M Maraux

A Gaillardet

A Gally

P Saas

T Cherrier
email: thomas.cherrier@efs.sante.fr

E F S Umr1098

Human primary neutrophil mRNA does not contaminate human resolving macrophage mRNA after efferocytosis

Keywords: resolving macrophage (M R ), apoptotic neutrophils (AN), monocyte-derived macrophages (MDM), Carboxyfluorescein succinimidyl ester human, resolving macrophage, apoptotic neutrophils, efferocytosis, inflammation resolution, RNA sequencing

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Macrophages constitute a heterogeneous population of innate immune cells. They are present in all tissues and play a role in development, tissue and metabolic homeostasis, tissue repair and host defence [START_REF] Wynn | Macrophage biology in development, homeostasis and disease[END_REF]. They are able to phagocyte pathogens, infected and dead cells and to display processed antigens in association with major histocompatibility class II complex [START_REF] Arango Duque | Macrophage cytokines: involvement in immunity and infectious diseases[END_REF].

They also produce various mediators like cytokines, chemokines, enzymes, growth factors, reactive oxygen intermediates or lipid mediators. During the past several decades, many macrophage subtypes have been identified and characterized based on their biological functions, phenotypes, secretomes, and metabolomes. So far, two major distinct states have been described [START_REF] Shapouri-Moghaddam | Macrophage plasticity, polarization, and function in health and disease[END_REF][START_REF] Mills | M-1/M-2 macrophages and the Th1/Th2 paradigm[END_REF]:

-Classically activated macrophages (also called M1-like) that exhibits powerful antimicrobial and anti-tumoral activity. They are induced by inflammatory stimuli, such as lipopolysaccharide, TNFα and IFN-γ.

-Alternatively activated macrophages (also called M2-like) that have enhanced phagocytic abilities, can promote tissue repair and possess pro-angiogenic and pro-fibrotic properties.

They are generated in respond to certain cytokines, such as IL-4, IL-10 or IL-13. Thereafter the M2 state has been subdivided into four categories depending on the inducing stimuli (3): M2a-like generated with IL-4 and IL-13, M2b-like induced by stimulation with immune complexes and TLR-agonists, M2c-like generated with glucocorticoids, TGF-β and IL-10, and M2d-like (or tumor-associated macrophage [TAM]-like) induced by TLR agonists through the adenosine receptor.

As illustrated above, various stimuli can induce a plethora of phenotypes and functions. Moreover, macrophages are highly plastic cells and these stimuli can reprogram them by leading to phenotype and function switches. "M1-like" macrophages can adopt an "M2-like" phenotype with the appropriate stimuli and vice versa [START_REF] Davis | Macrophage M1/M2 polarization dynamically adapts to changes in cytokine microenvironments in Cryptococcus neoformans infection[END_REF]. Despite considerable advances, some macrophage phenotypes remain poorly characterized like resolution-phase macrophages (M R ).

Phagocytosis of apoptotic corpses, a process called efferocytosis, is a stimulus inducing profound phenotypical changes in macrophages. Efferocytosis gives birth to a new type of macrophage: the M R [START_REF] Elliott | Efferocytosis signaling in the regulation of macrophage inflammatory responses[END_REF][START_REF] Gordon | Macrophage clearance of apoptotic cells: A critical assessment[END_REF][START_REF] Szondy | Antiinflammatory Mechanisms Triggered by Apoptotic Cells during Their Clearance[END_REF]. Efferocytosis is therefore considered as a major trigger of inflammation resolution, since it allows inflammatory macrophage reprograming toward a pro-resolving profile [START_REF] Szondy | Antiinflammatory Mechanisms Triggered by Apoptotic Cells during Their Clearance[END_REF]. Since the end of the 90s, several papers have shed light on some critical features of M R [START_REF] Fadok | Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF[END_REF][START_REF] Morioka | Efferocytosis induces a novel SLC program to promote glucose uptake and lactate release[END_REF][START_REF] Bonnefoy | Factors Produced by Macrophages Eliminating Apoptotic Cells Demonstrate Pro-Resolutive Properties and Terminate Ongoing Inflammation[END_REF][START_REF] Park | Continued clearance of apoptotic cells critically depends on the phagocyte Ucp2 protein[END_REF][START_REF] Wang | Mitochondrial fission promotes the continued clearance of apoptotic cells by macrophages[END_REF][START_REF] Kourtzelis | DEL-1 promotes macrophage efferocytosis and clearance of inflammation[END_REF][START_REF] Zhang | Efferocytosis fuels requirements of fatty acid oxidation and the electron transport chain to polarize macrophages for tissue repair[END_REF]. For example, M R have been demonstrated to regulate several innate and adaptive immune cells [START_REF] Bonnefoy | Plasmacytoid dendritic cells play a major role in apoptotic leukocyte-induced immune modulation[END_REF][START_REF] Kleinclauss | Intravenous apoptotic spleen cell infusion induces a TGF-betadependent regulatory T-cell expansion[END_REF]. However, most of these studies were conducted in various mouse models. Up to now, data about human M R are quite sparse, and these cells remain overlooked. Global approaches like RNA sequencing may provide crucial information on these cells.

An in vitro full human system based on the co-culture of human primary apoptotic neutrophils (AN) and primary monocyte-derived macrophages (MDM) was set up several decades ago and provided some features on human M R [START_REF] Fadok | Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF[END_REF]. However, we do not hitherto know if this single-specie model is suitable for mRNA investigations like RNA sequencing. Indeed, mRNA arisen from human primary apoptotic neutrophils may contaminate M R mRNA. To alleviate this issue, macrophages and apoptotic bodies from different species can be mixed to induce efferocytosis [START_REF] Morioka | Efferocytosis induces a novel SLC program to promote glucose uptake and lactate release[END_REF]. Nonetheless this method may lead to some flaws since interspecies sequence similarities can be high. For example, mouse and human genomes share 40% of nucleotide alignment (18). A study has shown that the mapping of 60 RNA-Sequencing data sets performed in Chinese hamster versus the human genome has a total mapped rate of 22.8% [START_REF] Le | An evaluation of public genomic references for mapping RNA-Seq data from Chinese hamster ovary cells[END_REF]. Moreover, ingestion of apoptotic bodies from another specie may preclude some physiological mechanisms. For these reasons, a multi-species system, despite presenting some advantages for RNA investigations, may not represent an optimal model.

With this work, we answer to the crucial question: can ingested apoptotic neutrophil mRNA contaminate macrophage mRNA in a single-specie model, thus precluding further transcriptomic investigation in human M R ? To achieve this goal, we have reconstituted a long been used model by co-culturing primary human AN with human primary MDM to induce efferocytosis and human M R generation. We show here that human primary neutrophils, whether apoptotic or not, exhibit degraded mRNA after using a specific cell purification protocol. By Q-PCR, we show that the remaining amplifiable neutrophil mRNA are degraded and non-detectable, contrary to PBMC or Jurkat cell mRNA, after ingestion by MDM. Moreover, we confirmed that macrophages are efficiently reprogramed by efferocytosis and that purifying and mixing apoptotic neutrophils with allogenic MDM did not induce neutrophil activation, nor inflammatory macrophage responses, as previously shown [START_REF] Fadok | Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF[END_REF][START_REF] Huynh | Phosphatidylserinedependent ingestion of apoptotic cells promotes TGF-beta1 secretion and the resolution of inflammation[END_REF][START_REF] Fadok | Differential effects of apoptotic versus lysed cells on macrophage production of cytokines: role of proteases[END_REF]. Together our data establish that this well-known co-culture system allows global mRNA approaches to expand our knowledge of the elusive human M R .

Material and Methods

Cell purification and culture

Leukocyte-platelet concentrates and cytapheresis kits were obtained from healthy volunteers after informed consent signature (authorization number #AC-2015-2408). Samples were collected from the French Blood Transfusion Center (EFS Bourgogne Franche-Comté, Besançon, France)

and processed the same day. Several blood donors were used during this study.

Human primary neutrophils were isolated at room temperature from 50 mL of fresh leukocyteplatelet concentrates using a Ficoll-Paque density gradient (ρ = 1.077 g/mL, Eurobio) followed by sedimentation in a 3% dextran solution (Sigma). 25 mL of undiluted concentrate were layered over 15 mL of Ficoll-Paque in a 50 mL conical tube. After centrifugation at 900g, 20 min, the red blood cell's containing layer was collected and washed twice in PBS (centrifugation at 300g, 5min) and resuspended in 15 mL of PBS. Cell suspension was then diluted in a 3% dextran solution at a 1:3 ratio. After gentle homogenization, neutrophils were allowed to sediment for one hour. The upper layer, containing neutrophils, was collected and washed once in PBS. Red blood cells were lysed by incubation of the neutrophil pellet in a hypertonic solution (NH4Cl (0.077 M), EDTA (0.633 mM), KHCO3 (5 mM)). After an incubation of 5 min, PBS was added (to achieve 50 mL) in order to stop the reaction. Cells were centrifuged (300g, 3 min), and purified neutrophils were resuspended in complete medium (RPMI 1640-GlutaMAX-HEPES (Gibco), 10% fetal bovine serum (Gibco), 1X penicillin/streptomycin (Thermo-Fisher) , 1X nonessential amino acid (Lonza), 1X Sodium Pyruvate (Lonza)) and incubated at 37°C, 5% CO2. Neutrophils were further handled carefully to avoid any mechanical stress.

