression and function. The IB3-1 (ΔF508/W1282X) cell line is associated with the greatest amounts of inflammation. The W1282X mutation is associated with severe disease clinically, that can be attributed to little to no protein expression. Inflammation in the IB3-1 cell line in vitro does not correlate with ENaC protein expression. The underlying mechanism of an ENaC/NLRP3 axis driving inflammation in cells with CF-associated mutations may not be common to all mutation classes. Notably, all of the cells (monocytes and epithelia) had at least one ΔF508 allele. Whether the loss of control of ENaC expression and function observed in ΔF508 CFTR expressing cells is unique to this more common mutation will require further study. It is important to note that inhibition of ENaC alleviated NLRP3 inflammasome-mediated inflammation in all cell lines (Figure 5E–F). While inflammation in CF is believed to be driven predominantly by infection, many CFTR mutant animal models have shown that airway inflammation and bronchiectasis can occur under sterile conditions (Montgomery et al., 2017; Keiser et al., 2015; Rao and Grigg, 2006). For instance, in a CFTR knockout ferret model, inflammation, bronchiectasis and mucus accumulation can develop in the absence of infection (Rosenow, 2018). A recent study from the Australian Respiratory Early Surveillance Team for CF highlights the association between pulmonary inflammation and structural lung disease in young children with and without infection (Rosenow et al., 2019). IL-1β is detectable in bronchoalveolar lavage (BAL) fluid from children with CF and is strongly correlated with neutrophil counts, independent from detectable infection (Montgomery et al., 2018). The increased IL-1β neutrophil production in BAL fluid from patients with CF appears to be driven via NLRP3 (McElvaney et al., 2018).

Autoinflammation is defined as an exaggerated inflammatory response, driven by dysregulated innate immune cells, in the absene of antigen-driven T-cells, B-cells, or associated autoantibodies (Peckham et al., 2017; McDermott et al., 1999; McDermott and Aksentijevich, 2002; McGonagle and McDermott, 2006; Stoffels and Kastner, 2016). Adaptive immune cells may be recruited in response to the downstream consequences of autoinflammation, with increased susceptibility to infection, and progression to autoimmunity and hyperinflammation (Wekell et al., 2016). In fact, we have shown that endoplasmic reticulum (ER) stress present in CF innate immune cells, including neutrophils, monocytes and M1 macrophages, causes an exaggerated inflammatory response (Lara-Reyna et al., 2019). Based on these data and cited literature, CF displays features of autoinflammatory disease, in part driven by aberrant ionic fluxes and recurrent infections. The NLRP3 inflammasome can be primed by proinflammatory cytokines, such as TNF, although bacterial components do provide a far more potent stimulus (Swanson et al., 2019; McElvaney et al., 2019).

Targeting the exaggerated inflammatory response without simultaneously predisposing individuals to infection remains an elusive goal which carries inherent (potential) risk. This was highlighted by the termination of a study investigating a leukotriene B4- (LTB4) receptor antagonist for treatment of lung disease in CF following a disproportionate incidence of respiratory serious adverse events (Konstan et al., 2014). Treating inflammation remains a priority and multiple trials targeting various anti-inflammatory pathways are ongoing (Cystic Fibrosis Foundation, 2017). There is evidence in the literature of endotoxin tolerant monocytes from patients with CF, with reduced cytokine expression in response to repetitive endotoxin exposure (del Campo et al., 2011; del Fresno et al., 2009; del Fresno et al., 2008). However, we propose that peripheral monocytes such as those used in this study, are not constantly exposed to common pathogenic antigens that exist within the lung microenvironment during infections. In fact, one may propose a scenario of trained immunity, where innate immune cells hyper-respond to the recurrent infections with greater destructive consequences. Endotoxins are not the only inflammatory stimulus for NLRP3 inflammasome priming that exist in the inflammatory milieu of the CF lung. Epithelial derived cytokines, neutrophilic extracellular traps (NETs), necrotic cell death and subsequent damage associated molecular patterens (DAMPs) may all induce transcription of IL-1β/IL-18 and other inflammasome components, independent of endotoxins. A caveat of our study is that we have not tested the full array of DAMPs and pathogen associated molecular patterns (PAMPs) that exist in the CF lung that may trigger the intrinsic predisposition to NLRP3 inflammasome activation observed in this study.

The data here suggest that IL-18 may be a useful therapeutic target, by preventing adaptive cell airway infiltration whilst maintaining a potent IL-1β response to infection (Gabay et al., 2018); however, NLRP3 activation exercises a protective role in animal models of induced colitis (Allen et al., 2010), running contrary to the expectation that reduced NLRP3 expression might reduce inflammation in the bowel. Furthermore, IL-18 has an epithelial protective role in promoting repair of gut epithelium (Zaki et al., 2010), and more ex vivo studies and CF disease models are required to elucidate the benefits or hazards of IL-18 blockade. Anakinra is a viable therapeutic option for CF, particularly with its short half-life and daily treatment regime allowing a more controlled dosage during any inevitable infections. In fact a phase IIa clinical trial to evaluate safety and efficacy of subcutanous administration of anakinra in patients with cystic fibrosis is in progress (EudraCT Number: 2016-004786-80). It is notable that inhibition of TLR4 signalling was the most effective means of blocking NLRP3 activation in our study, which suggests that targeting this pathway may be a therapeutic option in CF (Keeler et al., 2019; Greene et al., 2008). These novel data, along with our observation that decreased intracellular K+ levels upon stimulation with ATP in cells with CF-associated mutations, correlates with the characteristic and excessive ENaC-mediated Na+ transport, suggest that CF-associated mutations lower the threshold for NLRP3 assembly, rather than priming this key intracellular component of innate immune defenses.

Through inhibition of amiloride-sensitive Na+ channels, we were able to reduce NLRP3 inflammasome activation, and associated IL-18 and IL-1β secretion, in vitro. Notably, ENaC inhibition did not modulate IL-18 and IL-1β secretion in monocytes from individuals with SAID, indicating the proposed ENaC-NLRP3 axis is unique to CF-associated mutations. These findings highlight the importance of excessive ENaC-mediated intracellular Na+ as a disease mechanism in CF, and also highlight its potential as a therapeutic target. Targeting amiloride-sensitive Na+ channels such as ENaC to restoring airway surface liquid and mucociliary clearance, has been previously attempted, using amiloride in the 1990 s, with little efficacy, due to amiloride’s short half-life and limited effectiveness (Graham et al., 1993).

