Oliveira et al. BMC Molecular and Cell Biology
https://doi.org/10.1186/s12860-023-00476-3(2023) 24:11

BMC Molecular andCell Biology

Open Access

RESEARCH

Pre‑treatment with IL‑6 potentiates β‑cell
death induced by pro‑inflammatory cytokines
V. R. Oliveira, C. C. Paula, S. Taniguchi and F. Ortis*

Abstract
Background Type I Diabetes mellitus (T1D) is characterized by a specific destruction of β-cells by the immune
system. During this process pro-inflammatory cytokines are released in the pancreatic islets and contribute for β-cells
demise. Cytokine-induced iNOS activation, via NF-κB, is implicated in induction of β-cells death, which includes ER
stress activation. Physical exercise has been used as an adjunct for better glycemic control in patients with T1D, since
it is able to increase glucose uptake independent of insulin. Recently, it was observed that the release of IL-6 by skeletal muscle, during physical exercise, could prevent β-cells death induced by pro-inflammatory cytokines. However,
the molecular mechanisms involved in this beneficial effect on β-cells are not yet completely elucidated. Our aim wasto evaluate the effect of IL-6 on β-cells exposed to pro-inflammatory cytokines.
Results Pre-treatment with IL-6 sensitized INS-1E cells to cytokine-induced cell death, increasing cytokine-induced
iNOS and Caspase-3 expression. Under these conditions, however, there was a decrease in cytokines-induced p-eIF2-αbut not p-IRE1expression, proteins related to ER stress. To address if this prevention of adequate UPR response isinvolved in the increase in β-cells death markers induced by IL-6 pre-treatment, we used a chemical chaperone(TUDCA), which improves ER folding capacity. Use of TUDCA increased cytokines-induced Caspase-3 expression
and Bax/Bcl-2 ratio in the presence of IL-6 pre-treatment. However, there is no modulation of p-eIF2-α expression by
TUDCA in this condition, with increase of CHOP expression.
Conclusion Treatment with IL-6 alone is not beneficial for β-cells, leading to increased cell death markers andimpaired UPR activation. In addition, TUDCA has not been able to restore ER homeostasis or improve β-cells viability
under this condition, suggesting that other mechanisms may be involved.
Keywords β-cells, NF-κB, Pro-inflammatory cytokines, IL-6, ER stress and TUDCA
Background
Type I Diabetes mellitus (T1D) is an autoimmune diseasecharacterized by a genetic predisposition associated withenvironmental factors, that results in an autoimmune
attack against insulin-producing β-cells, leading to its
dysfunction and death [1–6].

*Correspondence:
F. Ortis
fortis@usp.br
Department of Cell and Developmental Biology, Institute of Biomedical
Sciences, University of São Paulo, São Paulo, Brazil

This specific attack against β-cells occurs through aprocess known as insulitis, in which cells of the immune
system infiltrate pancreatic islets and secret pro-inflammatory cytokines, such as Interleukin (IL)-1β, Interferon(IFN)-γ and Tumoral Necrosis Factor (TNF) [3, 7]. Leading, among others, to the expression of Inducible Nitric
Oxide Synthase (iNOS), with consequently productionof nitric oxide (NO) and of Endoplasmic Reticulum (ER)stress induction, triggering β-cells death [1–3, 8–11]. The
activation of ER stress induces a cell survival response
known as Unfolded Protein Response (UPR), which has
the function of recovering homeostasis of ER. However,if the ER homeostasis is not recovered, pro-apoptotic© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​
mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Oliveira et al. BMC Molecular and Cell Biology(2023) 24:11

