# Protein tyrosine phosphatase delta is a STAT3-phosphatase and suppressor of metabolic liver disease

Armando Andres Roca Suarez, Frank Jühling, Julien Moehlin, Laurent Mailly, Alessia Virzì, Nicolas Brignon, Sarah C. Durand, Marine A. Oudot, Eugénie Schaeffer, Romain Martin, et al.

► To cite this version:

Armando Andres Roca Suarez, Frank Jühling, Julien Moehlin, Laurent Mailly, Alessia Virzì, et al. Protein tyrosine phosphatase delta is a STAT3-phosphatase and suppressor of metabolic liver disease. eGastroenterology, 2025, 3 (1), pp.e100159. 10.1136/egastro-2024-100159. hal-05021484

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# Protein tyrosine phosphatase delta is a STAT3-phosphatase and suppressor of metabolic liver disease

Armando Andres Roca Suarez,<sup>1,2,3,4</sup> Frank Jühling,<sup>1</sup> Julien Moehlin,<sup>1</sup>
Laurent Mailly,<sup>1,5</sup> Alessia Virzi,<sup>1</sup> Nicolas Brignon,<sup>1,5</sup> Sarah C Durand,<sup>1</sup>
Marine A Oudot,<sup>1</sup> Eugenie Schaeffer,<sup>1</sup> Romain Martin,<sup>1,5</sup> Laura Meiss-Heydmann,<sup>1</sup>
Charlotte Bach,<sup>1</sup> Zakaria Boulaitouf,<sup>1</sup> Lea Girard,<sup>1</sup> Emma Osswald,<sup>1</sup>
Carole Jamey,<sup>6</sup> Daniel Brumaru,<sup>6</sup> Nassim Dali-Youcef,<sup>6,7,8,9</sup> Atish Mukherji,<sup>1</sup>
Maria Saez-Palma,<sup>2,3,4</sup> Barbara Testoni <sup>ID</sup>,<sup>2,3,4</sup> Fabien Zoulim <sup>ID</sup>,<sup>2,3,4,10</sup>
Bhuvaneswari Koneru,<sup>11</sup> Naoto Fujiwara,<sup>11</sup> Yujin Hoshida,<sup>11</sup> Emanuele Felli,<sup>1,12</sup>
Patrick Pessaux,<sup>1,12</sup> Michel L Tremblay,<sup>13,14,15</sup> Romain Parent,<sup>2,3,4</sup>
Catherine Schuster,<sup>1</sup> Thomas F Baumert,<sup>1,16</sup> Joachim Lupberger <sup>ID</sup><sup>1</sup>

**To cite:** Roca Suarez AA, Jühling F, Moehlin J, et al. Protein tyrosine phosphatase delta is a STAT3-phosphatase and suppressor of metabolic liver disease. *eGastroenterology* 2025;3:e100159. doi:10.1136/egastro-2024-100159

► Prepublication history and additional supplemental material for this paper are available online. To view these files, please visit the journal online (https://doi.org/10.1136/egastro-2024-100159).

Received 30 October 2024

Accepted 08 January 2025

## ABSTRACT

**Objective** Impaired hepatic expression of protein tyrosine phosphatase delta (PTPRD) is associated with increased STAT3 transcriptional activity and reduced survival from hepatocellular carcinoma in patients with chronic hepatitis C virus infection. However, the PTPRD-expressing hepatic cell types, signalling pathways responsive to PTPRD and their role in non-viral liver disease are largely unknown.

**Methods** We studied PTPRD expression in single-cell and bulk liver transcriptomic data from mice and humans, and established a Ptprd-deficient mouse model for metabolic dysfunction-associated steatohepatitis (MASH). Identified pathways were validated by perturbation studies in human hepatocytes and PTPRD substrates by pull-down assays. The clinical relevance was further explored in a cohort with metabolic disease by ranking patients according to PTPRD expression and analysing its association with metabolic disease markers.

**Results** The analysis of individuals ranked according to PTPRD expression and Ptprd-deficient mice, showed that PTPRD levels were associated with hepatic glucose/lipid signalling and peroxisome function. Hepatic PTPRD expression is impaired in aetiologies of chronic liver diseases that are associated with metabolic disease.

We further validated PTPRD as a STAT3 phosphatase in the liver, acting as a regulator of peroxisomal fatty acid metabolism. During MASH, low PTPRD led to increased liver steatosis in Ptprd+/- mice and a pronounced unfolded protein response, which impacts insulin signalling. Accordingly, silencing of PTPRD blunted insulin-induced AKT phosphorylation. Patients with obesity and low hepatic PTPRD expression exhibit increased levels of metabolic risk factors.