In conclusion, we have shown that hypersensitive NLRP3 inflammasome activation in CF induces proinflammatory serum and cellular profiles. Overexpression of β-ENaC, in the absence of CFTR dysfunction as well as dysregulation of amiloride-sensitive Na+ channel activity in CF, further potentiates LPS-induced NLRP3 inflammasome activity. Both ENaC and NLRP3 are potential therapeutic targets for reducing inflammation in patients with CF.

Materials and methods

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

Clinical characteristics of patients

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Patients with CF, systemic autoinflammatory diseases (SAID), non-CF bronchiectasis (NCFB) and healthy controls (HC) were recruited from the Department of Respiratory Medicine and Research laboratories at the Wellcome Trust Benner Building at St James’s Hospital. All SAID patients were on Anakinra treatment, when blood samples were obtained. The study was approved by Yorkshire and The Humber Research Ethics Committee (17/YH/0084). Informed written consent was obtained from all participants at the time of the sample collection. Demographics are shown in Supplementary file 1. 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).

PBMC and monocyte isolation

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Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using the density gradient centrifugation. Whole blood were mixed with equal volume of PBS, carefully layered onto of Lymphoprep (Axis-Shield, Dundee, UK) and centrifuged at 1100xg for 20 min without brakes. The white buffy layer was removed and washed twice in PBS by centrifuging at 1100xg for 10 min. PBMC pellet was resuspended in complete RPMI medium (RPMI medium containing 10% heat inactivated foetal bovine serum, 50 U/ml penicillin, 50 μg/ml streptomycin).

Monocytes were isolated by negative selection from PBMCs using the monocyte isolation kit II (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Pelleted PBMCs were resuspended in 30 μl of buffer per 107 cells (autoMACS Rinsing Solution containing 0.5% BSA). This was mixed with 10 μl FcR Blocking Reagent followed by 10 μl of biotin-conjugated antibodies and incubated at 4°C for 10 min. Next 30 μl of buffer were added together with 20 μl of anti-biotin microbeads and incubated for an additional 15 min at 4°C. This whole mixture was washed with 2 ml of buffer and centrifuged at 300 xg for 10 min. The cell pellet was resuspended in 500 μl of buffer and past down a MS column (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) on a magnetic stand. PBMCs (2 × 106/ ml) and monocytes (1 × 106/ ml) were allowed to adhere overnight prior to experimentation.

Cell line culture

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Human cell lines BEAS-2B (ATCC CRL-9609), CuFi-1 (ATCC CRL-4013), CuFi-4 (ATCC CRL-4015) and IB3-1 (ATCC CRL-2777) were purchase from ATCC (UK) which ensures STR profiling of the cell lines used. BEAS-2B and IB3-1 were cultured in LHC basal medium (Thermo Fisher Scientific, Loughborough, UK) supplemented with 10% FBS, 50 U/ml penicillin and 50 μg/ml streptomycin). CuFi-1 and CuFi-4 were grown on Cell+ surface plates or flasks (Sarstedt, Leicester, UK) with LHC-9 medium (Thermo Fisher Scientific, Loughborough, UK). All cells were cultured in a humidified incubator at 37°C, 5% CO2. Cells were used at 1 × 106/ ml. Cell lines were routinely tested for mycoplasma using MycoAlertTM Mycoplasma Detection Kit Lonza catalog#: LT07-118, and were all negative.

Cell stimulations

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Cells were pre-treated with the following compounds where indicated prior to NLRP3 stimulation; MCC950 (15 nM, Cayman Chemical, Cambridge, UK) for 1 hr, YVAD (2 μg/ mL, Invivogen, San Diego, California) for 1 hr, OxPAPC (30 μg/ mL, Invivogen) for 1 hr, Amiloride (10 μM, 100 μM, Cayman Chemical, Cambridge, UK) for 1 hr, EIPA (10 μM, Cayman Chemical) for 1 hr, SPLUNC1-derived peptide, S18 (25 μM, gift from Spyryx Biosciences, Inc) for 4 hr or ouabain (100 nM, Cayman Chemical, Cambridge, UK) for 24 hr. Inflammasome stimulation was achieved using either LPS (10 ng/mL, Ultrapure EK, Invivogen) for 4 hr with the addition of ATP (5 mM, Invivogen, San Diego, California) for the final 30 min of stimulation, poly(dA:dT) dsDNA (1 μg/mL with Lipofectamine 2000, Invivogen, San Diego, California) for 1 hr, TcdB (10 ng/mL, Cayman Chemical, Cambridge, UK) for 1 hr or flagellin (10 ng/mL with Lipofectamine 2000, Invivogen, San Diego, California) for 1 hr for the final 1 hr of the LPS stimulation. ATP was dissolved in pre-warmed at 37°C medium (100 mM stock) and immediately (~2 min) added to the cells. All incubations were done in a humidified incubator at 37°C, 5% CO2. Supernatant, RNA and protein were collected and stored immediately following stimulation.

Cytokine quantification using ELISA

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Cytokines from patient sera and cell cultured media were detected by ELISAs (IL-1 beta Human Matched Antibody Pair, human IL-18 Matched Antibody Pair, IL1RA Human Matched Antibody Pair, TNF alpha Human Matched Antibody Pair and IL-6 Human Matched Antibody Pair) (ThermoFisher Scientific, Loughborough, UK), as per the manufactures recommendations. In general, ELISA plates were coated with 100 μl cytokine capture antibody in PBS overnight at 4°C. The plates were washed three times with PBST (PBS containing 0.5% Tween 20) and the wells blocked in 300 μl assay buffer (0.5% BSA, 0.1% Tween 20 in PBS) by incubating for 1 hr. The plates were washed twice with PBST and 100 μl of sera/culture supernatants, together with appropriate standards, were added to wells in duplicates. Immediately 50 μl of detection antibody were added to all wells and incubated for 2 hr. After the incubation the plates were washed five times with PBST and 100 μl of tetramethybenzidine (TMB) substrate solution (Sigma, Poole, UK) were added to all wells and incubated for 30 min. Colour development was stopped by adding 100 μl of 1.8N H2SO4. And absorbance measured at 450 nm and reference at 620 nm. Note all incubation steps were done at room temperature with continual shaking at 700 rpm. All data points are an average of duplicate technical replicates for each independent experiment.