pathways are activated [9, 12–14], as we observe forcytokine-exposed β-cells [9, 13–16].
T1D patients treatment consists of administration ofexogenous insulin combined with the practice of physicalexercise, which helps to restore glycemic control [17–19]
and increase glucose uptake independent of insulin [20–22]. Beside this effect, it has being reported that physical exercise has an anti-inflammatory effect, leading to
improvement in insulin secretion and β-cells protection[23–25]. However, the mechanism and pathways involvedin this protection are not yet well defined. In the lastyears, attention has been focused on Interleukin 6 (IL6) [26, 27], which when is released by skeletal muscles,
it can stimulate the release of anti-inflammatory factors
such as Interleukin 10 (IL-10) and inhibit the productionof pro-inflammatory cytokines such as TNF [20, 28–30].
The protective anti-inflammatory effect of IL-6 in pancreatic β-cells was shown by Paula and collaborators[24, 25], in which islets of human and trained animals
were resistant to cell death induced by pro-inflammatory cytokines, with a decrease in Caspase-3 and iNOS
expression. Moreover, serum from physical trained subjects protected human and rodent β-cells against ER
stress and apoptosis [25]. In addition, blocking of IL-6
partially prevented these beneficial effects [24, 25].
Since IL-6 is a pleiotropic cytokine, that is describedto have pro- and anti-inflammatory effects [31], it isimportant to understand the specific pathways that
could be modulated by IL-6 in β-cells, to prevent the

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deleterious effect of pro-inflammatory cytokines. Thisknowledge may help to design therapeutic protocols forT1D treatment or even prevention. Thus, here we preexposed the rat β-cell line, INS-1E, to IL-6 and thentreated these cells with a combination of pro-inflammatory cytokines, to mimetic the insulites environment.
We then investigate the effects of this pre-treatment
on the expression of pro-apoptotic markers involved incytokine induced β-cells death.

Results
Pre‑treatment with IL‑6 increases cytokine‑induced
pancreatic β‑cells death markers

Exposure of INS-1E cells to pro-inflammatory
cytokines (IL-1β + IFN-γ) leads to cell death, mainly by
apoptosis, with increase of Caspase-3 (the active form)
and iNOS expression [3, 11], as observed here (Fig. 1).
Pre-exposure of these cells to IL-6 increased
cytokines-induced Caspase-3 expression (Fig. 1 A-B),
although IL-6 alone had no effect on Caspase-3 expression. Similarly, induction of iNOS expression bycytokines was increased by IL-6 pre-exposure, with noeffect of IL-6 alone on iNOS expression (Fig. 1 C-D).
Of note, the same effect was observed when IL-6 was
only used during the pre-exposure time, 24 h before
cytokines exposure, or was kept also after this period
together with the cytokine mix.

Fig. 1 IL-6 increases IL-1β and IFN-γ induced Caspase-3 and iNOS expression in INS-1E cells. Cells were exposed to IL-6 (IL6) or untreated (Ctrl). After
24 h cells were exposed to IL-1β and IFN-γ in the absence (Cyto) or in the presence of pre-treatment with IL-6 (IL6 + Cyto) or continuous treatment
of IL-6 (IL6 + IL6 + Cyto). A Western blot for Caspase-3 and α-Tubulin. B The barplot showing the means values obtained in six independent
experiments corrected by the housekeeping protein α-Tubulin. One-way ANOVA followed by Sidak’s correction. *p < 0,05, **p < 0,001 vs Ctrl,&p < 0,05 vs Cyto. C Western blot for iNOS and α-Tubulin. D The barplot showing the mean values obtained in four independent experiments
corrected by the housekeeping protein GAPDH. One-way ANOVA followed by Sidak’s correction. **p < 0,001 vs Cyto

Oliveira et al. BMC Molecular and Cell Biology(2023) 24:11

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Pre‑treatment with IL‑6 protect β‑cells
against cytokine‑induced UPR protein expression

increased mRNA expression of CHOP (Fig. 4B) neitherof Bax/Bcl-2 ratio (Fig. 4C).

Cytokine-induced iNOS in rat β-cells leds to NO production and ER stress activation, which contribute to β-cells
death [3, 13, 16]. Thus, we evaluate expression of phosphorylated form of eIF2-α (p-eIF2-α) and IRE1 (p-IRE1),
two ER stress markers induced by cytokines in β-cells[13, 14, 16].
As expected, exposure of β-cells to cytokines increase
expression of p-eIF2-α and p-IRE1 (Fig. 2 A-B and C-D,
respectively). Although IL-6 alone did not modulate
expression of this ER stress marker, it prevented induction of p-eIF2-α by cytokines (Fig. 2).
The deleterious effect of IL‑6 on pancreatic β cells
is not prevented by increasing of ER protein folding
capacity

