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.

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).

Figure 7

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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 B_4- (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.

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

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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.

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).

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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”.

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

   "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

   "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

   "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

   "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

   "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

   "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

   "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

    "This ORCID iD identifies the author of this article:" 0000-0002-1015-0745

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