Polymerization of misfolded Z alpha-1 antitrypsin protein lowers CX3CR1 expression in human PBMCs

Proteins can lose their structure and form polymers because of mutations or changes in their immediate environment which can lead to cell damage and disease. Interestingly, polymers formed by a variety of proteins can reduce the levels of CX3C chemokine receptor 1 (CX3CR1 for short) that controls the behaviour of immune cells and is implicated in a range of illnesses.

Inherited ZZ alpha-1 antitrypsin deficiency is a rare genetic condition that highly increases the risk of liver and lung diseases. This disorder is characterised by mutant alpha-1 antitrypsin proteins (AAT for short) reacting together to form polymers; yet it remains unclear how the polymers affect different cells or organs, and lead to diseases. To investigate this question, Tumpara et al. examined whether polymers of mutant AAT influence the level of the CX3CR1 protein in specific classes of immune cells.

Experiments revealed that in people with AAT deficiency, certain blood immune cells express lower levels of CX3CR1. Regardless of age, clinical diagnosis, or treatment regimen, all individuals with ZZ alpha-1 antitrypsin deficiency had AAT polymers circulating in their blood: the higher the levels of polymers measured, the lower the expression of CX3CR1 recorded in the specific immune cells. When Tumpara et al. added polymers of mutant AAT to the immune cells of healthy donors, the expression of CX3CR1 dropped in a manner dependent on the polymer concentration. According to microscopy data, AAT polymers occurred inside cells alongside the CX3CR1 protein, suggesting that the two molecular actors interact. In the future, new drugs that remove these polymers, either from inside cells or as they circulate in the body, could help patients suffering from conditions associated with this abnormal protein aggregation.

Interactions between the chemokine receptors and chemokines, but also other proteins, peptides, lipids, and microbial products, play a critical role in the recruitment of inflammatory cells into injured/diseased tissues (Bachelerie et al., 2014). Many human diseases involve altered surface expression of chemokine receptors, which can lead to a defective cell migration and inappropriate immune response. Most of the human peripheral blood mononuclear cells (PBMCs) express CX3CR1 (Bazan et al., 1997), also known as the G-protein-coupled receptor 13 (GPR13) or fractalkine receptor, a mediator of leukocyte migration and adhesion. In the central nervous system, CX3CR1 is largely expressed by microglial cells (brain macrophages) (Ransohoff, 2009), which are involved in neurodegenerative diseases like Alzheimer’s disease. The major role of CX3CR1-expressing cells is to recognize and enter tissue following CX3CL1 (fractalkine or also called neurotactin) gradient, and to crawl or ‘patrol’ in the lumen of blood vessels (Auffray et al., 2007). Since CX3CR1/CX3CL1 axis is also involved in the synthesis of anti-inflammatory cytokines and has a significant role in cytoskeletal rearrangement, migration, apoptosis, and proliferation, its dysregulation is associated with the development of cardiovascular diseases, kidney ischemia–reperfusion injury, cancer, chronic obstructive pulmonary disease (COPD), neurodegenerative disorders, and others (Harrison et al., 1998; Ning et al., 2004; Rius et al., 2013). Some studies indicate that CX3CR1 deficiency contributes to the severity of infectious diseases (Bonduelle et al., 2012) and promotes lung pathology in respiratory syncytial virus-infected mice (Das et al., 2017). Animals with deletion of CX3CR1 show impaired phagocytosis (Thome et al., 2015), which is vital to prevent unwanted inflammation. It is clear that CX3CR1-expressing cells have tissue-specific roles in different pathophysiological conditions. Nevertheless, a comprehensive knowledge on the regulation of CX3CR1 expression is still missing.