We next used a chemical chaperon, TUDCA, that waspreviously shown to improve ER protein folding and rescue cells from ER stress [14, 32, 33], to evaluate if thiswould help to protect β-cells against IL-6 pre-treatment
leading to cytokine-induced cell death worsening.
Our results showed that TUDCA addition to conditionof pre-treatment with IL-6 and exposure to cytokines
seems to further increase Caspase-3 expression (Fig. 3
A-B), in spite of not being able to modulate the expression of p-eIF2-α (Fig. 3 C-D) or BiP mRNA (Fig. 4A) in
this conditions. In addition, TUDCA did not prevent

Discussion
Physical exercise is an important tool in the treatment of
numerous diseases, including T1D. Particularly, in the
T1D the exercises have been argued as a beneficial factor,
closely associated with the increase of β-cells viability andits physiological functions [23–25]. It is known that during physical exercise myokines are produced by skeletal
muscle that can result in beneficial effects, among them,IL-6 has been shown to stimulate anti-inflammatory factors release and inhibit of pro-inflammatory cytokines
production [20, 28–30]. However, most of these findings
are based on the use of serum from exercised animals,
which can induce the activation of several pathways inaddition to IL-6, leading to a combined beneficial effectto the β-cells in the pro-inflammatory insult that occurs
during T1D [23–25]. Therefore, it is important to evaluate the effects of IL-6 alone in pancreatic β-cells exposed
to pro-inflammatory cytokines.
It is known that the cytokine-induced nitric oxide production, through NF-κB induced iNOS, is associated withthe blocking of the ER calcium pump (SERCA2b) [16,34] and causes stress in the ER, contributing to β-cells
death [3, 13, 16]. Our data showed that pre-treatment
with IL-6 leads to a higher sensitivity of INS-1E cells todeath induced by pro-inflammatory cytokines, inducing
increased iNOS and Caspase-3 expression. These data

Fig. 2 IL-6 decreases IL-1β and IFN-γ induced p-eIF2-α and p-IRE1 expression in INS-1E cells. Cells were exposed to IL-6 (IL6) or untreated (Ctrl).
After 24 h cells were exposed to IL-1β and IFN-γ in the absence (Cyto) or in the presence of pre-treatment with IL-6 (IL6 + Cyto) or continuous
treatment of IL-6 (IL6 + IL6 + Cyto). A Western blot for p-eIF2-α and α-Tubulin (note that α-Tubulin image is the same used in Fig. 1A, since they arefrom the same blot where caspase 3 and eiF2α were evaluated). B The barplot showing the mean values obtained in five independent experiments
corrected by the housekeeping protein α-Tubulin. One-way ANOVA followed by Sidak’s correction. *p < 0,05 vs Ctrl. C Western blot for p-IRE1 andα-Tubulin. D The barplot showing the mean values obtained in four independent experiments corrected by the housekeeping protein α-Tubulin

Oliveira et al. BMC Molecular and Cell Biology(2023) 24:11

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Fig. 3 TUDCA increases Caspase-3 but decreases p-eIF2-α expression in INS-1E cells. Cells were exposed to IL-6 (IL6) or untreated (Ctrl). After
24 h cells were exposed to TUDCA in the absence (TUDCA) or in the presence of pre-treatment with IL-6 (IL6 + TUDCA) and IL-1β and IFN-γ(TUDCA + Cyto) or pre-treatment with IL-6 and IL-1β and IFN-γ (IL6 + TUDCA + Cyto). Cells also were exposed to IL-1β and IFN-γ in the absence(Cyto) or in the presence of pre-treatment with IL-6 (IL6 + Cyto). A Western blot for Caspase-3 and α-Tubulin. B The barplot showing the meanvalues obtained in six independent experiments corrected by the housekeeping protein α-Tubulin. One-way ANOVA followed by Sidak’s correction.
**p < 0,001 vs Ctrl. C Western blot for p-eIF2-α and α-Tubulin. D The barplot showing the mean values obtained in five independent experiments
corrected by the housekeeping protein α-Tubulin. One-way ANOVA followed by Sidak’s correction. *p < 0,05, ***p < 0,0001 vs Ctrl