ASC protein aggregates (specks)

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Methodology as previously published (Rowczenio et al., 2018). Briefly, patients’ sera or culture media were incubated with 5 µL of phycoerythrin anti-ASC (TMS-1) antibody (Biolegend, London, UK) for 1 hr, and analysed on the LSRII flow cytometer instrument (BD Biosciences, California).

Caspase-1 activity

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A colorimetric assay (Caspase-1 Colorimetrix Assay, R and D Systems, Abingdon, UK) measured caspase-1 activity, via cleavage of a caspase-specific peptide conjugated to a colour reporter molecule, p-nitroalinine (pNA), performed on protein lysates and serum. All data points are an average of duplicate technical replicates for each independent experiment. Protein concentrations in the lysate were determined by BCA assay.

Detection of mRNA by RT-qPCR

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Cells were washed in PBS, pelleted and immediately lysed in 1 ml TRIzol Reagent (Ambion Life technologies, Paisley, UK) and RNA extracted using the PureLink RNA mini kit (Ambion, Life technologies, Paisley, UK). Chloroform (200 μl) were added to each sample and mixed vigorously for 15 s and left to stand for 2 min at room temperature. These were centrifuged at 12000 xg for 15 min at 4°C. The top clear phase was transferred to a fresh tube and mixed with equal volume of 70% ethanol. This mixture was transferred to a spin cartridge, with a collection tube, and centrifuged at 12000 xg for 15 s at room temperature. The waste was disposed of and the spin cartridge was centrifuged one more time. The spin cartridge was washed 700 μl of wash buffer I and centrifuged at 12000 xg for 15 s. A second wash with 500 μl wash buffer II (containing ethanol) were added to the spin cartridge and centrifuged at 12000 xg for 15 s followed by further spin for 1 min. RNA was recovered by adding 30 μl of RNase free water to the spin cartridge, incubated for 1 min and centrifuged for 2 min.

The High Capacity cDNA Reverse Transcription kit (Applied Biosystems, California) was used to convert the RNA to cDNA according to the manufacturer’s instructions. TaqMan assays were done in the QuantStudio 5 Real-Time PCR instrument (Thermo Fisher Scientific, Loughborough, UK).

The Taqman primers used in this study are detailed below:

GeneAssay IDDye
SCNN1BHs01548617_m1FAM
IFNGHs00989291_m1FAM
HPRT1Hs02800695_m1FAM

Transient transfection

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BEAS-2B cells were transiently transfected with 10 μg of sodium channel epithelial one beta subunit (SCNN1B) cDNA (Addgene, Teddington, UK) or pcDNA3.1 vector only control (gift from Professor NM Hooper, Manchester University) using Lipofectamine 2000 (Thermo Fisher Scientific, Loughborough, UK) for 48 hr, as per manufacturers’ instructions. Cells were harvested, lysed in RIPA buffer and protein concentrations were determined using the Pierce bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Loughborough, UK).

Western blotting

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Samples were made up in dissociation buffer [1x dissociation buffer (100 mM Tris-HCl, 2% (w/v) sodium dodecyl sulfate, 10% (v/v) glycerol, 100 mM dithiothreitol, 0.02% (w/v) bromophenol blue, pH 6.8] and heated at 95°C for 5 min, and equal protein concentration was loaded and resolved by 10% SDS-PAGE on Tris-glycine gels and then transferred to Hybond PVDF membranes (GE Healthcare, Buckinghamshire, UK). Following electrotransfer in Towbin buffer (25 mM Tris, 192 mM glycine, and pH 8.3, 20% methanol) at 100 V for 1 hr, the membranes were blocked for 1 hr in blocking solution (PBS containing 0.1% Tween 20% and 5% (w/v) non-fat milk). After three washes in PBST (PBS with 0.5% Tween 20), primary antibodies were incubated with PVDF membrane overnight at 4°C. The membrane was washed three times with PBST and secondary antibody-HRP conjugate were added and incubated for 2 hr with constant rocking at room temperature. The membrane was washed five times with PBST and 3 ml of ECL detection system (Immobilon chemiluminescent HRP substrate, Millipore, UK) was added onto the membrane for 5 min, before being imaged with the ChemiDoc Imaging system (Bio-Rad, Hertfordshire, UK). Primary antibodies used: rabbit anti-SCNN1B (Avia Systems Biology, San Diego; 1/500 dilution), rabbit anti-actin-β (GeneTex, Nottingham, UK) at 1/20000 dilution. Secondary antibodies used: anti-rabbit IgG horseradish peroxidase-conjugated (Cell Signalling Technology, Hertfordshire, UK) were diluted at 1/4000. All antibodies were diluted in PBS containing 0.1% Tween 20% and 2% BSA.

Fluorometric determination of na+ and K+ concentration

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Na+ and K+ sensitive dyes, SBFI (S-1263) and PBFI (P-1266) (Molecular Probes, Paisley, UK), respectively, were used as cell permeant selective ion indicators for the fluorometric determination of Na+ and K+ concentrations. Monocytes and HBECs were allowed to adhere in black 96-well cell culture plates overnight. Cells were then incubated with various stimulants, as indicated in figure legends, before being washed and incubated in the appropriate low serum media (see above) (1%) for 1 hr. The dyes (10 mM final concentration) were loaded with Pluronic F-127 (Sigma) and incubated for 100 min. All wells were washed with NaCl solution. ATP (5 mM) was then added to the wells prior to measuring fluorescence. Excitation at 344 nm and 400 nm with emission at 500 nm was measured immediately to calculate the percentage change in fluorescence compared to an untreated control. All data points are an average of duplicate technical replicates for each independent experiment.

Statistics

All analyses were performed using GraphPad Prism v 7. Bar graphs were expressed as mean standard error of the mean (S.E.M). The Kruskal-Wallis test with Dunn’s multiple comparison or the Mann Whitney test was performed when comparing non-parametric populations. A two-way ANOVA statistical test with Tukey’s multiple comparison post-hoc analysis was performed when calculating variance between samples (p values * =≤ 0.05, ** =≤ 0.01, *** =≤ 0.001 and ****=≤0.0001). A p<0.05 was considered significant. Statistical tests used are indicated in the figure legends. p-values are measured using a 2-sided hypothesis.