**Conclusion** Our data revealed an important regulatory role of the hepatic PTPRD-STAT3 axis in maintaining glucose/lipid homeostasis, which is recapitulated in clinical manifestations of metabolic liver disease.

## WHAT IS ALREADY KNOWN ON THIS TOPIC

⇒ Hepatic expression of protein tyrosine phosphatase delta (PTPRD) is impaired in the context of hepatitis C virus (HCV) infection, resulting in lower patient survival from hepatocellular carcinoma.

⇒ Little is known regarding the implication of PTPRD in non-viral liver disease.

## WHAT THIS STUDY ADDS

⇒ Hepatic PTPRD expression is impaired in aetiologies of chronic liver disease that are associated with metabolic disease.

⇒ Ptprd deficiency in mice fed with a high-fat diet promotes hepatic steatosis and altered glucose homeostasis.

## HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

⇒ Strategies rescuing PTPRD function in diseased livers could therefore represent novel therapeutic options to accompany measures aiming to eliminate the underlying liver disease aetiology and restore metabolic liver homeostasis.

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© Author(s) (or their employer(s)) 2025. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ Group.

For numbered affiliations see end of article.

**Correspondence to**
Dr Joachim Lupberger;
joachim.lupberger@unistra.fr

## INTRODUCTION

Chronic liver disease (CLD) represents a significant public health burden, with approximately 2 million deaths per year worldwide.¹ This stems from the presence of numerous risk factors such as viral hepatitis and the Western lifestyle that favour the progression from chronic inflammation, steatosis, fibrosis, cirrhosis and ultimately to the development of hepatocellular carcinoma (HCC).² ³ Although the disease aetiologies may vary, the similarities in the course of liver disease progression suggest the involvement
of common dysregulated signalling pathways driving this pathological process.⁴ ⁵ Therefore, a more detailed understanding of the shared molecular alterations associated with CLD is needed to set the basis for the novel preventive or therapeutic interventions that are urgently needed.

Transient phosphorylation of proteins is a key process in cellular signal transduction, which is tightly regulated by protein kinases and phosphatases, allowing a balance between the amplitude, rate and duration of a given transcriptional programme following its activation/inhibition.⁶ Hence, it is no surprise that a vast number of diseases are caused by aberrant protein phosphorylation through dysregulated kinase or phosphatase function.⁷ In the context of liver disease, we have previously demonstrated that hepatitis C virus (HCV) infection impairs the hepatic expression of protein tyrosine phosphatase delta (*PTPRD*), resulting in an increased activity of the signal transducer and activator of transcription 3 (STAT3) pathway and lower patient survival from HCC.⁸ Interestingly, the impairment of *PTPRD* persists even after viral cure, being linked to an epigenetic viral footprint that suggests a long-lasting impact of dysregulated *PTPRD* on liver homeostasis.⁹ Although encouraging findings in hepatoma cell culture suggest *PTPRD* as a STAT3 phosphatase in liver cancer,¹⁰ little is known about the impact of *PTPRD* expression in a diseased liver of non-viral aetiology, nor whether *PTRPD* directly interacts with STAT3 in the liver.

In the present work, we performed an unbiased characterisation of *PTPRD*-responsive signalling pathways in the liver microenvironment. Our analysis revealed that hepatic *PTPRD* is impaired in those CLD aetiologies associated with metabolic disease. Moreover, our findings shed light on the role of *PTPRD* in regulating multiple aspects of liver metabolism, such as glucose/lipid homeostasis, and clinical manifestations of metabolic disease.

# MATERIALS AND METHODS

## Animal experiments

Sperm from C57BL/6 *Ptprd*-deficient mice, originally developed by Uetani *et al*,¹¹ was obtained from McGill University (Montréal, Canada) for subsequent in vitro fertilisation and repopulation at the Institut Clinique de la Souris (ICS, Illkirch, France). Genotyping of *Ptprd* mice was performed using Phusion High-Fidelity Polymerase (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and previously described primers (online supplemental table 1).¹² *Ptprd* knockout mice (*Ptprd*--/-) exhibit a perturbed brain development, which affects feeding behaviour.¹¹ Therefore, the analysis was restricted to wild-type (*Ptprd*+/-) and *Ptprd* heterozygous (*Ptprd*+/-) mice. *Ptprd*+/- (n=22) and *Ptprd*+/- (n=13) male mice aged 8 weeks received a single intraperitoneal injection of diethylnitrosamine (100 mg/kg) (Sigma-Aldrich, St. Louis, Missouri, USA) and were subsequently fed with choline-deficient, L-amino acid-defined, high-fat diet (CDA-HFD)

(A06071302, Research Diet, New Jersey, USA) for 16 weeks. Glucose levels were determined in blood collected from the tail vein using a handheld Accu-Chek active glucometer (Roche diagnostics, Basel, Switzerland) after overnight fasting. Oil red and Sirius red staining was performed following sacrifice of the mice after 24 weeks (eight additional weeks of normal diet following CDA-HFD) using Certistain dry dye Oil red O and Direct red 80, respectively (Sigma-Aldrich). Red-stained particle quantification was performed using Fiji ImageJ (V.1.52s). H&E staining was performed in the same samples.