Human monocytes were isolated from fresh cytapheresis kit using a Ficoll-density gradient followed by positive CD14 + cell selection (human CD14 + MicroBeads, Miltenyi). CD14 + magnetic separation was performed according the manufacturer recommendations. For mixing experiments, apoptotic cells were harvested, centrifuged at 300g, 5 min, resuspended in X-Vivo15 (Gibco) and mixed with macrophages at a 5:1 ratio for the indicated period. MDM, cultured in a dedicated well, were previously harvested and counted to evaluate the number of apoptotic cells required for each experiment. After 6 h or 24 h, the non-engulfed apoptotic cells were then removed by extensive PBS washes, and macrophages were harvested by scrapping in a PBS-10 mM EDTA solution and further processed. For LPS stimulation experiments, apoptotic neutrophils were removed after 24 h and LPS (Lipopolysaccharides from Escherichia coli O55:B5, Sigma) was added in X-vivo15 medium to MDM at 100 ng/mL for 24 h.

Phenotyping and phagocytosis test

For phenotyping experiments, CD14 + cells were seeded in a 6-well plate at a concentration of 1 million cells per mL in 2.5mL. After 7 days of differentiation, MDM were harvested, washed in PBS and 100 000 cells were resuspended in 100 µL PBS-3% FCS with 2µL of the appropriate antibodies (see table 1). After 15 min incubation at 4°C in dark, cells were washed one time with PBS, and resuspended in PBS before flow cytometry analysis (SP6800 Spectral Analyzer, Sony or LSR Fortessa, Becton Dickinson).

For phagocytosis experiments, CD14 + cells were seeded in a 12-well plate at a concentration of 1 million cells per mL in 1mL. After 7 days of differentiation, MDM, cultured in a dedicated well, were counted to evaluate the number of neutrophils required for each mixing experiment.

Apoptotic cells were labelled with 10 µM CFSE (Invitrogen, C34554) for 10 min at 37°C or with pHrodo (Thermo-Fisher, P35372). Reaction was stopped by complete medium addition (to achieve 50mL), and cells were centrifuged at 300g, 3 min, resuspended in X-vivo15 and mixed at a 5:1 ratio with MDM. MDM were harvested 24 h later and labelled, as described above with a CD206-BV421 antibody. Gating strategy was specified in each appropriate experiment.

Viability assessment

Cells were harvested at the indicated time point, and 100 000 cells were resuspended in 100 µL of Annexin-V Binding Buffer (BD, 556454), incubated with 5µL of FITC-Annexin-V (BD, 556419) and 5µL of 7AAD (BD, 559925), for 15 min at room temperature in the dark. Cells were centrifuged at 300g, 5 min and resuspended in 100 µL of Annexin-V Binding Buffer. Viability was assessed by flow cytometry within one hour (SP6800 Spectral Analyzer, Sony or LSR Fortessa, Becton Dickinson). Gating strategy was: definition of a SSC vs. FSC gate > doublet discrimination > Annexin V-FITC vs. 7AAD dot plot.

Cytokine measurement by ELISA

Experiments were conducted in a 6-well plate containing a final volume of 2.5 mL of X-vivo15.

Two mL of supernatants were collected at the indicated time points and centrifuged 5 min at 300g to remove any cells. Samples were stored at -80°C while awaiting cytokine measurement.

ELISA were conducted according to manufacturer's instructions on 100 µL of supernatant. IL-6, TNFα, IL-10 and IL1-β kits were purchased from Biolegend and TGF-β kit from R&D Systems.

Microplates were read with a Sunrise absorbance microplate reader (Tecan) at 450 nm and results

were normalized to cell number.

RNA quality and Q-PCR experiments

Experiments were conducted in a 6-well plate. At the indicated time points, cells were harvested, centrifuged 5 min at 300g and the dry pellets were stored at -80°C, while awaiting RNA extraction. RNA was extracted with NucleoSpin® RNA Plus kit (Macherey-Nagel) according to manufacturer's instructions. RNA quantity was measured using a Nanodrop spectrophotometer (Thermo-Fisher). RNA quality was assessed using an Experion™ Automated Electrophoresis System according to manufacturer's instructions.

For Q-PCR experiments, RNA was first retrotranscribed using RevertAid H Minus First Strand cDNA Synthesis Kit with random primers, according to manufacturer's instructions (Thermo-Fisher, K1632). 12 µL of RNA template was engaged in each reverse transcription reaction. Q-PCR experiments were then conducted on a CXF96 real-time detection system (Biorad) using either Taqman probes (Elane probes, Hs00236952_m1 (Applied Biosystem) ; for one reaction: mastermix: 1µL of fluo mix probe, 10 µL of TaqMan Universal Master Mix II with UNG (Applied Biosystem), 5 µL H2O. DNA template: 4 µL) or Sybr Green (SsoAdvanced™ Universal SYBR® Green Supermix, (BioRad) based probes (CD66b, GAPDH, MT-ND6, IL-6, TNFα, CD3E and TRAC probes, see table 2) (for one reaction: mastermix: 10µL of syber green Supermix, 0,06 µL of each primer at 100 µM, 6µL H2O and 4µL of DNA template). GAPDH gene expression was used to normalize results when necessary.

Elastase assay

Elastase activity was assessed by Neutrophil Elastase (NE) Activity Assay Kit (BioVision, K383-100) according to manufacturer's instructions. Briefly, supernatants were harvested at the indicated time points and centrifuged at 300g, 5 min to remove any cells. 50 µL of cell culture supernatant was mixed with 50 µL of NE substrate mix. Fluorescence was read at 380 nm in kinetic mode for 10 min using EnVision® Xcite Multilabel Reader (PerkinElmer).

Statistical analysis

Unless stated otherwise, graph values are presented as means ± standard deviations, calculated on at least three independent experiments. Depending on the results of normality tests, statistical significance was determined using two-tailed Student's t-test, one-sample t-test or one-way ANOVA test with a Tukey's adjustment.

Results

Efferocytosis is induced in a co-culture of primary human apoptotic neutrophils

and primary human monocyte-derived macrophages.

The co-culture of AN and MDM was set up several decades ago [START_REF] Fadok | Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF[END_REF]. Nonetheless, some protocols, used here, may slightly differ from those used in the original articles. First, we have to determine if, in our hand and with our protocols, this model still recapitulate key features. Since human neutrophils enter spontaneous apoptosis in vitro [START_REF] Akgul | Molecular control of neutrophil apoptosis[END_REF], we first determined the optimal time point for the further efferocytosis experiments. Neutrophils were isolated from leukocyte-platelet concentrates using a Ficoll-density gradient followed by sedimentation in a 3% dextran solution.

A purity of nearly 92% was achieved using the method (Supplementary Fig. 1A). We then analysed their viability in a time course by flow cytometry after Annexin-V/7AAD staining (Fig. 1A). About 30% of freshly isolated neutrophils are apoptotic. After 13 h in culture, this percentage raise up to 77% and it slightly decreases at the later time points. On the other hand, the percentage of necrotic cells starts to increase at 24 h. The optimal time point for adding neutrophils to MDM culture to trigger efferocytosis is at 13 h after isolation, since it exhibits the highest percentage of apoptotic neutrophils (AN) and the lowest percentage of necrotic cells.

We then determined the efferocytosis efficiency in our co-culture system. Human macrophages were generated by M-CSF induced differentiation of human primary CD14 + cells during 7 days.

MDM phenotype was verified by flow cytometry using CD14, CD163, MERTK, CD80 and CD206 labelling to assess differentiation efficiency (Supplementary Fig. 1B). Generated MDM were subsequently co-cultured with CFSE-labelled AN for 6 or 24 h and labelled with CD206-BV421 antibody. Phagocytosis efficiency was assessed by flow cytometry (Fig. 1B). MDM containing at least one neutrophil was defined as CD206 + /CFSE + cells. After 6 h of co-culture, about 64% (±16.7%) of MDM have ingested at least one neutrophil and after 24 h, about 61% (±18%) (Fig. 1C-D). We checked that MDM viability is not impacted in this co-culture system (supplementary Fig. 1C). We also verified that AN are efficiently engulfed and do not just stick 3.2. Human primary neutrophils contain less mRNA than MDM and these mRNA are highly degraded.

In order to ensure that neutrophil mRNA cannot contaminate macrophage mRNA after efferocytosis, we determined first the mRNA quantity per cell in neutrophils and macrophages.

Freshly isolated and 13 h cultured neutrophils exhibit the same mRNA per cell ratio but macrophages contain 7.31 fold more mRNA per cell (Fig. 2A). We then analysed the detectability of these mRNA. We chose to quantify by Q-PCR two highly expressed mRNA arisen from the nuclear gene GAPDH and the mitochondrial gene MT-ND6. When normalized to the retrotranscribed mRNA quantity, GAPDH and MT-ND6 mRNA are respectively about 7700 and 5600 fold less detectable in AN compared to MDM (Fig. 2B,C). But GAPDH and MT-ND6 mRNA detectability remains equivalent in freshly isolated and in 13 h post-isolation cultured neutrophils (Fig. 2B,C). These experiments suggest that neutrophil mRNA are degraded even right after cell isolation.