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Decision letter

  1. Jos WM van der Meer
    Reviewing Editor; Radboud University Medical Centre, Netherlands
  2. Satyajit Rath
    Senior Editor; Indian Institute of Science Education and Research (IISER), India
  3. Jos WM van der Meer
    Reviewer; Radboud University Medical Centre, Netherlands
  4. Siroon Bekkering
    Reviewer; Radboud University, Netherlands

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "ENaC-mediated sodium influx exacerbates NLRP3-dependent inflammation in Cystic Fibrosis" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Jos van der Meer as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Satyajit Rath as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Siroon Bekkering (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

The reviewers agree this is an interesting paper exploring NLRP3 inflammasome activation in Cystic fibrosis. In general, the experiments are well carried out and convincing.

The authors compare the IL-1 and IL-18 (as well as caspase-1 and ASC specks) in CF production directly to patients with Autoinflammatory syndromes (as well as to patients with non CF bronchiectases and Healthy controls), and in principle this gives a good impression of the magnitude of autoinflammation in CF.

The authors convincingly show that the responses in CF are NLRP3 inflammasome-specific as they are blocked by an NLRP3-specific small molecule inhibitor, and because AIM2, NLRC4, or Pyrin inflammasome responses are not elevated. Mechanistically, the authors find that there are significant changes in levels of intracellular Na+ (increased) and K+ (decreased) in CF monocytes and CF mutant HBECs when NLRP3 is activated. This is associated with increased expression of epithelial sodium channels (ENaC) suggesting that CFTR mutations drive the dysregulation of ENaC, which via sodium influx leads to increased potassium efflux,which is a central trigger of NLRP3 activation. Critically, using specific inhibitors of ENaC attenuates the hyperactivation of NLRP3 observed in CF monocytes and CF mutant HBECs, but does not affect healthy control cells.

Although the paper is generally well presented, there is some lack of precision, which is reflected by the large number of comments.

Essential revisions:

1) Is it possible that ER stress driven by CFTR mutations contributes to NLRP3 activation? Has this been evaluated by the authors?

2) In all experiments, the LPS or ATP only controls are missing. This is of special importance in the monocyte stimulation experiments, since monocytes can activate the inflammasome independent of ATP as shown by Gaidt et al., 2016. If the controls were included in the experiment but not shown, that should be mentioned or shown in the supplementary.

3) Figure 1: What is OxPac? Is this supposed to be OxPAPC?

4) As said the comparison with autoinflammatory syndromes (SAID) is a major asset to the paper, but it should be realised that SAID is a mixed bag. Although these are all IL-1beta disorders, the pathophysiology differs, as well as the magnitude of the inflammatory response. The authors could be a bit more critical on this.

5) In the Introduction, the rationale of the comparison with SAID and NCFB should be explained.

6) In line with comment #2, it is clear that there are some outliers in the SAID group in Figure 3 A and B. Are these the same patients? What was their underlying disorder? This information should be included at least in the legend to Figure 3.

7) Patients with SAID are often found to have ex-vivo IL-1beta production in the absence of an inflammatory stimulus. The question is whether this is the case in CF. It is a pity that in Figure C and D unstimulated cultures are not depicted. Are these measurements available? The blank measurements in Figure 2B are a bit of a concern as the IL-1beta concentrations are in the range of low dose LPS stimulation (and hence contamination).

8) In some experiments, all monocytes (CF, SAID and NCFB) are studied. In others, only monocytes from CF. The comparison with SAID monocytes is especially interesting in the Na/K experiments and for example the IFNgamma experiments. Why are they missing there?

9) IL-1beta measurements in serum are most often below or close to the detection level of the commercial ELISA assays. The authors should inform us about the level of detection with their use of the assay. This also holds for the other ELISAs used.

10) In line with the previous comment: the serum cytokine concentrations measured are extremely high, even for healthy controls. Please check whether the assays were performed correctly, and calculations done right. Usually concentrations of circulating IL-6 are usually around 1 pg/ml (and not 50-200), IL-1beta below 1 pg/ml (here values ranging from 5 – 90pg/ml are reported). Are CRP values known for the participants?

11) In subsection “Proinflammatory cytokines and ASC specks are elevated in CF sera, and are comparable to patients diagnosed with systemic autoinflammatory disease (SAID)” it suddenly becomes clear that the SAID patients were on Anakinra treatment. This should have been mentioned in the patient section.

12) In connection with the previous comment: by blocking the IL-1 receptor, Anakinra will inhibit IL-1beta induced by IL-1(β and α). In general, this effect is rather limited if one looks at circulating IL-1beta. Of course, secondary cytokines like IL-6 and IL-8 will be lower. Thus, the sentence” All SAID patients were on active recombinant IL-1Ra (anakinraR) therapy, which will have reduced serum IL-1b levels.” should be rephrased.

13) Subsection “Proinflammatory cytokines and ASC specks are elevated in CF sera, and are comparable to patients diagnosed with systemic autoinflammatory disease (SAID)”: how do the authors know that they detect endogenous IL-1Ra and not Anakinra?

14) It is hard to believe that the genotypes of the SAID patients (with the exception of the Schnitzler patient) are not known (Suppl Table).

15) In the PBMC stimulation experiments (in the Materials and methods as well as in the figure legends) details are missing. How long were the cells stimulated, how many cells, and how much stimulus?

16) The ATP preparation is missing in the Materials and methods section, how was ATP prepared for these experiments? This is of great importance as described by Stoffels et al., 2015. Please add methods.

17) Although the discussion on the disparity between IL-1 and IL-18 production is interesting (Discussion section), the reasons why the cell lines do not show IL-1beta production should also be discussed.

18) In the same vein, there should be some discussion of the responses of the different mutation HBEC cell lines. Generally, there seems to be the most significant effects with the IB3-1 line but this does not correlate with the level of b-ENaC observed by Western blotting (Figure 4G).

19) The Discussion section again starts with an introduction. Instead it should start mentioning the main findings of the paper, and deal with the aims that were set at the end of the Introduction. So at least skip the first paragraph and rephrase the sentences that follow.