show us that pre-treatment with IL-6 alone does nothave a beneficial effect on INS-1E cells viability exposed
to pro-inflammatory cytokines. This apparently contradiction of our results and the beneficial effects of pretreatment with IL-6 observed by Paula [24] may be due tothe different cytokine combination used here. Paula used
a combination of IL-1β and IFNγ, here added also TNF,
since these three are major cytokines players during insulites [3]. This may indicate that TNF may induce otherdeleterious pathways that would lead to a pro-apoptotic
effect of IL-6 in these cells.
It has recently been shown serum from exercise
human or mouse prevent the death of human and rat
β-cells exposed to tapsigargin or CPA (inhibitors of the
SERCA2b pump), showing an effect of myocins in the
ER stress pathways [25]. Our data shows that the pretreatment of INS-1E cells with IL-6 alone decreases
IL-1β and IFN-γ-induced expression of p-eIF2-α
and p-IRE1, two important UPR markers, which are
induced during ER stress provoked in β-cells exposed tocytokines [14, 16, 35].
Although pre-treatment with IL-6 decrease partially
UPR response it still increases expression of Caspase-3
and iNOS expression, indicating increase of β-cells deathin this condition. One possible hypothesis is that IL-6
could be decreasing the β-cells ability to activate UPR
pathways, losing the ability to improve ER homeostasis,

precipitating β-cells demise. It is known that although
the activation of UPR in response to IL-1β and IFN-γ
contributes to the death of β-cells [3, 9, 13, 14, 16], thesepathways are primarily cellular responses to recover function and maintain cell viability [12, 13]. Thus, ER stress ischaracterized by a cellular response to the accumulationof poorly folded proteins within this organelle, however,it can lead to cell death if fails to restore homeostasis
in the ER [12]. Of note, exposure of β-cells to cytokines
induces a deficient UPR, which leads to activation of cell
death pathways in this cells [7, 11–14, 16, 35, 36].
To solve the problem of poorly folded proteins, one of
UPR mechanisms is to increase the expression of chaperones, that bind to unfolded proteins preventing themfrom aggregating [33, 37, 38]. The use of chemical chaperones to restore ER homeostasis have being reported,with beneficial results in protecting β-cells from ERstress induced conditions [14, 32, 39]. Administration
of TUDCA, a chemical chaperone, has been shown to
reduce apoptosis of β-cells in a TD1 mice model andrestore ER homeostasis [24], in addition, this protection
has also been shown in human β-cells against cytokineinduced apoptosis [14].
Thus, we used TUDCA to assess whether an increasein the folding capacity of poorly folded proteins in the
ER could improve the viability of INS-1E cells exposed toIL-6 and IL-1β + IFN-γ. The treatment with TUDCA did

Oliveira et al. BMC Molecular and Cell Biology(2023) 24:11

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Fig. 4 TUDCA prevent increase in BiP expression induced by cytokines + IL6 but not CHOP expression and Bax/Bcl2 ratio in INS-1E cells. Cells were
exposed to IL-6 (IL6) or untreated (Ctrl). After 24 h cells were exposed to TUDCA in the absence (TUDCA) or in the presence of pre-treatment with
IL-6 (IL6 + TUDCA). Cells also were exposed to IL-1β and IFN-γ (Cyto) or not (-). Expression of BiP A and CHOP B and Bax/Bcl-2 C was evaluated by
RT-qPCR. The bar plot shows the mean values obtained in three independent experiments normalized by Rn18s and control by method 2-ΔΔCT.
One-way ANOVA followed by Sidak’s correction. **p < 0,001 vs Ctrl

not prevent β-cells death under these conditions, indicating that perhaps other pathways may be involved inthis process. Furthermore, there was an increase in Bax/
Bcl2 mRNA ratio under these conditions, corroborating with lack of cell death prevention. We also observedthat under control conditions, administration of TUDCA
decrease expression of BiP (Fig. 4A), which could be due
to TUDCA working as a chemical chaperone, decreasing the need for BiP expression. However, since BiP is
an important chaperone for maintaining ER homeostasis and its decrease can lead to ER stress activation [13,14], this could contribute for the deleterious effects whenthese cells are exposed to cytokines in combination with
IL-6, since this treatment also lead to impaired ER stressresponses, preventing activation of p-eIF2α.
Taking together, this data may indicate that the positive effects by IL-6 on β-cells, observed by other authors,
may involve negative modulation of the ER stress, which,
together with the activation of additional pathways,
induced by other regulators, such as other myocines present in the serum of trained animals, may prevent β-cells
death.