To test this hypothesis, we checked mRNA quality using Experion Automated Electrophoresis System. Neutrophil mRNA, whatever the time point after neutrophil isolation, exhibits strong smears contrary to macrophage mRNA after electrophoresis (Fig. 2D). Moreover, the RNA quality indicator (RQI), measured on a scale from 1 to 10, is between 1.74 and 2.21 for neutrophil mRNA depending on the time point and is above 9 for macrophage mRNA (Fig. 2E).

This demonstrates that human primary neutrophil mRNA are severely degraded after isolation according to our protocol.

Collectively, these experiments argue that human primary neutrophils contain less mRNA than MDM, and that these neutrophil mRNA are less detectable due to extensive degradation, even in freshly isolated neutrophils, after a purification based on our protocol.

Human primary neutrophil specific mRNA are not detectable post-efferocytosis.

During efferocytosis, the engulfed corpse is trapped in a phagosome called efferosome which subsequently fuse with lysosome leading to apoptotic cell destruction [START_REF] Martin | Macrophages clean up: efferocytosis and microbial control[END_REF]. Nonetheless, to our knowledge, the fate of ingested mRNA during efferocytosis remains unknown. To investigate this, we performed a co-culture of MDM and AN to induce efferocytosis. Six or 24 h later, macrophage mRNA were extracted and submitted to Q-PCR analysis to detect two specific mRNAs, arisen from Elane and CD66b genes, which are highly expressed in neutrophils but not in macrophages [START_REF] Amanzada | Myeloperoxidase and elastase are only expressed by neutrophils in normal and in inflamed liver[END_REF][START_REF] Lakschevitz | Identification of neutrophil surface marker changes in health and inflammation using high-throughput screening flow cytometry[END_REF]. As mentioned in Fig. 1C, at least 57% (±15%) of MDM have engulfed at least one apoptotic neutrophil at 24 h. However, Elane (Fig. 3A) and CD66b (Fig. 3B) mRNA levels are comparable in MDM co-cultured or not with neutrophils. Moreover, we also checked the TNFα mRNA level in MDM. MDM differentiated with M-CSF express low level of TNFα mRNA, whereas AN contain large amount of it (about 90 fold more compared to MDM).

Nonetheless, ingestion of AN by MDM did not induce an increase of TNFα mRNA level in efferocytic MDM (Fig. 3C). These data indicate that neutrophil mRNA does not contaminate MDM mRNA after efferocytosis.

Since apoptotic human PBMC and apoptotic Jurkat cells are also commonly used as apoptotic corpse to trigger efferocytosis, we investigated the fate of specific mRNA expressed in these cells in efferocytic MDM. We first assessed that apoptosis is efficiently induced. Human PBMC were irradiated (X-rays) and exhibit about 29.4% ± 8.5 of cells in apoptosis, whereas Jurkat cells were treated with camptothecin and exhibits about 40% ± 7.4 of cells in apoptosis (Supplementary Fig. 3A). These cells are efficiently engulfed by MDM: 75.1% ± 9.4 of MDM have engulfed at least one apoptotic PBMC and 84.6% ± 5.7 of MDM at least one apoptotic Jurkat cell (Supplementary Fig. 3B). We then measured by Q-PCR the expression level of two specific T cell transcripts: CD3E and TRAC mRNA. Contrary to specific AN transcripts, specific apoptotic Jurkat cell transcripts were significantly enriched in efferocytic MDM. Specific apoptotic PBMC transcripts were also enriched in some samples although not reaching globally the significant threshold (Fig. 3D). These results indicate that performing transcriptomic studies in MDM postefferocytosis with apoptotic human PBMC or apoptotic Jurkat cells may be risky and should be avoided.

Allogenic apoptotic neutrophil and monocyte-derived macrophage co-culture did not induce cell activation

Since our co-culture system of MDM and neutrophils are collected from two different donors, we checked if this allogenic context did not artificially influence the activation status of these cells.

We first measured neutrophil activation by quantifying Elastase activity in culture supernatant.

Elastase is a specific enzyme stocked in azurophilic granules of neutrophils released upon cell activation. Extracellular Elastase activity is by the way representative of the neutrophil activation state [START_REF] Lominadze | Proteomic analysis of human neutrophil granules[END_REF]. When neutrophils were activated by PMA, we observed a strong Elastase activity in cell culture supernatant (Fig. 4A). However, no Elastase activity was detected when MDM were cultured alone neither after a 24 h incubation with neutrophils (Fig. 4A). These data demonstrate that when co-cultured with MDM, activation of allogenic human primary neutrophils is not triggered.

Then, we also monitored the impact of neutrophils on MDM activation. Allogenic co-culture may induce macrophage pro-inflammatory responses. To rule out this possibility, we quantified by ELISA the amount of TNFα and IL-6, two pro-inflammatory cytokines, in culture supernatant.

The amount of TNFα and IL6 released by MDM is comparable, whether macrophages are cultured alone or with allogenic neutrophils (Fig. 4B). This imply that MDM are not activated when co-incubated with primary neutrophils.

Collectively, these data emphasize that allogenic primary MDM and primary neutrophils coculture did not trigger an artifactual activation of any of these cells.

Efferocytosis efficiently reprograms human monocyte-derived macrophages

Quantification of pro-inflammatory cytokine secretion after LPS stimulation is commonly performed to assess the efficiency of MDM reprograming after efferocytosis [START_REF] Fadok | Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF[END_REF]. MDM were cocultured or not with AN for 24 h, AN were subsequently removed and MDM were stimulated with LPS for 24 h. We then measured, by ELISA, the release of TNFα in cell supernatant. MDM which have previously engulfed AN secrete 2.94 times less TNFα compared to MDM cultured without AN, demonstrating that MDM are less prone to Toll like receptor 4 stimulation after efferocytosis and are therefore reprogramed. We also checked the TNFα mRNA level to decipher whether efferocytosis can affect or not TNFα transcription. There is no significant difference in TNFα mRNA level whether MDM were previously co-cultured or not with AN, indicating that the lower released of TNFα after LPS stimulation post-efferocytosis is not transcriptiondependent (Supplementary Fig. 5).

Discussion and conclusion

In vitro systems where macrophages and apoptotic cells are co-cultured to study efferocytosis allowed to unravel many features of M R , like identification of some murine markers or secreted mediators for example [START_REF] Fadok | Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF[END_REF][START_REF] Kourtzelis | DEL-1 promotes macrophage efferocytosis and clearance of inflammation[END_REF][START_REF] Odaka | Murine macrophages produce secretory leukocyte protease inhibitor during clearance of apoptotic cells: implications for resolution of the inflammatory response[END_REF][START_REF] Dalli | Specific lipid mediator signatures of human phagocytes: microparticles stimulate macrophage efferocytosis and pro-resolving mediators[END_REF][START_REF] Luciani | The ATP binding cassette transporter ABC1, is required for the engulfment of corpses generated by apoptotic cell death[END_REF]. However, up to now, involved pathways and comprehensive molecular changes induced by efferocytosis remain puzzling in mice, and even more in humans. Global approaches, like RNA sequencing, may help to clear up some mechanistic insights. Nonetheless, these approaches may be impeded by few hurdles like apoptotic corpses mRNA cross-contamination or induced allogenic immune reactions. Some studies try to alleviate the risk of mRNA cross-contamination by setting up a model where apoptotic corpses and macrophages are obtained from two different species [START_REF] Morioka | Efferocytosis induces a novel SLC program to promote glucose uptake and lactate release[END_REF][START_REF] Park | Continued clearance of apoptotic cells critically depends on the phagocyte Ucp2 protein[END_REF]. Despite 20 being useful, in this model some but not all transcripts can be discriminated due to high sequence similarity, thus precluding part of efferocytosis triggered mRNA variations (18,[START_REF] Le | An evaluation of public genomic references for mapping RNA-Seq data from Chinese hamster ovary cells[END_REF]. A twospecies model may also hinder some physiological mechanisms since in the body, a macrophage only engulfs apoptotic corpses deriving from its own specie. The nature of the apoptotic corpses employed is often "non physiological". Irradiated Jurkat T cells or thymocytes are commonly used as apoptotic cell source [START_REF] Park | Continued clearance of apoptotic cells critically depends on the phagocyte Ucp2 protein[END_REF][START_REF] Luciani | The ATP binding cassette transporter ABC1, is required for the engulfment of corpses generated by apoptotic cell death[END_REF]. In these cells, apoptosis is artificially triggered with irradiation, and they do not represent the main cell type engulfed physiologically during the resolution phase of inflammation, which is mainly apoptotic blood-derived neutrophils. In this sense, the type of ingested corpses and the way to induce apoptosis may influence the macrophage response (discussed in [START_REF] Saas | Concise Review: Apoptotic Cell-Based Therapies -Rationale, Preclinical Results and Future Clinical Developments[END_REF]). If we add the fact that human primary neutrophils exhibit a much higher apoptotic rate, compared to apoptotic Jurkat cells or apoptotic PBMC, and that apoptosis is triggered without chemical treatment or irradiation, the use of AN seems preferable.

We shed light here on the feasibility of mRNA investigations in a long been used full human model of efferocytosis. Obviously, we did not claim that this system perfectly mimics the in vivo response. But so far, regarding the complexity of in vivo investigations in humans, the co-culture of human AN and MDM is the most accurate and the closest possible in vitro system to reflect what occurs in human body at the resolution phase of inflammation. It has also the great advantage to allow investigations in human M R , which is an overlooked model compared to its murine counterpart.