20) The authors are very prudent discussing the potential therapeutic consequences (Discussion section). Anakinra would be worth trying, as it does not meet with major infectious complications (at least much less so than Canakinumab). In fact, it has been given successfully to patients with Chronic granulomatous disease (who also suffer from excess IL-1beta production, but at the same time have a serious immunodeficiency).

21) A scheme summarizing the mechanism and sequence of events would help the readers.

22) As a suggestion: the authors might discuss why CF patients that are constantly exposed to Pseudomonas spp do not show endotoxin-tolerant monocytes.

https://doi.org/10.7554/eLife.49248.019

Author response

We thank the reviewers for their positive comments and for their thorough evaluation of the manuscript.

Essential revisions:

1) Is it possible that ER stress driven by CFTR mutations contributes to NLRP3 activation? Has this been evaluated by the authors?

It is possible that ER stress is a contributing factor to NLRP3 activation. In a recent publication by our group, we have shown that ER stress is present in CF innate immune cells, affecting human bronchial epithelial cells (HBECs), neutrophils, monocytes and M1 macrophages. We found high levels of IL-6 and TNF in M1 macrophages, with an associated activation of the IRE1α-XBP1 pathway, which could be reversed by inhibition of the RNase domain of IRE1a (Lara-Reyna et al., 2019).

To expand on this, we have shown that inhibition of the RNase domain of IRE1α with 4μ8c in LPS/ATP stimulated CF monocytes reduces TNF levels significantly in healthy control (HC) (p=0.04, n=3) and in CF (p<0.0001, n=3), consistent with our observations in M1 macrophages. Interestingly, IL-1β levels were also significantly reduced in IRE1α inhibited CF monocytes following stimulation with LPS/ATP (p<0.001, n=3) but although reduced, did not reach significance in HC (P=0.772, n=3). This data is consistent with a recent report in which inhibition of IRE1α RNase domain with MKC8866 reduces IL1β levels in PBMCs stimulated with LPS/ATP (Talty et al., 2019). It seems that as well as modulating the unfolded protein response and managing ER stress, IRE1a signalling also promotes the efficiency of inflammasome assembly, and that blocking IRE1α seems to have a direct effect on inflammasome activation (Talty et al., 2019).

Author response image 1

Our previous work shows that IRE1α mRNA levels are increased in CF relative to HC, and IRE1α protein levels are increased in HBEC’S with CF-associated mutations relative to wild-type controls (Lara-Reyna et al., 2019). We can conclude from our new data that ER stress driven by CFTR mutations may, in part, contribute to NLRP3 activation seen in CF, but further work would be required to establish whether this effect is due to ER stress, as a result of CFTR-dependent IRE1α, upregulation or another factor(s).

We have added the following into the Discussion section:

“In fact, we have shown that endoplasmic reticulum (ER) stress present in CF innate immune cells, including neutrophils, monocytes and M1 macrophages, causes an exaggerated inflammatory response.”

2) In all experiments, the LPS or ATP only controls are missing. This is of special importance in the monocyte stimulation experiments, since monocytes can activate the inflammasome independent of ATP as shown by Gaidt et al., 2016. If the controls were included in the experiment but not shown, that should be mentioned or shown in the supplementary.

We have included LPS only controls for HC, CF, SAID and NCFB as a supplementary figure (Figure 2—figure supplement 2A,B). The manuscript by Gaidt et al., 2016 suggests that LPS alone can activate NLRP3 inflammasome through upstream activation of TLR4-TRIF-RIPK1-FADD-CASP8. They show an increase in IL-1β when monocytes were stimulated for 14 hours with 2 μg/ml LPS. They include a titration (9 different concentrations) with LPS from 2 μg/ml to 200 fg/ml and detected IL-1β cytokine production by ELISA. Unfortunately, they do not indicate the specific concentrations used – at the fourth titration of LPS alone, LPS does not induce IL-1β production in the absence of nigerecin, suggesting that low doses of LPS does not activate the non-classical inflammasome activation pathway.

As part of our experiments we did stimulate our monocytes with LPS (10ng/ml for 4h) alone (but did not include the data in the original manuscript) and observed no increase in IL-1β or IL-18 in HC, CF or NCFB patients under these conditions in the absence of ATP.

We have included the graphs below in Figure 2—figure supplement 2A,B. And included the following comment in the text (subsection “Increased NLRP3-dependent IL-1β/ IL-18 secretion in human monocytes with CF-associated mutations”):

“Under basal conditions primary monocytes, isolated from HC and CF, showed no significant difference in the secretion of IL-18 and IL-1β cytokines (Figure 2A, B) or when monocytes were stimulated with LPS alone across all patient groups (Figure 2—figure supplement 1A,B)”.

3) Figure 1: What is OxPac? Is this supposed to be OxPAPC?

We have amended this to OxPAPC throughout the manuscript. OxPAPC is a TLR2 and TLR4 inhibitor.

4) As said the comparison with autoinflammatory syndromes (SAID) is a major asset to the paper, but it should be realised that SAID is a mixed bag. Although these are all IL-1beta disorders, the pathophysiology differs, as well as the magnitude of the inflammatory response. The authors could be a bit more critical on this.

In fact, the variety of autoinflammatory disorders, described in this manuscript, was deliberately chosen to demonstrate the broad range of pathophysiology within this rare inflammatory disease spectrum, as the reviewers rightly state, thereby offering a complete and fair comparison between SAID and CF. We have also added a short description of the differences, in both the genetics and inflammatory response of autoinflammatory disease, into the manuscript and how CF might theoretically fit into this spectrum.

We have included the following into the Discussion section:

“It should be noted that the SAID patient cohort is comprised of a variety of autoinflammatory diseases that have differing molecular pathophysiology (Savic et al., in press) and varying degrees of innate driven inflammation.”

5) In the Introduction, the rationale of the comparison with SAID and NCFB should be explained.

We have revised the Introduction to outline this comparison:

“In order to fulfil these aims, monocytes and epithelial cells with characterised CF-associated mutations are directly compared to cohorts of NCFB and SAID. The NCFB cohort comprises of individuals with primary ciliary dyskinesia (PCD), a rare, ciliopathic, autosomal recessive genetic disorder affects the movement of cilia in the lining of the respiratory tract. Individuals with PCD suffer from reduced mucus clearance from the lungs, and susceptibility to chronic recurrent respiratory infections, as is the case with CF. By comparing monocytic- and epithelial- driven inflammation in CF and PCD, one is able to distinguish between inflammation due to recurrent infection, as is the case with both CF and NCFB, and inflammation that is downstream of CFTR/ENaC-mediated ionic disturbances, specific to CF.