Conclusions
Our data indicate that IL-6 pre-treatment sensitizes INS1E cells death induced by pro-inflammatory cytokines
IL-1β and IFN-γ. Increased iNOS expression in this condition, indicates involvement of NF-κB activation. This

pre-treatment, however, prevented cytokine-induced
important UPR pathways, namely p-eIF2-α, which may
negatively interfere in ER folding capacity and cell viability. Use of the chemical chaperone TUDCA showed thatthe deleterious effect of pre-treatment with IL-6 may
not seem to be related only to a worsening of the protein
folding capacity in the ER, but to other mechanisms not
yet demonstrated in the literature.

Methods
Cell culture

INS-1E cell line, an efficient model to study β-cells,
because of their similarity to primary mouse β-cells inboth gene expression and insulin secretion in the glucose response [40, 41], was used in this work. This rat
insulinoma cells, kindly provided by Prof. Dr. Claes B.
Wolheim and Prof. Dr. Pierre Maechler (University ofGeneva, Switzerland), was grown in RPMI 1640 culture
medium (Gibco/Invitrogen, Carlsbad, CA, USA) supplemented with 5% fetal bovine serum (FBS), 10 mM
HEPES, 100U/mL penicillin, 100 µg/mL streptomycin,
1 mM sodium pyruvate and 50 µM 2-mercaptoethanol.
The cultures of these cells were kept in an incubator(Autoflow IR Water-Jacketed ­CO2 Incubator, NuAire) at
37ºC, in a humidified atmosphere at 5% ­CO2.

Oliveira et al. BMC Molecular and Cell Biology(2023) 24:11

Cell treatment

The cells were plated (100.000 cells/ condition in 24
wells plate) and cultured for 48 h before any treatment,
to guarantee their adherence to the plaque and reduce
the effects of stress due to trypsinization. After 48 h,cells were exposed to IL-6 at a concentration of 100 ng/
mL (Life Technologies, Grand Island, NY, USA) for 24 hand then exposed to cytokine mix of IL-1β (Promega) at aconcentration of 10U/mL and IFN-γ (Promega) at a concentration of 14U/mL, for additional 24 h. This additional
exposure was performed in the presence or absence ofIL-6 and/or TUDCA (Calbiochem), at a concentration of
300 µM. Unexposed cells were used as a control.
Western blot

Total protein extracts were fractionated on sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDSPAGE) gel at concentrations of 8 or 14%. The proteins
were transferred by electrophoresis to nitrocellulose
membranes and the detection of specific bands was doneusing specific primary antibodies for the iNOS protein(#IO117), Caspase-3 (#9662, Cell Signaling), p-eIF-2α(#9721, Cell Signaling), p-IRE1 (#MABC742, Millipore),
and as loading control α-Tubulin (#9026, Sigma-Aldrich)and GAPDH (#25,778, Santa Cruz Biotechnology). The
membranes were incubated with secondary antibodies(horseradish peroxidase-labeled anti-IgG) anti-mouse
IgG (#170–5046, Bio-Rad) or anti-rabbit IgG (#170–5047,
Bio-Rad). The chemiluminescence (Ameersham ECL Plus
Western Blotting Detection Reagents (GE Healthcare Life
Sciences) and the detection of specific bands were madeusing the image documenter ImageQuant™ LAS 4000
(GE Healthcare Life Sciences). Quantification was performed by densitometry using the ImageQuant™ TL 8.1(GE Healthcare) and Image Lab 6.1 (Bio-Rad) program
and the values were normalized by the internal control
densitometry values (α-Tubulin and GAPDH).
Total RNA was isolated with TRIzol™ Reagent according
to the manufacturer’s protocol. Two μg of DNase-treated
total RNA (A260/280 ≥ 1,8), was used for cDNA synthesis, performed with SuperScript™ IV according to the
manufacturer’s protocol. Quantitative PCR was performedin triplicates with 10 ng of cDNA and 300 nM of specificprimers for the reference gene rat (Rn)18 s (F: 5’-TTC​CCA​
GTA​AGT​GCG​GGT​CAT-3’ and R: 5’-AGT​CAA​GTT​CGA​
CCG​TCT​TCTCA-3’), CHOP (F: 5’-CCA​CAC​CTG​AAA​
GCA​GAA​ACC-3’ and R: 5’- GCT​AGG​GAT​GCA​GGG​
TCA​AG-3’), BiP (F: 5’-GCT​AGG​GAT​GCA​GGG​TCA​
AG-3’ and R: 5’- AAG​GGT​CAT​TCC​AAG​TGC​GT-3’), Bax(F: 5’-AGC​TGC​AGA​GGA​TGA​TTG​CT-3’ and R: 5’-AGC​
CAC​CCT​GGT​CTT​GGA​T-3’) and Bcl2 (F: 5’- GGC​CTT​
Two‑step RT‑qPCR