In this system, even in fresh neutrophils, isolated according to our experimental conditions, transcript amplification is tricky, including for highly expressed genes like GAPDH and MT-ND6. All transcripts detected exhibit a high cycle quantification value (Cq) (data not shown).

This is surprizing since many labs performed Q-PCR experiments and RNAseq studies on fresh human neutrophils [START_REF] Jiang | RNA sequencing from human neutrophils reveals distinct transcriptional differences associated with chronic inflammatory states[END_REF][START_REF] Tutino | Circulating neutrophil transcriptome may reveal intracranial aneurysm signature[END_REF]. However, our protocol is slightly different from their own. For instance, we did not process neutrophils within an hour but within 4-5 hours after blood collection, since we are working on fresh leukocyte-platelet concentrates. Some other subtle differences in blood collection, cell purification and RNA extraction protocols may explain why we observe degraded mRNA in our "fresh" neutrophils. Therefore, our results absolutely do not challenge the reliability of previously published work on mRNA investigations in human neutrophils. It just emphases that this neutrophil purification protocol is suitable for setting up a model of efferocytosis to limit neutrophil mRNA contamination in M R , but should be avoided for mRNA investigations in human primary neutrophils. Indeed, we demonstrate here that AN mRNA does not contaminate MDM mRNA. This absence of contamination can be due to the low RNA quantity and quality in neutrophils after a purification based on our extraction protocol.

Even if we did not observe any statistically significant increase of Elane and CD66b mRNA in MDM after efferocytosis, we still can detect an extremely low background (Figure 3). Moreover, a highly expressed mRNA in neutrophils, like TNFα mRNA, does not lead to an increase of basal TNFα mRNA level in MDM post-efferocytosis. Engulfed cells transit via efferosome were they are ultimately degraded, meaning that the low neutrophil mRNA quality is probably even more altered. Furthermore, even if Elane and CD66b mRNA are specific of neutrophils we can still occasionally detect few amplifiable Elane and CD66b transcripts in MDM cultured alone (Figure 3). Collectively, considering the tremendous discrepancies in mRNA quality and quantity between AN and MDM, it is very unlikely that the reminiscent Elane and CD66b mRNA levels reflect a contamination with neutrophil mRNA. On the contrary, when using apoptotic Jurkat cells or human apoptotic PBMC, we were able to detect some T-cell specific transcripts like Another pitfall in this system may be the question of DNA horizontal transfer. Indeed, several manuscripts demonstrate that some portions of the apoptotic cell DNA may be transferred to the phagocyte cell, leading to the expression of some apoptotic cell genes and promoting tumorigenesis. These results were obtained in other cellular models with apoptotic bodies expressing viral oncogenes (Epstein-Barr virus or human papillomavirus) [START_REF] Bergsmedh | Horizontal transfer of oncogenes by uptake of apoptotic bodies[END_REF][START_REF] Holmgren | Horizontal transfer of DNA by the uptake of apoptotic bodies[END_REF]. Nonetheless it seems that DNA transfer is lost if it does not confer a selective advantage to the recipient cell [START_REF] Yan | Regulation of mammalian horizontal gene transfer by apoptotic DNA fragmentation[END_REF]. In our model, even if we cannot completely exclude DNA transfer during efferocytosis, AN specific transcripts we studied are not expressed in MDM, suggesting that even if DNA transfer occur, it does not lead to significant neutrophil gene expression. Further experiments are required to investigate the potential occurrence of DNA transfer, even if it seems here unlikely.

The co-culture system, used here, was characterized several decades ago [START_REF] Fadok | Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF[END_REF][START_REF] Fadok | Differential effects of apoptotic versus lysed cells on macrophage production of cytokines: role of proteases[END_REF]. Nonetheless, since we changed some parameters like cell purification methods (CD14 + magnetic sorting versus selection by adherence…) or apoptosis induction (spontaneous apoptosis but not UV-irradiation), we verified that in our hands, this model recapitulates some previously characterized key features: 1) Efferocytosis is induced in this model. At 6 h, most of MDM uptake at least one neutrophil. Although non-significant, we observed a slight decrease of CSFE positive MDM between 6 and 24 h. This suggests that all MDM able to phagocyte already reached satiety at 6 h (Figure 1C). Partial lysosomal destruction of CFSE may be responsible for this minor decreased of CSFE positive macrophage frequency. Using pHrodo labelling we confirmed that AN are internalized and not just docked at MDM surface. By the way, the use of an imaging flow cytometer may bring a more direct proof of apoptotic corpse engulfment when using apoptotic cell CFSE labelling.

2) There is no artefactual cell activation in this allogenic co-culture system. Indeed human primary neutrophils are very fragile and can be activated by a simple mechanical stress for example. Neutrophil granule release, containing Elastase in particular, may induce macrophage activation (36). However, we did not observe any increase of Elastase activity nor macrophage pro-inflammatory cytokine secretion in co-culture cell supernatants (Figure 4A-C), thus ensuring that this system did not suffer from these pitfalls.

3) Macrophages are efficiently reprogramed by efferocytosis (Figure 5). Indeed efferocytic MDM are less prone to release pro-inflammatory TNFα upon LPS stimulation. This mechanism seems to be transcription-independent, since TNFα mRNA level is not affected by efferocytosis.

Alteration of TLR signalling may be involved in this phenomenon, but further experiments are required to test this hypothesis.

With this work, we demonstrated the superiority of the AN-MDM co-culture system compared to apoptotic PBMC/apoptotic Jurkat cells-MDM and we shed light on the proof-of-concept of mRNA investigation feasibility in this full human model of resolving macrophage, thus opening the door to a better characterization of this essential actor in human inflammation resolution.

Figure 1 .

 1 Figure 1. Characterization of apoptotic neutrophil/monocyte-derived macrophage coculture (A) Neutrophil viability assessment. Purified primary neutrophils were cultured for the

Figure 2 .

 2 Figure 2. Human primary neutrophils exhibit low degraded mRNA content. (A) Human primary neutrophils contain less mRNA quantity than MDM. Freshly isolated or 13 h postisolation (PI) human primary neutrophil and MDM mRNA is isolated and quantified with NanoDrop spectrophotometer and normalized to cell number. Values are means ± SD. n=3 for neutrophils points and n=4 for MDM. Two-tailed unpaired t-test. *: p≤0.05, ns: non-significant. (B-C) Human primary neutrophil mRNA is less amplifiable than MDM mRNA. Freshly isolated or 13 h PI human primary neutrophil and MDM mRNAs were isolated and retrotranscribed into cDNA. NT-ND6 (B) and GAPDH (C) mRNA are then amplified by Q-PCR and normalized to the mRNA quantity previously retrotranscribed. Values are means ± SD. n=3 for neutrophils and n=4 for MDM. Two-tailed unpaired t-test. ****: p≤0.0001, ns: non-significant (D) Human primary neutrophil mRNA is degraded. Freshly isolated or 13/24/48h PI human primary neutrophil and MDM mRNAs were isolated. Ladder (L), MDM and neutrophil mRNAs were separated using Experion Automated Electrophoresis System. Picture is representative of three independent experiments. (E) Quantification of Experion Automated Electrophoresis experiments. Values are means ± SD. n=7 for neutrophils and n=4 for MDM. One-way ANOVA with Tukey's post-hoc test. ****: p≤0.0001, ns: non-significant.

Figure 3 .

 3 Figure 3. Human primary neutrophil mRNA are not detectable in macrophages after efferocytosis. MDM were co-cultured with or without AN for 6 or 24h. Non-engulfed neutrophils were removed by 5 PBS washing. MDM were subsequently harvested by scrapping and mRNA was extracted and retrotranscribed into cDNA. Elane mRNA (A), CD66b mRNA (B) and TNFα mRNA (C) were quantified by Q-PCR. Results were normalized to GAPDH expression. Values are means ± SD. n=at least 3 independent experiments (A), n=at least 4 independent experiments (B-C), n= 3 independent experiments. One-way anova with Tukey's post-hoc test. *: p≤0.05, ****: p≤0.0001, ns: non-significant. (D) Some human primary Jurkat and PBMC mRNA can be detected in macrophages after efferocytosis. MDM were co-cultured with or without apoptotic PBMC and apoptotic Jurkat cells for 24h. Non-engulfed apoptotic corpses were removed by 5 PBS washing. MDM were subsequently harvested by scrapping and mRNA was extracted and retrotranscribed into cDNA. TRAC mRNA (Left panel), CD3E mRNA (right panel) were quantified by Q-PCR. Results were normalized to GAPDH expression. Values are means ± SD. n= 3 independent experiments. Two-tailed unpaired t-test. *: p≤0.05, ns: non significant.

Figure 4 .

 4 Figure 4. Human primary neutrophil and MDM co-culture does not induce allogenic reactions. (A) Human primary neutrophils are not activated by co-culture with MDM. MDM are co-cultured with or without AN for 24 h. AN were stimulated with PMA (100nM, 1 h at 37°C) as a positive control. Elastase activity in cell supernatant was quantified using a fluorimetric test and normalized to 1 million cells. Values are means ± SD. n=at least 3 independent experiments. One-way ANOVA with Tukey's post-hoc test. ****: p≤0.0001. (B-C) MDM-AN did not induce MDM pro-inflammatory responses. MDM are co-cultured with or without AN for 24 h. Cell supernatants were collected and centrifuged at 300 g 5 min to remove any cells. IL6 (B) and TNFα (C) proteins were quantified by ELISA. Results were normalized to 1 million cells. Values are means ± SD. n=3. Two-tailed unpaired t-test. ****: p≤0.0001, ns: non-significant.