The SAID patient cohort is composed of an array of systemic autoinflammatory diseases that are defined by an innate immune driven inflammation. The variety of autoinflammatory disorders described in this manuscript demonstrates the broad range of pathophysiology within this rare inflammatory disease spectrum. Here we demonstrate that the intrinsic ionic defect in cells and individuals with CF-associated mutations predisposes hyperactivation of the NLRP3 inflammasome, leading to inappropriate and destructive innate immune driven inflammation, as found in autoinflammation.”

6) In line with comment #2, it is clear that there are some outliers in the SAID group in Figure 3A and B. Are these the same patients? What was their underlying disorder? This information should be included at least in the legend to Figure 3.

The outliers in the SAID group in Figure 3A and 3B are the same patients. The highest outlier corresponds to HIDS 1 patient (shown in the updated Supplementary file 1) and the second highest outlier corresponds to A20 (TNFIP3) haploinsuficiency. This information has been included in the legend for Figure 3.

“Outliers in SAID group for IL-1β and IL-1Ra correspond to HIDS 1 and A20 deficiency”

7) Patients with SAID are often found to have ex-vivo IL-1beta production in the absence of an inflammatory stimulus. The question is whether this is the case in CF. It is a pity that in Figure C and D unstimulated cultures are not depicted. Are these measurements available? The blank measurements in Figure 2B are a bit of a concern as the IL-1beta concentrations are in the range of low dose LPS stimulation (and hence contamination).

To address this question we have included IL-18 and IL-1β measurements from unstimulated and stimulated (LPS or LPS/ATP) from all patient groups (SAID, HC, CF and NCFB). See the attached data in response to comment 2. These data shows that there are elevated levels of IL-18 relative to HC but not IL-1β in the unstimulated SAID samples. Additionally, Ex-vivo production of IL-18 and IL-1β is not increased in the unstimulated CF samples.

The authors agree that the unstimulated levels of IL-1β are higher than expected and may indeed be assay artefact due to high background levels that is often the case with difficult to measure cytokines, such as IL-1β. However, the authors wish to emphasise that the key conclusion of these data is that there is no difference between the HC and CF cohorts in terms of NLRC4, Pyrin or AIM2 inflammasome activation, regardless of the precise cytokine concentration observed.

8) In some experiments, all monocytes (CF, SAID and NCFB) are studied. In others, only monocytes from CF. The comparison with SAID monocytes is especially interesting in the Na/K experiments and for example the IFNgamma experiments. Why are they missing there?

As the reviewers will appreciate, the availability of such rare samples is extremely limited. In light of this, we prioritised experiments to ensure the comparison between the innate inflammation that governs both SAID and CF diseases was complete as possible. Unfortunately, the trade-off was insufficient sample to perform the two experiments that the reviewers highlight here. However, we would be confident in hypothesising that the ionic fluxes of SAID patients would be similar to that of the HC cohort and the IFNγ measurements would match that of CF, if not with a more pronounced IFN signature. These are experiments that we plan to perform in the future as further SAID patient samples become available for follow-up projects.

9) IL-1beta measurements in serum are most often below or close to the detection level of the commercial ELISA assays. The authors should inform us about the level of detection with their use of the assay. This also holds for the other ELISAs used.

The detection range of the IL-1β assay used in this study was 31.2-2000 pg/ml with assay sensitivity <31.2 pg/ml. The few values that were below the limit of the ELISAs used were extrapolated using the standard curve. The authors acknowledge that IL-1β is challenging to measure in the serum, due to its poor bioavailability and stability. However, we are confident that the data are accurate and reflect the extent to which the inflammasome pathway is active within these patient cohorts. In addition, IL-18, IL-1Ra, caspase-1 and ASC complement the IL-1β measurements, supporting systemic inflammasome activation in CF and SAID cohorts. We have included a resource table as part of the methods so that each ELISA kit used can be identified (https://www.thermofisher.com/elisa/product/IL-1-β-Human-Matched-Antibody-Pair/CHC1213).

10) In line with the previous comment: the serum cytokine concentrations measured are extremely high, even for healthy controls. Please check whether the assays were performed correctly, and calculations done right. Usually concentrations of circulating IL-6 are usually around 1 pg/ml (and not 50-200), IL-1beta below 1 pg/ml (here values ranging from 5–90pg/ml are reported). Are CRP values known for the participants?

We can confirm that the assays were performed as per the manufacturer’s instructions and the analyses have been further double checked. Circulating serum cytokines can vary between individuals, disease states and assay used, discounting experimental variations. Various ‘physiological’ ranges exist in the literature but ranges of 13-227pg/ml for IL-1β, 42-203pg/ml for TNF, 13-149pg/ml for IL-6 and 112-294pg/mL for IL-1Ra have been observed (Sekiyama et al., 1994) and support the data in this manuscript. CRP levels have now been included with the revised Supplementary file 1.

11) In subsection “Proinflammatory cytokines and ASC specks are elevated in CF sera, and are comparable to patients diagnosed with systemic autoinflammatory disease (SAID)” it suddenly becomes clear that the SAID patients were on Anakinra treatment. This should have been mentioned in the patient section.

12) In connection with the previous comment: by blocking the IL-1 receptor, Anakinra will inhibit IL-1beta induced by IL-1(β and α). In general, this effect is rather limited if one looks at circulating IL-1beta. Of course, secondary cytokines like IL-6 and IL-8 will be lower. Thus, the sentence “All SAID patients were on active recombinant IL-1Ra (anakinraR) therapy, which will have reduced serum IL-1b levels.” should be rephrased.

We have included the following in the revised subsection “Clinical characteristics of patients”:

“All SAID patients were on Anakinra treatment, when blood samples were obtained”.

13) Subsection “Proinflammatory cytokines and ASC specks are elevated in CF sera, and are comparable to patients diagnosed with systemic autoinflammatory disease (SAID)”: how do the authors know that they detect endogenous IL-1Ra and not Anakinra?