Current findings suggest that divergent proteins with a common propensity to form extracellular oligomers interact with chemokine receptors and affect their expression levels. For example, Alzheimer’s peptide, Aβ, interacts with CX3CR1 and significantly reduces its expression in cultured microglial cells and in Alzheimer’s brain (Cho et al., 2011). Similarly, highly aggregated extracellular Tau protein binds to CX3CR1, promotes its internalization, and reduces expression in microglial cells (Bolós et al., 2017). In concordance, polymers of human Z alpha-1 antitrypsin (Z-AAT), resulting from protein misfolding due to the Z (Glu342Lys) mutation in SERPINA1 gene, lower CX3CR1 mRNA expression in human PBMCs, which parallels with increased intracellular CX3CR1 and Z-AAT protein levels.

Inherited alpha-1 antitrypsin deficiency (AATD) is a rare genetic condition caused by SERPINA1 gene mutations. Homozygous Z AATD mutation is the most clinically relevant among Caucasians (prevalence is about 1:2000-1:5000) that is characterized by low plasma levels of AAT protein (10–15% compared to the wild type, MM AAT, 1.3–2 g/l) and the presence of intracellular and circulating Z-AAT polymers (Tan et al., 2014). The liver is the major producer of AAT, therefore the accumulation of Z-AAT polymers in hepatocytes is a marker for diagnosing AATD (Janciauskiene et al., 2011). The intracellular Z-AAT polymers have also been identified in other AAT-expressing cells like monocytes and macrophages (Belchamber et al., 2020). The accumulation of polymers is harmful for AAT-producing cells, whereas the circulating Z-AAT polymers are not able to execute the tasks of AAT protein, a major inhibitor of serine proteases having a strong immunomodulatory potential. Based on the facts that: (i) circulating Z-AAT polymers contribute to the risk of developing pathologies (Parmar et al., 2002; Strnad et al., 2020), (ii) pathogenic oligomeric proteins affect CX3CR1 expression (Bolós et al., 2017), and (iii) CX3CR1/CX3CL1 axis plays a significant role in immunity (Imai and Yasuda, 2016), we aimed to investigate CX3CR1 expression in PBMCs of ZZ AATD individuals. For this, in collaboration with German Alpha1 Patient Association and Aachen University, was prepared RNA from freshly isolated PBMCs of 41 clinically stable ZZ AATD volunteers independently of their clinical diagnosis or treatment with intravenous AAT, a specific augmentation therapy (Janciauskiene and Welte, 2016). For comparison, PBMCs isolated from healthy volunteers having normal plasma AAT levels were used. Additionally, a limited amount of RNA sample was available from PBMCs isolated from a cohort of 12 ZZ AATD emphysema patients at Leiden University Medical Center, The Netherlands (Figure 1—figure supplement 1).

Independent of individual’s age, clinical diagnosis (healthy, lung or liver disease), or augmentation therapy, the CX3CR1 mRNA expression turned to be much lower in ZZ AATD PBMCs than in PBMCs from non-AATD controls (median [range]: 4.1 [2.7–5.5] vs. 18.5 [13–26.6], p<0.001) (Figure 1A). A previous study has shown that CX3CR1^−/− mice have significantly higher plasma levels of CX3CL1 than wild-type mice (Cardona et al., 2008). A diminished expression of CX3CR1 might be related to increased levels of soluble CX3CL1, an exclusive ligand for CX3CR1 (Bachelerie et al., 2014). However, the concentration of plasma CX3CL1 was low, did not differ between ZZ AATD and non-AATD individuals (Figure 1B), and did not correlate with CX3CR1 mRNA in PBMCs. The expression and release of CX3CL1 is generally low in the absence of inflammatory insults (Umehara et al., 2004) showing that at the time point of blood donation all volunteers were under stable clinical condition.

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Source files, containing original data for Figure 1A and B, to document CX3CR1 expression (A), and plasma levels of CX3CL1 in alpha-1 antitrypsin-deficient (AATD) and non-AATD individuals (B).

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Although CX3CR1 is preferentially expressed on monocytes, other cells also express this receptor (Landsman et al., 2009). Previous reports indicated that exogenous IL-15 is a negative regulator of CX3CR1 expression in human CD56⁺ NK cells (Barlic et al., 2003; Sechler et al., 2004). However, plasma levels of IL-15 were lower in ZZ AATD than in non-AATD (pg/ml, median [range]: 6.6 [5.9–6.9], n = 23 vs. 7.63 [6.63–8.1], n = 21, p=0.001), excluding a possible link between IL-15 and CX3CR1 mRNA levels.