Figure 5 .

 5 Figure 5. MDM are efficiently reprogramed by efferocytosis. MDM are co-cultured with or without AN for 24h. AN were then removed by PBS washing and MDM were stimulated with LPS (100 ng/mL for 24 hours). After LPS stimulation, cell supernatants were collected and centrifuged at 300 g, 5 min to remove any cells. TNFα proteins were quantified by ELISA. Results were normalized to MDM values for each donor. Values are means ± SD. n=3. Onesample t-test. **: p≤0.01.

CD3E

  and TRAC mRNA in MDM after efferocytosis. Indeed, these transcripts are highly expressed even in apoptotic PBMC and apoptotic Jurkat T cells, exhibiting low quantification cycle values (data not shown), and are easily amplified by Q-PCR. In contrast, as stated above, most neutrophil transcripts exhibit a low quantification cycle values, even with usually highly expressed genes like NT-ND6 and GAPDH. The discrepancies of these Cq values between PBMC and Jurkat cell, on one side, and AN, on the other side, may reside in RNA quality and quantity differences and most probably explain why AN mRNA are not detectable in efferocytic macrophages, contrary to some PBMC and Jurkat mRNA. This clearly establishes the potential risk of mRNA contamination in MDM post-efferocytosis, when using apoptotic human PBMC and apoptotic Jurkat T cells. Therefore, we discourage the use of these cells as apoptotic corpse in any transcriptomic studies of efferocytic MDM. The use of AN extracted from leukocyteplatelet concentrate must be prioritized for this application.

Table 1 :

 1 list of antibodies

	Antibodies	Supplier	Cat No
	CD16-APC	Miltenyi Biotec	130-106-763
	CD15-FITC	Sony	2109520
	PE-CF594-CD14	BD Biosciences	562335
	Alexa647-CD163	BD Biosciences	562669
	BV421-CD80	Sony	2126110
	BB515-CD206	BD Biosciences	564668
	PE-hMER	Sony	2438040
	BV421-CD206	BD Biosciences	564062

Table 2 :

 2 list of primers used in Q-PCR experiments

	Target	Sequence	specificity
	CD66b forward	TCAAAGCATTTGCAATCAGC	human
	CD66b reverse	GTGGGCAACTTCACAAAGGT	human
	GAPDH forward	GAA GGT GAA GGT CGG AGT C	human
	GAPDH reverse	GAA GAT GGT GAT GGG ATT TC	human
	TNF forward	AGGACCAGCTAAGAGGGAGA	human
	TNF reverse	CCCGGATCATGCTTTCAGTG	human
	IL6 forward	CACTGGCAGAAAACAACCTG	human
	IL6 reverse	TGGCTTGTTCCTCACTACTCT	human
	MT-ND6 forward	TGATTGTTAGCGGTGTGGTC	human
	MT-ND6 reverse	CCACAGCACCAATCCTACCT	human
	TRAC forward	ATTGCCCCTCTTCTCCCTCT	human
	TRAC reverse	GCAGTGTTTGGCAGCTCTTC	human
	CD3E forward	GTTGTCCCCCATCCCAAAGT	human
	CD3E reverse	GGTGGAGGGAGTAGGGGATT	human

  to MDM surface by labelling AN with pHrodo dye, a pH-sensitive dye non-fluorescent at neutral pH and brightly fluorescent in acidic environments. This labelling allows to discriminate internalized AN from AN bound to the cell surface. We obtain no statistical difference when AN are labelled with CFSE or with pHrodo (68.1% ±10.21 at 6 h and 80.03% ± 6.43 at 24 h of MDM were CD206 + /pHrodo + ; 80.13% ±3.8 at 6 h and 81.5% ±6.63 at 24 h of MDM were CD206 + /CFSE + ), indicating that AN were engulfed and processed into phagosomes (Supplementary Fig.2A, 2B and 2C). Moreover, these results indicate that CFSE labelling of AN allows the accurate assessment of phagocytosis efficiency. These data collectively show that efferocytosis is efficiently triggered in this co-culture.

Jaffray, C. 2000. Specific pancreatic enzymes activate macrophages to produce tumor necrosis factor-alpha: role of nuclear factor kappa B and inhibitory kappa B proteins,. J. Gastrointest. Surg. 4: 370-378.

Acknowledgments

We would like to thank all the members of the UMR1098 for helpful and stimulating discussions and in particular Dr. F. Bonnefoy and Dr. M.Couturier.

Funding

This work was supported by grants from the Agence Nationale de la Recherche (Labex LipSTIC, ANR-11-LABX-0021) and the Région de Bourgogne Franche-Comté (support to Labex LipSTIC [to PS] 2017 and to obtain the SH800 cell sorter [Sony]). TC received financial support from the Région de Bourgogne Franche-Comté (postdoctoral fellowship). MM received a PhD grant from the University of Bourgogne Franche-Comté.

Competing and declaration of interests:

The data presented in this manuscript are original, and have not been published or submitted elsewhere. All listed authors have approved the manuscript and agreed with the submission. In addition, we declare no financial interest related to this work.


Kelly Virecoulon Giudici
email: kellygiudici@gmail.com

Philipe De

PhD Souto Barreto

MD, PhD John Beard

Christelle Cantet

MD Islene Araujo De Carvalho

MD, PhD Yves Rolland

MD, PhD Bruno Vellas

PhD Kelly Virecoulon Giudici

Mapt / Dsa

Effect of long-term omega-3 supplementation and a lifestyle multidomain intervention on intrinsic capacity among community-dwelling older adults: Secondary analysis of a randomized, placebo-controlled trial (MAPT study)

Keywords: intrinsic capacity, cognition, functional performance, Alzheimer's, clinical trials, aging Abbreviations ω-3, omega-3, AD, Alzheimer's disease, ANOVA, analysis of variance, DHA, docosahexaenoic acid, EPA, eicosapentaenoic acid, GDS, Geriatric Depression Scale, IC, intrinsic capacity, MAPT, Multidomain Alzheimer Preventive Trial, MET, metabolic equivalent of task, MI, multidomain intervention, MMSE, Mini-Mental State Examination, PUFA, polyunsaturated fatty acids, SAS, Statistical Analysis Software, SPPB, Short Physical Performance Battery, WHO, World Health Organization

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The World Health Organization (WHO) has recently proposed a novel healthcare model for older people focusing on functional rather than disease-based perspectives. This approach is built around the construct of intrinsic capacity (IC), which is characterized as the composite of all of the physical and mental capacities of an individual (1). It has been suggested that IC includes five key domains: locomotion, vitality, cognition, psychological and sensory [START_REF] Cesari | Evidence for The Domains Supporting The Construct of Intrinsic Capacity[END_REF].

Clinical approaches aiming to delay the incidence and evolution of age-related functional decline and, therefore, to increase quality of life, are imperative in the present context of populational aging -the global population aged ≥60 years has doubled in the past four decades (from about 382 million in 1980 to 962 million in 2017), and is expected to double again by 2050, when it is projected to reach nearly 2.1 billion [START_REF]World Population Prospects: The 2017 Revision[END_REF]. The IC construct may provide new opportunities for identifying effective interventions to ensure these growing populations of older people will experience the healthiest ageing possible. However, while the IC concept has been theoretically proposed, there is only limited empirical evidence supporting how IC levels might be affected by specific health interventions. Some trials promoting lifestyle interventions [START_REF] Van Charante | Effectiveness of a 6-year multidomain vascular care intervention to prevent dementia (preDIVA): a cluster-randomised controlled trial[END_REF][START_REF] Lehtisalo | Dietary changes and cognition over 2 years within a multidomain intervention trial-The Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability (FINGER)[END_REF][START_REF] Bae | The effect of a multicomponent intervention to promote community activity on cognitive function in older adults with mild cognitive impairment: A randomized controlled trial[END_REF] or omega-3 (ω-3) polyunsaturated fatty acids (PUFA) supplementation [START_REF] Van De Rest | Effect of fish oil on cognitive performance in older subjects: a randomized, controlled trial[END_REF][START_REF] Tajalizadekhoob | The effect of low-dose omega 3 fatty acids on the treatment of mild to moderate depression in the elderly: a double-blind, randomized, placebocontrolled study[END_REF][START_REF] Sinn | Effects of n-3 fatty acids, EPA v. DHA, on depressive symptoms, quality of life, memory and executive function in older adults with mild cognitive impairment: a 6-month randomised controlled trial[END_REF][START_REF] Smith | Fish oil-derived n-3 PUFA therapy increases muscle mass and function in healthy older adults[END_REF][START_REF] Baleztena | Association between cognitive function and supplementation with omega-3 PUFAs and other nutrients in ≥ 75 years old patients: A randomized multicenter study[END_REF] (known for its anti-inflammatory effects) have been performed in order to verify potential benefits in cognitive performance [START_REF] Van Charante | Effectiveness of a 6-year multidomain vascular care intervention to prevent dementia (preDIVA): a cluster-randomised controlled trial[END_REF][START_REF] Lehtisalo | Dietary changes and cognition over 2 years within a multidomain intervention trial-The Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability (FINGER)[END_REF][START_REF] Bae | The effect of a multicomponent intervention to promote community activity on cognitive function in older adults with mild cognitive impairment: A randomized controlled trial[END_REF][START_REF] Van De Rest | Effect of fish oil on cognitive performance in older subjects: a randomized, controlled trial[END_REF][START_REF] Baleztena | Association between cognitive function and supplementation with omega-3 PUFAs and other nutrients in ≥ 75 years old patients: A randomized multicenter study[END_REF], depression prevention [START_REF] Sinn | Effects of n-3 fatty acids, EPA v. DHA, on depressive symptoms, quality of life, memory and executive function in older adults with mild cognitive impairment: a 6-month randomised controlled trial[END_REF] or treatment [START_REF] Tajalizadekhoob | The effect of low-dose omega 3 fatty acids on the treatment of mild to moderate depression in the elderly: a double-blind, randomized, placebocontrolled study[END_REF], and physical function [START_REF] Smith | Fish oil-derived n-3 PUFA therapy increases muscle mass and function in healthy older adults[END_REF] over aging, but no trial has yet examined the effect on declines in IC as a whole. Such analysis would provide a fuller assessment of the influence of these interventions on healthy aging.