Anakinra (17.3 KD) differs from the sequence of IL-1Ra (23-25 KD) by one methionine at the Nterminus. Anakinra is also not glycosylated. Whether these factors affect the ELISA assay’s ability to detect anakinra is unknown to the authors and therefore agree this there is a possibility that some of the IL-1Ra serum levels in the SAID patient cohort displayed in Figure 3C may be anakinra ‘contamination’.

We have included a statement in the figure legend making this point clear.

“Of note, an undetermined amount of detected IL-1Ra is attributed to circulating Anakinra (recombinant IL-1Ra) specifically in the SAID cohort.”

14) It is hard to believe that the genotypes of the SAID patients (with the exception of the Schnitzler patient) are not known (Supplementary Table).

The authors regret to have not included this information in the original paper and have now included the known genotypes for the SAID patients in the revised Supplementary file 1.

15) In the PBMC stimulation experiments (in the Materials and methods as well as in the figure legends) details are missing. How long were the cells stimulated, how many cells, and how much stimulus?

Two million PBMCs were used for this experiment. This is detailed in subsection “PBMC and monocyte isolation”. The following details have been included in Figure 2—figure supplement 1C, D legend where PBMCs were used.

“PBMCs were unstimulated or stimulated with LPS (10ng/ml, 4 hours) or LPS (10ng/ml, 4 hours) and ATP (5mM) for the final 30 minutes.”

16) The ATP preparation is missing in the Materials and methods section, how was ATP prepared for these experiments? This is of great importance as described by Stoffels et al., 2015. Please add methods.

The following details on ATP preparation have been included (subsection “Cell stimulations”):

“ATP was dissolved in pre-warmed at 37°C medium (100 mM stock) and immediately (~2 minutes) added to the cells”.

17) Although the discussion on the disparity between IL-1 and IL-18 production is interesting (Discussion section), the reasons why the cell lines do not show IL-1beta production should also be discussed.

We feel we have discussed this disparity between the cell lines adequately in the Discussion section. We have also moved the paragraph referred to in the comment to follow directly on from this discussion as it fits better here.

18) In the same vein, there should be some discussion of the responses of the different mutation HBEC cell lines. Generally, there seems to be the most significant effects with the IB3-1 line but this does not correlate with the level of b-ENaC observed by Western blotting (Figure 4G).

We have expanded on this with the following addition (Discussion section):

“Cystic fibrosis is a monogenic disease, yet consists of varying degrees of pathology depending on the precise CFTR mutation and the extent to which the encoded CFTR protein is subsequently transcribed, translated, folded within the ER, expressed on the plasma membrane, its stability on the surface and its ability to conduct chloride and bicarbonate. There are well documented examples of different classes of CFTR mutations that fail at each one of the above stages of CFTR expression and function. The IB3-1 (ΔF508/W1282X) cell line is associated with the greatest amounts of inflammation. The W1282X mutation is associated with severe disease clinically, that can be attributed to little to no protein expression. Inflammation in the IB3-1 cell line in vitro does not correlate with ENaC protein expression. The underlying mechanism of an ENaC/NLRP3 axis driving inflammation in cells with CFassociated mutations may not be common to all mutation classes. Notably, all of the cells (monocytes and epithelia) had at least one ΔF508 allele. Whether the loss of control of ENaC expression and function observed in ΔF508 CFTR expressing cells is unique to this more common mutation will require further study. It is important to note that inhibition of ENaC alleviated NLRP3 inflammasome-mediated inflammation in all cell lines (Figure 5 E-F)”.

19) The Discussion section again starts with an introduction. Instead it should start mentioning the main findings of the paper, and deal with the aims that were set at the end of the Introduction. So at least skip the first paragraph and rephrase the sentences that follow.

We have rephrased the discussion appropriately with the following (Discussion section):

“Here we have described our findings that IL-1-type cytokines were elevated in both monocytes and serum of patients with CF, but two of the major inflammasome-independent cytokines, TNF and IL-6, were not significantly elevated in these patients. In contrast to patients with SAID, these data suggest that the proinflammatory cytokine response in CF has a predominant NLRP3 inflammasome constitution. Furthermore, on NLRP3-inflammasome activation, IL-18 secretion was upregulated in the CF-associated mutant cell lines, IB3-1 (p<0.0001) and CuFi-1 (p<0.0001) relative to the BEAS-2B control, and these levels were reduced by treatment with small molecule inhibition of NLRP3-inflammasome signalling, thereby confirming the NLRP3-inflammasome as a major source of the elevated IL-18 inflammatory cytokine in these cells. Perhaps the most notable feature is the uncovering of a molecular link between enhanced ENaC-dependent Na+ influx, observed in cells with CF-associated mutations, and the exacerbated NLRP3 inflammasome activation. By pretreating these cells with CF-associated mutations with small molecule inhibitors of ENaC, the exaggerated IL-1-type cytokine response in vitro was diminished (Figure 7)”.

20) The authors are very prudent discussing the potential therapeutic consequences (Discussion section). Anakinra would be worth trying, as it does not meet with major infectious complications (at least much less so than Canakinumab). In fact, it has been given successfully to patients with Chronic granulomatous disease (who also suffer from excess IL-1beta production, but at the same time have a serious immunodeficiency).

Anakinra may have a role in the management of CF. And we have previously used the drug to manage a complex case of athropathy in CF (unpublished). Despite significant improvements in joint symptoms there was no improvement in lung function. Therefore, modifying the aberrant IL-18 as well as IL-1b response may be necessary to downregulate autoinflammation in CF. A phase 2 trial using anakinra in CF is is progress (EudraCT Number: 2016-004786-80).

We have proposed this in the revised Discussion section:

“Anakinra is a viable therapeutic option for CF, particularly with its short half-life and daily treatment regime allowing a more controlled dosage during any inevitable infections. In fact, a phase IIa clinical trial to evaluate safety and efficacy of subcutanous administration of anakinra in patients with cystic fibrosis is in progress (EudraCT Number: 2016-004786-80).”

21) A scheme summarizing the mechanism and sequence of events would help the readers.

We have included the following schematic diagram and legend in the paper, as figure 7.

“Figure 7: 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.”

22) As a suggestion: the authors might discuss why CF patients that are constantly exposed to Pseudomonas spp do not show endotoxin-tolerant monocytes.