Because ZZ AATD individuals, differently from non-AATD, have about 90% lower blood concentration of Z-AAT protein, which may influence cellular microenvironment (Ramos et al., 2010), a relationship between CX3CR1 and Z-AAT plasma levels cannot be excluded. However, no correlation was found between CX3CR1 mRNA in PBMCs and plasma levels of Z-AAT measured by nephelometry (data not shown). Next, plasma Z-AAT polymers were measured, as the biomarkers of all carriers of the Z allele (Tan et al., 2014; Janciauskiene et al., 2002). As anticipated, only minor amounts of polymers were detected in plasma of non-AATD individuals while plasma of ZZ AATD contained high amounts of polymers (µg/ml, mean [SD]: 4.1 [6], n = 18 vs. 1399.8 [750], n = 20, respectively). Since most of the ZZ AATD individuals received intravenous augmentation therapy with plasma purified AAT protein, ZZ AATD individuals were segregated into subgroups who receive or not receive therapy. There were no significant differences in Z-AAT polymer levels between the subgroups: (µg/ml, median [range]: non-augmented 1506.6 [854–1781], n = 17 vs. augmented 1348.5 [779.5–1529], n = 23, respectively). A previous study used a sandwich ELISA based on 2C1 antibody and found that circulating Z-AAT polymers range between 8.2 and 230.2 μg/ml in ZZ AATD (Tan et al., 2014) whereas much higher circulating levels of Z-AAT polymers were detected by using the single monoclonal antibody (LG96)-based ELISA. These discrepancies can be due to the differences between antibody specificities. For example, 2C1 showed high affinity for polymers formed by heating M- or Z-AAT at 60°C (Miranda et al., 2010) while LG96 antibody recognizes naturally occurring/native Z-AAT polymers without requiring sample heating. To answer, why some individuals have higher plasma levels of Z-AAT polymers than monomers (measured by nephelometry) is of great importance for the further studies.

Most interestingly, in ZZ AATD individuals, was found a trend toward an inverse relationship between CX3CR1 mRNA in PBMCS and plasma Z-AAT polymers (r² = −0.31, n = 38, p=0.055) (Figure 1C). This latter finding prompted more extensive investigation whether Z-AAT polymers affect CX3CR1 expression when added to healthy donor PBMCs for 18 hr, ex vivo. Lipopolysaccharide (LPS, from Escherichia coli, 1 µg/ml) was included as a known reducer of CX3CR1 expression (Pachot et al., 2008; Sica et al., 1997). Indeed, polymeric Z-AAT in a concentration-dependent manner lowered CX3CR1 mRNA (Figure 2—figure supplement 2) whereas repeated experiments using Z-AAT at a constant concentration of 0.5 mg/ml reduced CX3CR1 mRNA more than twice as compared to non-treated controls (Figure 2A). Accordingly, LPS and polymer containing Z-AAT preparation significantly decreased surface expression of CX3CR1, specifically in CD14⁺ monocytes and NK cells (Figure 3).

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Source file, containing original data for Figure 2A, to document reduced CX3CR1 expression in peripheral blood mononuclear cells (PBMCs) treated with Z alpha-1 antitrypsin (Z-AAT) or lipopolysaccharide (LPS) (A).

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Source files, containing original data for Figure 3A,B, to document reduced CX3CR1 surface expression in monocytes (A) and NK cells (B) after treatment with Z alpha-1 antitrypsin (Z-AAT) or lipopolysaccharide (LPS) (A).