Based on this novel concept proposed by the WHO, this non pre-specified secondary analysis of the Multidomain Alzheimer Preventive Trial (MAPT) aimed to investigate the effect of a 3-year intervention of ω-3 polyunsaturated fatty acid supplementation and a multidomain intervention (composed by physical activity counselling, cognitive training and nutritional advice), combined or alone, on IC decline among community-dwelling older adults without major neurocognitive disorders. We hypothesized that ω-3 supplementation and a lifestyle multidomain intervention may slow down age-related IC declines over time among older adults, compared to placebo.

Methods

The MAPT Study was a multicentre, randomized, placebo-controlled trial, originally created to investigate the efficacy of a 3-year ω-3 PUFA supplementation and a multidomain intervention (nutritional counseling, physical activity advice and cognitive training) in preventing cognitive decline. MAPT was launched in May 2008 and followed a total of 1,680 individuals over 3 years. The final study visit was completed by 1,286 participants (76.5%) [START_REF] Vellas | MAPT Study: a multidomain approach for preventing Alzheimer's disease: design and baseline data[END_REF][START_REF] Andrieu | Effect of longterm omega 3 polyunsaturated fatty acid supplementation with or without multidomain intervention on cognitive function in elderly adults with memory complaints (MAPT): a randomised, placebo-controlled trial[END_REF].

Ethical disclosure

Volunteers were recruited by the investigating physicians, and signed an informed

Randomization

Participants were randomly assigned (1:1:1:1) to one of the following groups: multidomain intervention plus ω-3 supplementation; multidomain intervention plus placebo; ω-3 supplementation only; placebo only. Briefly, a computer-generated randomization procedure was used with block sizes of eight and stratification by center. A clinical research assistant (not involved in the assessment of participants) used a centralized interactive voice response system to allocate participants in each group; randomization concealment was guaranteed. All participants and study staff were blinded to ω-3 or placebo assignment (both sets of capsules looked and tasted identical; placebo capsules contained flavored paraffin oil).

Due to the nature of the multidomain intervention, this component was not blinded in the study, but outcome assessors were blinded to group assignment. Full description of the randomization procedure has been previously described [START_REF] Andrieu | Effect of longterm omega 3 polyunsaturated fatty acid supplementation with or without multidomain intervention on cognitive function in elderly adults with memory complaints (MAPT): a randomised, placebo-controlled trial[END_REF].

Intervention

The multidomain intervention consisted of preventive consultations with a physician (at baseline, 12 and 24 months) and of group sessions. In the first two months of the trial, a total of 12 small group 2-hour sessions were done (two sessions per week in the first month of follow-up, and one session per week in the second). Each session comprised cognitive stimulation (60 minutes of reasoning and memory training), physical activity (45 minutes of advice and demonstrations of exercising), and nutritional counseling (15 minutes based on guidelines established by the Programme National Nutrition Santé -the French National Nutrition and Health Programme) [START_REF] Hercberg | The French National Nutrition and Health Program: 2001-2006-2010[END_REF]. From the third month on, participants in the multidomain intervention groups monthly attended a 1-hour session to reinforce the key messages. Furthermore, 2-hour sessions were held at 12 and 24 months of follow-up.

Omega-3 PUFA supplementation consisted of a daily intake of two capsules of the active supplement V0137, an oil blend containing natural fish oil with minimally 65% of docosahexaenoic acid (DHA) and maximally 15% of eicosapentaenoic acid (EPA), leading to a dose per capsule of 400 mg of DHA and ≤ 112.5 mg of EPA. The total daily dose represented a level that exceeded the daily recommended intake (i.e., 250 mg of DHA and 250 mg of EPA), without exceeding the maximum daily intake limit of 2,000 mg (according to the French Food Safety Authority). All participants were allowed to continue consuming fish and other natural sources of ω-3 during the trial. Placebo capsules were made of flavored paraffin oil, and tasted like the others.

Adherence to the interventions was assessed every 6 months. For ω-3 supplementation, the number of capsules returned by participants was counted. For the multidomain intervention, adherence was based on the percentage of attended sessions.

Participants were considered adherent by having an attendance rate of at least 75% in group sessions and/or taking at least 75% of the prescribed capsules, as appropriate. Further details can be found in Andrieu at al. [START_REF] Andrieu | Effect of longterm omega 3 polyunsaturated fatty acid supplementation with or without multidomain intervention on cognitive function in elderly adults with memory complaints (MAPT): a randomised, placebo-controlled trial[END_REF].

Outcome: intrinsic capacity

Four IC domains (cognition, locomotion, psychological and vitality) were evaluated to create a unique outcome of IC. Cognition was assessed by a composite Z-score based on the sum of Z-scores from four cognitive tests divided by 4: the ten orientation items of the Mini-Mental State Examination, Digit Symbol Substitution Test, free and total recall of the Free and Cued Selective Reminding test, and Category Naming Test [START_REF] Andrieu | Effect of longterm omega 3 polyunsaturated fatty acid supplementation with or without multidomain intervention on cognitive function in elderly adults with memory complaints (MAPT): a randomised, placebo-controlled trial[END_REF]. Combined, these tests assess episodic memory, timed executive function, and global cognition. Locomotion was evaluated by the Short Physical Performance Battery (SPPB), an instrument evaluating mobility aspects (chair stand test, usual gait speed test and total balance test) with scores varying from 0 to 12 (in which higher is better) [START_REF] Guralnik | A short physical performance battery assessing lower extremity function: association with self-reported disability and prediction of mortality and nursing home admission[END_REF]. The psychological domain was measured by the Geriatric Depression Scale (GDS), a 15-item questionnaire able to identify depressive symptoms in older adults and whose scale varies from 0 to 15 points (higher is worse) [START_REF] Sheikh | Geriatric Depression Scale (GDS): recent evidence and development of a shorter version[END_REF]. Vitality, a domain that can be considered to reflect physiologic reserves, was defined as hand grip strength (assessed in kg by a handheld dynamometer), a vital sign also used to build this domain by Beard at al. [START_REF] Beard | The structure and predictive value of intrinsic capacity in a longitudinal study of ageing[END_REF].

A composite IC Z-score was defined as the sum of each domain's Z-scores (standardised with baseline means and standard deviation for each test from the intention-totreat population) divided by 4, with the psychological domain (GDS score) weighted as -1 (since higher scores indicate domain impairment, unlike the others), and all others weighted as 1. All measurements were performed by trained health professionals (neuropsychologists, physicians and nurses), and variables were assessed at baseline, 6, 12, 24 and 36 months of follow-up. Analyses were performed on a modified intention-to-treat basis (ie, including all randomly assigned participants with a cognitive composite score at baseline who completed at least one post-baseline visit), whose details are described elsewhere [START_REF] Andrieu | Effect of longterm omega 3 polyunsaturated fatty acid supplementation with or without multidomain intervention on cognitive function in elderly adults with memory complaints (MAPT): a randomised, placebo-controlled trial[END_REF].

Statistical analysis

Original sample size calculation for MAPT Study can be found in [START_REF] Andrieu | Effect of longterm omega 3 polyunsaturated fatty acid supplementation with or without multidomain intervention on cognitive function in elderly adults with memory complaints (MAPT): a randomised, placebo-controlled trial[END_REF]. From the 1,680 participant originally included, one participant in the polyunsaturated fatty acid group withdrew consent, and 154 had no cognitive assessment available after baseline. In addition, due to missing values for at least one IC domain in baseline, 80 subjects were not included in the present study, leading to a final sample of 1,445 individuals.