Page 6 of 8

CTT​TGA​GTT​CGG​TG-3’ and R: 5’- ATA​TAG​TTC​CAC​
AAA​GGC​ATC​CCA​G) with PowerUp™ SYBR™ Greenaccording to the manufacturer’s protocol in the RotorGene Q. Data was analyzed with the 2-ΔΔCT method, as
described previously [42].
Statistical analysis

The results are presented as means ± SEM of the indicated number of independent experiments and were analyzed statistically, using GraphPad Prism 7 program (La
Jolla, CA, USA), by ANOVA followed by Sidak’s correction, as indicated. A < 0.05 p value was considered statistically significant.
Abbreviations
T1D	Type one Diabetes
IL-1β	Interleukin-1β
IFN-γ	Interferon-γ
TNF	Tumoral necrosis factor
NF-κB	Nuclear factor kappa B
iNOS	Inducible nitric oxide synthaseNO	Nitric oxide
ER	Endoplasmic reticulum
UPR	Unfolded protein response
TUDCA	Tauroursodeoxycholic
IL-6	Interleukin 6
IL-10	Interleukin 10
p-eIF2-α	Phospho-Eukaryotic initiation factor 2-αp-IRE1	Phospho-Inositol-requiring enzyme 1
FBS	Fetal bovine serum
SDS-PAGE	Sodium dodecyl sulphate–polyacrylamide gel electrophoresis
IgG	Immunoglobulin G
SERCA2b	Sarcoplasmic/endoplasmic reticulum calcium ATPase 2b
CPA	Cyclopiazonic acid
RT-qPCR	Reverse transcriptase quantitative polymerase chain reaction

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12860-​023-​00476-3.
Additional file 1.
Additional file 2.
Acknowledgements
We thank JF Vettorazzi, L Barreto, Professors AC Boschero and EM Carneiro(from UNICAMP-SP) for providing TUDCA and Adriane Pereira Fernandes
Araújo for her excellent technical support. We also thank Professor AR Carpinelli for giving space in his laboratory where some experiments were done,
and FFM Paula for the important discussion.
Authors’ contributions
VRO, CCP, ST and FO contributed to the experimental design, collection, analysis and interpretation of the data. VRO and FO wrote the manuscript. The final
manuscript was read and approved by all authors.
Funding
This work was supported by grants from the São Paulo Research Foundation(FAPESP): 2017/16513–8; 2017/04580–2; 2019/26062–9; Coordination for
the Improvement of Higher Education Personnel (CAPES); National Instituteof Science and Technology in Bioanalytic (INCTBio) – FAPESP 2014/50867–3
and National Counsel of Technological and Scientific Development (CNPq):465389/2014–7.

Oliveira et al. BMC Molecular and Cell Biology(2023) 24:11

Availability of data and materials
All data generated or analysed during this study are included in this publishedarticle [and its supplementary information files].

Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Received: 7 July 2022 Accepted: 15 March 2023

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