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By contrast, cellular levels of CX3CR1 protein increased in PBMCs treated with Z-AAT polymers or LPS (used as a positive control) as compared to non-treated controls (Figure 2B). The CX3CR1 protein was present in detergent resistant lipid raft fraction of PBMCs treated with Z-AAT (Figure 2—figure supplement 3A). Total cell lysates and lipid raft fractions from Z-AAT-treated PBMCs, in contrast to those prepared from M-AAT-treated or non-treated PBMCs, contained high amounts of AAT polymers (Figure 2C, Figure 2—figure supplement 3B). The laser scanning confocal microscopy of double-labeled specimens showed a co-localization of Z-AAT polymers with CX3CR1 protein (Figure 2D). Furthermore, 3D reconstruction of cross sections visualized larger Z-AAT aggregates surrounded by cellular extensions in a cap-like formation, suggesting that cells may react differently depending on the size of Z-AAT polymers (Figure 2E). It cannot be excluded that Z-AAT polymers, similar like polymers of Tau protein, interact with CX3CR1 and get internalized (Chidambaram et al., 2020). This may determine the fate of CX3CR1 mRNA expression, that is, sequestered intracellularly and not returning to the cell surface, CX3CR1 protein may induce signaling pathways lowering CX3CR1 expression. To achieve a definitive answer how Z-AAT or other types of protein polymers regulate CX3CR1 levels, detailed mechanistic studies are required. In general, along with transcriptional regulation, chemokine receptors trafficking is of great importance to understand (Kershaw et al., 2009).

Under the same experimental conditions, monomeric M-AAT had no effect on CX3CR1 mRNA expression relative to housekeeping gene HPRT1 (mean [SD]: 24.9 [2.9] controls, n = 5 vs. 23.7 [1.3], n = 5, NS), and protein levels (Figure 2B). Likewise, monomeric Z-AAT protein does not affect CX3CR1 mRNA and protein levels, and heat-induced polymers of M-AAT showed no effect on CX3CR1 expression as well (Figure 2—figure supplement 4). Probably, specific conformational properties and/or molecular sizes of Z-AAT polymers are required for their interaction with CX3CR1. For example, cell surfaces express CX3CL1 as a constitutive oligomer (three to seven molecules), which is essential for efficient interaction with CX3CR1 (Hermand et al., 2008; Ostuni et al., 2020). Numerous chemokines tend to self-associate that determines their activity (Proudfoot et al., 2003), and therefore certain Z-AAT polymers may resemble chemokine structures competing for the same receptor(s). In some experimental models, Z-AAT polymers expressed strong chemotactic properties (Parmar et al., 2002; Lomas and Mahadeva, 2002). When chemokine receptors are engaged in chemotaxis, they can be removed from the cell surface by the ligand–receptor internalization (Springer, 1994), which might explain a decrease of CX3CR1 in ZZ PBMCs. Interestingly, the soluble form of CX3CL1, even when used at a high concentration of 500 ng/ml, does not antagonize Z-AAT polymer effects on CX3CR1 mRNA and protein levels, and by itself showed no effect on CX3CR1 mRNA or protein levels (Figure 2—figure supplement 5), although some studies reported that CX3CL1 reduces CX3CR1 expression (Pachot et al., 2008; White et al., 2014). In solution CX3CL1 remains monomeric, even at high concentrations (Hermand et al., 2008; Mizoue et al., 1999) whereas, as mentioned above, membrane-bound CX3CL1 occurs as oligomer. These two forms of the CX3CL1 perform differential roles (Winter et al., 2020), and therefore it cannot be excluded that oligomeric, but not a soluble, form of CX3CL1 would compete with Z-AAT for CX3CR1 interaction in vivo.