Descriptive statistics (means and standard deviation; frequencies and percentages) was used to characterize the study population. The normality of the distribution of each variable was assessed with the Shapiro-Wilk test, and logarithmic transformation was used if necessary. Linear mixed effects regression analyses (with a random intercept and a random slope at the level of the participant and random intercept for the center effect) were used to determine the effect of interventions (multidomain intervention plus ω-3; multidomain intervention plus placebo; or ω-3 supplementation alone) compared to placebo on IC Z-score variation over the 3-year follow-up. Maximum likelihood tests were used to assess the linearity of trajectories by testing terms for time² and time³, which were retained in the model due to being significant. Time was used as a continuous variable, and fixed effects included in the model were: randomized groups (multidomain intervention plus ω-3; multidomain intervention plus placebo; ω-3 supplementation alone; and placebo alone), time, time² and time³, and the interactions between randomization group and time, time² and time³. P-values were adjusted for multiple comparisons with the Hochberg procedure to account for the fact that three intervention groups were compared with the placebo group in each analysis.

Sensitivity analysis considering time as a categorical variable was performed (with a random intercept at the level of the participant and the centers). In addition, sensitivity analysis merging the two multidomain groups (multidomain intervention plus ω-3 and multidomain intervention plus placebo) and the two non-multidomain intervention groups (ω-3 supplementation alone and placebo alone) was performed, as also another merging the two ω-3 groups and the two placebo groups. We also performed another sensitivity analysis restricted to the per-protocol population, which excluded all participants who did not fulfil the inclusion or exclusion criteria, those who did not receive at least one dose of ω-3 supplement or who did not attend at least one multidomain intervention session; those who did not receive the intervention they were allocated to, changed treatment arm during the trial (i.e., crossover), or took ω-3 supplements from other sources during follow-up. Analyses were performed with the Statistical Analysis Software (SAS) version 9.4 (Cary, NC, USA), and results were considered significant if p<0.05.

Results

Characterization of the sample

Adherence to intervention and baseline characteristics of participants according to randomized groups are presented in Table 1. From the total of 1,445 subjects (mean age 75.3 years, SD = 4.4), 64.2% (n = 927) were female.

Intrinsic capacity Z-score variation according to intervention groups

After 3 years, IC Z-score decreased among all groups, but no between-group differences (comparing each intervention group with participants taking placebo) were found (Table 2). In sensitivity analysis considering time as a categorical variable, significant withingroup declines on IC Z-score were first identified at 24 months for all groups. Between-group differences of each intervention group compared with participants taking placebo were not significant (Table 3). Results remained similar in sensitivity analysis restricted to the perprotocol population (n = 1,360) (Supplementary Table 1).

Sensitivity analysis merging the two multidomain intervention groups and the other two non-multidomain intervention groups found a decrease in IC Z-score after 3 years in both (multidomain intervention groups: -0.14, CI: -0.19 to -0.10; p < 0.0001; non-multidomain intervention groups: -0.19, CI: -0.24 to -0.15; p < 0.0001), however no significant betweengroup mean differences were observed (0.05, 95% CI: -0.01 to 0.11; p = 0.115) (Supplementary Table 2). Similarly, sensitivity analysis merging the two ω-3 supplementation groups and the other two placebo groups found no significant between-group mean differences (-0.01, 95% CI: -0.07 to 0.05; p = 0.722) (Supplementary Table 3).

Discussion

This study found that a multidomain lifestyle intervention (based on physical activity counselling, cognitive training and nutritional advice) and ω-3 supplementation, combined or alone, had no significant effect on preventing IC decline among community-dwelling older adults without major neurocognitive disorders over a 3-year follow-up. Within-group results from the sensitivity analysis using time as a categorical variable showed that IC score decrease was first identified at 24 months, among all groups.

The concept of IC has emerged as an innovative approach in health care [START_REF] Cesari | Evidence for The Domains Supporting The Construct of Intrinsic Capacity[END_REF].

Accordingly, increased interest in identifying predictors of IC decline has brought attention to potential protective factors associated with healthy aging. In this sense, trials promoting lifestyle changes, focused on physical activity, cognitive function stimulation and/or good nutritional habits, despite some inconclusive findings (4, [START_REF] Andrieu | Prevention of sporadic Alzheimer's disease: lessons learned from clinical trials and future directions[END_REF], have been associated with slower cognitive and/or functional decline [START_REF] Lehtisalo | Dietary changes and cognition over 2 years within a multidomain intervention trial-The Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability (FINGER)[END_REF][START_REF] Bae | The effect of a multicomponent intervention to promote community activity on cognitive function in older adults with mild cognitive impairment: A randomized controlled trial[END_REF]; such previous findings open the way to further exploring the effects of multidomain lifestyle interventions on combined IC domains.

In the present study, our intervention did not have an effect in a combined IC outcome.

Previous analyses were either unable to show any effect of our intervention in cognition, locomotion and psychological domains, separately [START_REF] Hercberg | The French National Nutrition and Health Program: 2001-2006-2010[END_REF]. The fact that MAPT interventions were characterized by low-intensity procedures (in particular for physical activity, which was not composed of exercise training sessions, but simply on counselling) and targeting mostly cognitive function may have contributed to this finding. It is possible that stronger interventions (e.g. with exercise training and controlled diet) are needed to identify an impact on IC trajectories. Moreover, the constitution of the IC Z-score was constrained by data availability. It is also possible that MAPT population was composed of globally healthy subjects, for whom the 3-year time frame, although being a large interval for behavioral interventions, was not large enough to detect important declines on IC domains.

In sensitivity analysis comparing merged multidomain intervention groups vs. merged non-multidomain intervention groups, no between-group differences on IC Z-score decline were found at the end of follow-up. Although not observing a protective effect of intervention on IC, specificities on IC Z-score variations in the present study should be noted. In the main analysis and in all sensitivity analyses, declines in IC score were lower in the multidomain groups. It should be considered that, as previously published, the multidomain groups of MAPT had a better 3-year evolution of physical activity than the placebo group, with a significant and clinically meaningful between-group difference, equivalent to about 70 minutes per week of brisk walking, reached by the multidomain plus placebo group [START_REF] Barreto P De | Effects of multidomain lifestyle intervention, omega-3 supplementation or their combination on physical activity levels in older adults: secondary analysis of the Multidomain Alzheimer Preventive Trial (MAPT) randomised controlled trial[END_REF]; this may have contributed to our findings. Indeed, exercise trials with older adults have shown the effect of exercising in improving functional performance [START_REF] Seguin | Improved physical fitness among older female participants in a nationally disseminated, community-based exercise program[END_REF][START_REF] Pahor | Effect of structured physical activity on prevention of major mobility disability in older adults: the LIFE study randomized clinical trial[END_REF][START_REF] Rodriguez-Mañas | Effectiveness of a multimodal intervention in functionally impaired older people with type 2 diabetes mellitus[END_REF], hand grip strength [START_REF] Htut | Effects of physical, virtual reality-based, and brain exercise on physical, cognition, and preference in older persons: a randomized controlled trial[END_REF], cognitive function [START_REF] Htut | Effects of physical, virtual reality-based, and brain exercise on physical, cognition, and preference in older persons: a randomized controlled trial[END_REF][START_REF] Bademli | Effects of Physical Activity Program on cognitive function and sleep quality in elderly with mild cognitive impairment: A randomized controlled trial[END_REF], and reducing depressive symptoms [START_REF] Catalan-Matamoros | Exercise improves depressive symptoms in older adults: An umbrella review of systematic reviews and meta-analyses[END_REF][START_REF] Von Berens | Effect of exercise and nutritional supplementation on health-related quality of life and mood in older adults: the VIVE2 randomized controlled trial[END_REF], i.e. the components of our IC Z-score. This topic deserves further investigation and should be a target of future research.

Although we were not able to show a protective effect of ω-3 supplementation in intrinsic capacity, multiple pathways sustain the hypothesis on how ω-3 would possibly act in this sense, however there is still a lack of conclusive evidence in this scenario. Despite the recognized capacity of ω-3 in improving vascular endothelial function and arterial stiffness (both important mechanistic determinants of cerebral blood flow), studies evaluating ω-3 administration and cognitive performance among adults and older adults had provided mixed evidence so far [START_REF] Joris | Cerebral Blood Flow Measurements in Adults: A Review on the Effects of Dietary Factors and Exercise[END_REF][START_REF] Derbyshire | Brain Health across the Lifespan: A Systematic Review on the Role of Omega-3 Fatty Acid Supplements[END_REF]. Other abilities of ω-3 are believed to help fight depressive symptoms, as interacting with serotonergic and dopaminergic neurotransmission through influencing membrane fluidity and regulating signal transduction in brain cells [START_REF] Grosso | Omega-3 fatty acids and depression: scientific evidence and biological mechanisms[END_REF]. The protective role of ω-3 PUFA on functional performance, in turn, is thought to be owed to its ability to reduce mitochondrial oxidant emissions, increase post absorptive muscle protein synthesis, and enhance anabolic responses to exercise, as shown in a study with older adults [START_REF] Lalia | Influence of omega-3 fatty acids on skeletal muscle protein metabolism and mitochondrial bioenergetics in older adults[END_REF]. In accordance, evidence linking ω-3 and increased muscle strength after supplementation among older adults [START_REF] Smith | Fish oil-derived n-3 PUFA therapy increases muscle mass and function in healthy older adults[END_REF] underlies how this nutrient could impact vitality. Although long-term effects were not observed among our sample, these mechanisms, taken together, illustrate how ω-3 supplementation may help protecting IC and encourage further exploring.