We have included the following the revised Discussion section:

“There is evidence in the literature of endotoxin-tolerant monocytes from patients with CF, with reduced cytokine expression in response to repetitive endotoxin exposure [70-72]. However, we propose that peripheral blood monocytes, such as those used in this study, are not constantly exposed to common pathogenic antigens that exist within the lung microenvironment during infections. In fact, one may propose a scenario of trained immunity, whereby innate immune cells hyper-respond to the recurrent infections, with greater destructive consequences. We also suggest that endotoxins are not the only inflammatory stimulus for NLRP3 inflammasome priming that exist in the inflammatory milieu of the CF lung. Epithelial-derived cytokines, neutrophilic extracellular traps (NETs), necrotic cell death and subsequent DAMPs may all induce transcription of IL-1β /IL-18 and other inflammasome components, independent of endotoxins. A caveat to our study is that we have not tested the full array of DAMPs and PAMPs that exist in the CF lung that may trigger an intrinsic predisposition to NLRP3 inflammasome activation observed in this study”.

https://doi.org/10.7554/eLife.49248.020

Article and author information

Author details

  1. Thomas Scambler

    Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, United Kingdom
    Contribution
    Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing—original draft, Writing—review and editing
    Contributed equally with
    Heledd H Jarosz-Griffiths
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2468-0218
  2. Heledd H Jarosz-Griffiths

    1. Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, United Kingdom
    2. Leeds Institute of Medical Research, University of Leeds, Leeds, United Kingdom
    3. Leeds Cystic Fibrosis Trust Strategic Research Centre, University of Leeds, Leeds, United Kingdom
    Contribution
    Data curation, Formal analysis, Investigation, Methodology, Writing—original draft, Writing—review and editing
    Contributed equally with
    Thomas Scambler
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5154-4815
  3. Samuel Lara-Reyna

    1. Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, United Kingdom
    2. Leeds Institute of Medical Research, University of Leeds, Leeds, United Kingdom
    Contribution
    Data curation, Formal analysis
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9986-5279
  4. Shelly Pathak

    Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, United Kingdom
    Contribution
    Data curation, Formal analysis
    Competing interests
    No competing interests declared
  5. Chi Wong

    1. Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, United Kingdom
    2. Leeds Institute of Medical Research, University of Leeds, Leeds, United Kingdom
    3. Leeds Cystic Fibrosis Trust Strategic Research Centre, University of Leeds, Leeds, United Kingdom
    Contribution
    Data curation, Formal analysis, Project administration
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2108-1615
  6. Jonathan Holbrook

    1. Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, United Kingdom
    2. Leeds Institute of Medical Research, University of Leeds, Leeds, United Kingdom
    3. Leeds Cystic Fibrosis Trust Strategic Research Centre, University of Leeds, Leeds, United Kingdom
    Contribution
    Writing—review and editing
    Competing interests
    No competing interests declared
  7. Fabio Martinon

    1. Leeds Cystic Fibrosis Trust Strategic Research Centre, University of Leeds, Leeds, United Kingdom
    2. Department of Biochemistry, University of Lausanne, Lausanne, Switzerland
    Contribution
    Conceptualization, Supervision, Investigation, Visualization, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6969-822X
  8. Sinisa Savic

    1. Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, United Kingdom
    2. Leeds Cystic Fibrosis Trust Strategic Research Centre, University of Leeds, Leeds, United Kingdom
    3. Department of Clinical Immunology and Allergy, St James’s University Hospital, Leeds, United Kingdom
    Contribution
    Conceptualization, Investigation, Visualization, Writing—original draft, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7910-0554
  9. Daniel Peckham

    1. Leeds Institute of Medical Research, University of Leeds, Leeds, United Kingdom
    2. Leeds Cystic Fibrosis Trust Strategic Research Centre, University of Leeds, Leeds, United Kingdom
    3. Adult Cystic Fibrosis Unit, St James’ University Hospital, Leeds, United Kingdom
    Contribution
    Conceptualization, Resources, Supervision, Funding acquisition, Investigation, Visualization, Writing—original draft, Project administration, Writing—review and editing
    Contributed equally with
    Michael F McDermott
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7723-1868
  10. Michael F McDermott

    1. Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, United Kingdom
    2. Leeds Cystic Fibrosis Trust Strategic Research Centre, University of Leeds, Leeds, United Kingdom
    Contribution
    Conceptualization, Resources, Funding acquisition, Investigation, Writing—original draft, Project administration, Writing—review and editing
    Contributed equally with
    Daniel Peckham
    For correspondence
    M.McDermott@leeds.ac.uk
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1015-0745

Funding

Cystic Fibrosis Trust (SRC009)

  • Heledd H Jarosz-Griffiths
  • Chi Wong
  • Jonathan Holbrook
  • Fabio Martinon
  • Sinisa Savic
  • Daniel Peckham
  • Michael F McDermott

University of Leeds (110 University Scholarship)

  • Thomas Scambler

Consejo Nacional de Ciencia y Tecnología (CONACyT)

  • Samuel Lara-Reyna

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

The authors would like to thank all the patients and research nurses, particularly Lindsey Gillgrass and Anne Wood, of the Adult Cystic Fibrosis Unit at St. James’s Hospital, Leeds. This work is supported by a grant (SRC009) from the Cystic Fibrosis Trust, a 110 Anniversary University of Leeds Scholarship (TS), LIRMM scholarship and CONACyT (SLR).

Ethics

Human subjects: Patients with CF, systemic autoinflammatory diseases (SAID), non-CF bronchiectasis (NCFB) and healthy controls (HC) were recruited from the Department of Respiratory Medicine and Research laboratories at the Wellcome Trust Benner Building at St James's Hospital. The study was approved by Yorkshire and The Humber Research Ethics Committee (17/YH/0084). Informed written consent was obtained from all participants at the time of the sample collection.

Senior Editor

  1. Satyajit Rath, Indian Institute of Science Education and Research (IISER), India

Reviewing Editor

  1. Jos WM van der Meer, Radboud University Medical Centre, Netherlands

Reviewers

  1. Jos WM van der Meer, Radboud University Medical Centre, Netherlands
  2. Siroon Bekkering, Radboud University, Netherlands

Publication history

  1. Received: June 11, 2019
  2. Accepted: September 17, 2019
  3. Accepted Manuscript published: September 18, 2019 (version 1)
  4. Version of Record published: September 27, 2019 (version 2)

Copyright

© 2019, Scambler et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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