The CX3CR1 helps to define the major subsets of human monocytes because classical monocytes express much lower levels of CX3CR1 than non-classical monocytes (Ziegler-Heitbrock et al., 2010). After in vitro challenge with LPS for longer periods (like for 18 hr), human monocytes are known to increase in the mRNA and membrane expression of CD14, a receptor for LPS (Landmann et al., 1996). The enhancement of CD14 expression after treatment of PBMCs with Z-AAT strikingly resembled LPS (Figure 4 and Figure 4—figure supplement 1). This raised a suspicion that Z-AAT preparations might contain endotoxin. According to the limulus amebocyte lysate test, endotoxin levels of Z-AAT preparations were below detection limit (0.01 EU/ml). Moreover, LPS significantly induced expression of TNFα, IL-6, and IL-1β while polymer containing Z-AAT preparations had no effect (Figure 4—figure supplement 2). Besides, LPS but not Z-AAT significantly increased release of cytokines (IL-1β, pg/ml, median [range]: LPS 1342.9 [1008–1834] vs. Z-AAT 3.2 [2.5–5.9] vs. controls 2.5 [2.1–3.7], n = 4 independent experiments; TNFα, ng/ml, mean [SD]: LPS 19.5 [2.5] vs. Z-AAT vs. controls, undetectable, n = 4 and IL-6, ng/ml, median [range]: LPS 15903.5 [14,626–17,262] vs. Z-AAT 5.4 [2.9–6.1] vs. control [1.7 (1.0–2.3), n = 4]). Therefore, the effect of Z-AAT preparations on CD14 is valid and unrelated to a potential LPS contamination. Although both Z-AAT polymers and LPS induce CD14 expression and similarly affect CX3CR1 expression and protein levels, data imply that Z-AAT polymers and LPS do not share the same signaling mechanisms.

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Source files, containing original data for Figure 4A, B to document CD14 gene expression in peripheral blood mononuclear cells (PBMCs) (A) and CD14 surface expression in monocytes (B).

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As a side note, it has been reported that CD14⁺⁺ monocytes have the lowest expression of CX3CR1 (Appleby et al., 2013). Low and high surface CX3CR1 levels are suggested to delineate two functional subsets of murine blood monocytes: ‘inflammatory’ and ‘resident monocytes’ (Geissmann et al., 2003). This dichotomy appears conserved in humans as CD14⁺ CD16⁻, and CD14^low CD¹⁶⁺ monocytes resemble ‘inflammatory’ and ‘resident’ monocytes. Previous study demonstrated that peripheral blood monocytes of clinically healthy young adults (30 years of age) with ZZ AATD have significantly higher mRNA and surface expression of CD14 as compared to age matched MM subjects (Sandström et al., 2008). Authors thought that the higher CD14 expression reflects early pathological processes whereas according to the current findings this phenomenon seems to relate with the circulating Z-AAT polymers.

During steady state, non-classical monocytes expressing CX3CR1 patrol healthy tissues through crawling on the resting endothelium but these monocytes are required for a rapid tissue invasion at the site of infection or inflammation (Auffray et al., 2009; Cros et al., 2010). Previous work evidenced that the non-classical subset of monocytes, characterized by high expression of CX3CR1, is almost absent in ZZ AATD emphysema patients (Stolk et al., 2019). Moreover, plasma levels of AAT polymers were found to correlate with the levels of endothelium-related markers like sE-selectin and sICAM-1 (Aldonyte et al., 2004). Beyond, in a small cohort of ZZ AATD emphysema patients, was found a strong inverse association between lung function, based on percentage (%) predicted transfer factor for carbon monoxide (TLCO%pred) and forced expiratory volume in 1 s (FEV1%pred), and plasma levels of Z-AAT polymers: (TLCO%pred [r² = −0.75, n = 9, p=0.02] and FEV1%pred [r² = −0.82, n = 9, p=0.006]). Thus, higher levels of Z-AAT polymers and lower numbers of CX3CR1-positive cells may favor the development of lung injury and disease. A decrease in the expression of CX3CR1 on human monocytes has been shown in patients with atopic dermatitis (Echigo et al., 2004) and septic shock (Pachot et al., 2008).

To date, many functional aspects of the CX3CR1–CX3CL1 axis have been suggested, including the adhesion of immune cells to vascular endothelial cells, chemotaxis, the crawling of the monocytes that patrol on vascular endothelial cells, the retention of monocytes of the inflamed endothelium to recruit inflammatory cells, and the survival of the macrophage. Considering the above, these different aspects of interactions between PBMCs and Z-AAT or other polymers occurring due to genetic or post-translational protein modifications require further investigations in dedicated clinical and experimental studies.