To the best of our knowledge, this is the first study to investigate the potential effect of lifestyle intervention and ω-3 supplementation (combined or alone) in the variation of IC over 3 years among community-dwelling older adults. Evaluating the complex construct of intrinsic capacity based on multiple outcomes combed into a composite Z-score allowed having an important panorama of functional reservoir among older people. The 3-year followup included multiple assessments of outcomes, enabling to closely follow variation in IC over time, as also to explore long-term associations. As a limitation of the study, it should be noted that MAPT study was originally designed to test the effectiveness of interventions in cognitive function, not in all IC domains. In addition, information regarding the sensory domain of IC was not available for the population investigated, so was not included in the IC outcome. As typically observed in clinical trials, adherence decreased with time. Lastly, MAPT participants were particularly highly educated (what suggests a high level of cognitive reserve) and with high physical activity levels, so findings should be cautiously compared with more vulnerable elderly populations.

Conclusions

Our study showed that a 3-year ω-3 supplementation and multidomain lifestyle intervention had no effect on intrinsic capacity compared to placebo among a sample of communitydwelling French older adults without major neurocognitive disorders. In spite of the negative results, the novel approach of this secondary analysis should encourage future studies focused on investigating potential protective factors of intrinsic capacity, especially trials providing stronger interventions (as exercise training and controlled diet) and embracing all five intrinsic capacity domains, in order to set the way for evidence-based preventive strategies in clinical care and public health. All authors read and approved the final manuscript submitted for publication.

Contributors

Highlights

• Intrinsic capacity (IC) is the composite of all the physical and mental capacities of an individual.

• The intrinsic capacity Z-score has four domains: locomotion, cognition, psychological and vitality.

• Changes in intrinsic capacity were evaluated during and after a 3-year intervention comprising nutritional and physical activity advice, cognitive training and omega-3 supplementation.

• After 3 years, intrinsic capacity Z-score had decreased among all groups. This trial did not find effects of the intervention on the composite intrinsic capacity Z-score.

• Declines in the intrinsic capacity score were first seen after 24 months.

  consent. The trial protocol (NCT00672685) was approved by the Advisory Committee for the Protection of Persons participating in Biomedical Research of the Toulouse University Hospital (CPP SOOM II) and was authorized by the French Health Authority. Study population Inclusion criteria for joining MAPT study comprised: being ≥ 70 years old; with a Mini-Mental State Examination (MMSE) score ≥ 24 (i.e., without major neurocognitive disorders); presenting at least one of the following criteria: spontaneous memory complaint expressed to their general practitioner, inability to carry out an instrumental activity of daily living, or slow walking speed (<0.8 m/s measured by a 4-meter usual walking test). Subjects in use of ω-3 supplements (extra-dietary) over the last 6 months prior to inclusion and/or taking ω-3 at that moment were not included. Data collection was held between May 2008 and February 2011 (recruitment) until March 2014 (end of the 3-year follow-up) in 13 French health centers participating in the study.

Kelly

  Virecoulon Giudici conceived and designed the present study, performed the review of related literature and statistical analyses, and wrote the article. Philipe de Souto Barreto conceived and designed the present study, and contributed to the statistical analyses, interpreting data and revising the article critically for important intellectual content. John Beard conceived and designed the present study, and contributed to data interpretation and revising the article critically for important intellectual content. Christelle Cantet contributed to the statistical analyses, interpreting data and revising the article critically for important intellectual content. Islene Araujo de Carvalho contributed to data interpretation and revising the article critically for important intellectual content. Yves Rolland gathered data and contributed to data interpretation and revising the article critically for important intellectual content. Bruno Vellas conceived the main MAPT project, conceived and designed the present study, gathered data, and contributed to data interpretation and revising the article critically for important intellectual content.

Table 1 .

 1 Characteristics of the studied sample and adherence to intervention according to randomized groups of MAPT study.

	Total	Mutidomain intervention + ω-3 supplementation	Mutidomain intervention + placebo	ω-3 supplementation	Placebo
	n = 1445	n = 356	n = 374	n = 359	n = 356
	mean (SD)*	mean (SD)*	mean (SD)*	mean (SD)*	mean (SD)*

Table 2 .

 2 Mixed-effect linear regression analysis for 3-year change from baseline in intrinsic capacity Z-score according to randomized intervention groups among community-dwelling older adults without major neurocognitive disorders (time considered as a continuous variable).

	Estimated mean within-group 3-year change from baseline † (95%
	CI); P-

value Group difference in intrinsic capacity Z-score between each intervention group and the placebo group after 3 years

						Multidomain plus ω-3	Multidomain plus placebo vs.	ω-3 supplementation vs.
						supplementation vs. placebo		placebo			placebo
		Multidomain plus 3 supplementation	Multidomain plus placebo	ω-3 supplementation	Placebo	Mean group difference (95% CI)	Raw P-value	Adjusted P-value*	Mean group difference (95% CI)	Raw P-value	Adjusted P-value*	Mean group difference (95% CI)	Raw P-value	Adjusted P-value*
	Intrinsic capacity Z-score	-0.16 (-0.22, -0.10); <0.0001	-0.13 (-0.19, -0.07); <0.0001	-0.19 (-0.25, -0.10); <0.0001	-0.20 (-0.26, -0.14); <0.0001	0.04 (-0.05, 0.12)	0.392 0.7844	0.07 (-0.02, 0.16)	0.121	0.363	0.01 (-0.08, 0.10)	0.835	0.835

CI: confidence interval; †NegaGve values indicate IC Z-score impairment. * Adjusted for multiple comparisons with the Hochberg procedure to account for the fact that three intervention groups were compared with the placebo group in each analysis.

Table 3 .

 3 Mixed-effect linear regression analysis for variation in intrinsic capacity Z-score over a 3-year follow-up according to randomized intervention groups among community-dwelling older adults without major neurocognitive disorders.

	Estimated

mean within-group 3-year change from baseline* (95% CI); P-value Group difference in intrinsic capacity Z-score between each intervention group and the placebo group over time

		Multidomain plus ω-3 supplementation	Multidomain plus placebo	ω-3 supplementation	Placebo	Multidomain plus ω-3 supplementation vs. placebo	Multidomain plus placebo vs. placebo	ω-3 supplementation vs. placebo
	Intrinsic capacity Z-score (time as categorical variable)
	6 months	0.01 (-0.04, 0.05);	0.00 (-0.04, 0.05);	-0.02 (-0.06, 0.03);	-0.04 (-0.08, 0.01);	0.04 (-0.02, 0.11);	0.04 (-0.02, 0.10);	0.02 (-0.05, 0.08);
		0.703	0.873	0.472	0.127	0.178	0.229	0.570
	12 months	0.01 (-0.04, 0.06);	0.04 (-0.01, 0.09);	-0.03 (-0.08, 0.02);	0.02 (-0.03, 0.07);	-0.01 (-0.07, 0.06);	0.02 (-0.04, 0.09);	-0.05 (-0.11, 0.02);
		0.635	0.096	0.258	0.467	0.856	0.520	0.188
	24 months	-0.08 (-0.13, -0.03);	-0.06 (-0.11, -	-0.14 (-0.19, -0.09);	-0.11 (-0.16, -0.06);	0.02 (-0.05, 0.09);	0.04 (-0.03, 0.11);	-0.04 (-0.11, 0.03);
		0.001	0.01); 0.015	<0.0001	<0.0001	0.527	0.210	0.292
	36 months	-0.14 (-0.19, -0.09);	-0.11 (-0.16, -	-0.17 (-0.22, -0.12);	-0.18 (-0.23, -0.13);	0.04 (-0.03, 0.11);	0.06 (-0.01, 0.13);	0.01 (-0.06, 0.08);
		<0.0001	0.06); <0.0001	<0.0001	<0.0001	0.234	0.071	0.744
	CI: confidence interval; *Negative values indicate IC Z-score impairment.

Acknowledgements

We would like to thank the investigators from CHU de Toulouse, Centre Hospitalier Lyon-Sud, Hôpital de Tarbes, Hôpital de Foix, Hôpital de Castres, CHU de Limoges, CHU de Bordeaux, Hôpital de Lavaur, CHU de Montpellier, Hôpital Princesse Grace, Hôpital de Montauban, CHU de Nice, and CHU de Dijon for their participation in this study.

Funding

The MAPT study was supported by grants from the French Ministry of Health (PHRC 2008(PHRC , 2009)), University Hospital Center of Toulouse / Gérontopôle, Pierre Fabre Research Institute (manufacturer of the omega-3 supplement), Exhonit Therapeutics (biological sample collection) and Avid Radiopharmaceuticals (PET-amyloid measurement). The promotion of this study was supported by the University Hospital Center of Toulouse. The data sharing activity was supported by the Association Monegasque pour la Recherche sur la maladie d'Alzheimer (AMPA) and the UMR 1027 Unit INSERM-University of Toulouse III. No sponsor placed any restriction on this work or had any role in the design of the study, data collection, data analyses or interpretation, or in the preparation, review, or approval of the manuscript.

Ethical approval

Volunteers were recruited by the investigating physicians, and signed an informed consent.

The trial protocol (NCT00672685) was approved by the Advisory Committee for the Protection of Persons participating in Biomedical Research of the Toulouse University Hospital (CPP SOOM II) and was authorized by the French Health Authority.

Provenance and peer review

This article was not commissioned and was externally peer reviewed.

Research data (data sharing and collaboration)

There are no linked research data sets for this paper. Data will be made available on request.

Conflict of interest

The authors declare that they have no conflict of interest.