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures.

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Author details

1. Srinu Tumpara

   Department of Respiratory Medicine, Hannover Medical School, Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Center for Lung Research (DZL), Hannover, Germany

   Contribution

   Data curation, Validation, Investigation, Methodology, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0363-3914

2. Matthias Ballmaier

   Cell Sorting Core Facility Hannover Medical School, Hannover, Germany

   Contribution

   Data curation, Investigation, Visualization, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1352-5995

3. Sabine Wrenger

   Department of Respiratory Medicine, Hannover Medical School, Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Center for Lung Research (DZL), Hannover, Germany

   Contribution

   Data curation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9733-6162

4. Mandy König

   8sens.biognostic GmbH, Berlin, Germany

   Contribution

   Data curation, Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

5. Matthias Lehmann

   8sens.biognostic GmbH, Berlin, Germany

   Contribution

   Data curation, Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

6. Ralf Lichtinghagen

   Institute of Clinical Chemistry, Hannover Medical School, Hannover, Germany

   Contribution

   Data curation, Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

7. Beatriz Martinez-Delgado

   Department of Molecular Genetics, Institute of Health Carlos III, Center for Biomedical Research in the Network of Rare Diseases (CIBERER), Majadahonda, Spain

   Contribution

   Data curation, Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6834-350X

8. Elena Korenbaum

   Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany

   Contribution

   Data curation, Investigation, Visualization, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

9. David DeLuca

   Department of Respiratory Medicine, Hannover Medical School, Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Center for Lung Research (DZL), Hannover, Germany

   Contribution

   Data curation, Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0141-9116

10. Nils Jedicke

    Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany

    Contribution

    Data curation, Investigation, Writing - review and editing

    Competing interests

    No competing interests declared

11. Tobias Welte

    Department of Respiratory Medicine, Hannover Medical School, Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Center for Lung Research (DZL), Hannover, Germany

    Contribution

    Conceptualization, Resources, Investigation, Writing - review and editing

    Competing interests

    reports grants from German Ministry of Research and Education, during the conduct of the study; personal fees from Grifols, CSL Behring, outside the submitted work

12. Malin Fromme

    Medical Clinic III, Gastroenterology, Metabolic Diseases and Intensive Care, University Hospital RWTH Aachen, Aachen, Germany

    Contribution

    Data curation, Investigation, Writing - review and editing

    Competing interests

    No competing interests declared

13. Pavel Strnad

    Medical Clinic III, Gastroenterology, Metabolic Diseases and Intensive Care, University Hospital RWTH Aachen, Aachen, Germany

    Contribution

    Conceptualization, Resources, Data curation, Funding acquisition, Investigation, Writing - review and editing

    Competing interests

    reports grants and personal fees from CSL Behring, grants and personal fees from Grifols Inc, personal fees from Dicerna Inc, grants from Vertex, grants from Arrowhead, outside the submitted work

14. Jan Stolk

    Department of Pulmonology, Leiden University Medical Center, Member of European Reference Network LUNG, section Alpha-1-antitrypsin Deficiency, Leiden, Netherlands

    Contribution

    Conceptualization, Resources, Data curation, Supervision, Validation, Investigation, Writing - original draft, Project administration, Writing - review and editing

    Competing interests

    reports grants from CSL Behring, grants from Kamada LtD, during the conduct of the study

15. Sabina Janciauskiene

    1. Department of Respiratory Medicine, Hannover Medical School, Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Center for Lung Research (DZL), Hannover, Germany
    2. Department of Pulmonology, Leiden University Medical Center, Member of European Reference Network LUNG, section Alpha-1-antitrypsin Deficiency, Leiden, Netherlands

    Contribution

    Conceptualization, Resources, Data curation, Supervision, Funding acquisition, Validation, Investigation, Visualization, Writing - original draft, Project administration, Writing - review and editing

    For correspondence

    janciauskiene.sabina@mh-hannover.de

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-7687-5258

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