## Transcriptomic datasets

Liver transcriptomic and clinical data were obtained from the NCBI Gene Expression Omnibus (GEO) database, accession numbers GSE61260 (n=38, microarray, control individuals with histological absence of cirrhosis and malignancy),¹³ GSE130991 (n=737, microarray, patients with obesity, non-treated only),¹⁴ GSE10140 (n=82, microarray, patients with cirrhosis from mixed aetiologies),¹⁵ GSE230397 (n=69 HBV-infected patients, n=9 normal controls, RNA sequencing (RNA-seq)),¹⁶ GSE185477 (n=4, single-cell/nuclei RNA-seq, healthy individuals),¹⁷ GSE166504 (n=16, single-cell RNA-seq, mice fed with chow of high-fat high-fructose diet (HFHFD))¹⁸ and GSE247128/GSE243977 (single-nuclei RNA-seq, healthy livers from neurologically deceased donors (n=24) and explants from patients with primary biliary cholangitis (PBC, n=3) and primary sclerosing cholangitis (PSC, n=10)).¹⁹

## Cell isolation and culture

The immortalised liver progenitor cell (LPC) line HepaRG (CVCL_9720) was differentiated (dHepaRG) with medium containing 1.8% dimethyl sulfoxide into a mix of liver epithelial cells (hepatocyte-like and biliary-like cells).²⁰ ²¹ Primary human hepatocytes (PHH) were isolated and cultured as previously described.²² For the silencing of *PTPRD*, PHH were reverse transfected with small interfering RNA (siRNA) targeting *PTPRD* (Ontarget-plus, Smartpool, Horizon Discovery), using lipofectamine RNAiMAX (Thermo Fisher Scientific) for 72 hours according to the protocol suggested by the manufacturer. Postsilencing, PHH were serum starved for 6 hours and stimulated with human insulin (Sigma-Aldrich, 100 nmol/L) for 10 min prior to cell lysis. dHepaRG cells were silenced with si*PTPRD* and/or si*STAT3* during 48 hours and were subsequently treated with 800 µM oleic acid and 400 µM palmitic acid (Sigma-Aldrich) for 24 hours.

## RNA-seq data processing

For the analysis of RNA-seq data from C57BL/6 mice and dHepaRG cells, reads were mapped onto the mm10 of the *Mus musculus* genome or hg38 of the human genome using STAR (V.2.5.3a). Quantification was performed using HTSeq (V.0.6.1p1) and read counts were normalised using the median-of-ratios method. Sequencing and
data processing were performed by the GenomEast platform of the *Institut de Génétique et de Biologie Moléculaire et Cellulaire* (Illkirch, France) and the Research Center for Molecular Medicine (Vienna, Austria).

## Bioinformatics analyses

Differential expression analysis of RNA-seq data from C57BL/6 mice and HepaRG cells was performed using the DESeq2 R package. P values were computed using the Wald test and adjusted for multiple testing using the Benjamini-Hochberg method.²³ Preranked gene set enrichment analysis (GSEA) was performed using gene sets belonging to the Molecular Signatures Database (V.6.2).²⁴ For the analysis of microarray data from GSE61260, probes were collapsed using the Collapse-Dataset tool available at GenePattern and samples were ranked using *PTPRD* as a continuous class label. Single-sample (ss)GSEA was performed to correlate the gene sets *Reactome diabetes pathways* and *Kegg insulin signalling pathway* with *PTPRD* expression. For the analysis of clinical and transcriptomic data from GSE130991, samples were ranked according to *PTPRD* expression and the 20% of patients with the highest (n=147) and lowest (n=147) expression were used to plot waist circumference, triglycerides, fasting blood glucose, haemoglobin A1c (HbA1c) and homeostasis model assessment 2 (HOMA2) index. Insulin resistance (IR) index was calculated using the HOMA2 calculator (https://www.dtu.ox.ac.uk/homacalculator/). Virtual cell sorting from bulk RNA-seq data was conducted using CYBERSORTx (https://cibersortx.stanford.edu/) and previously described cell type-specific signatures.²⁵,²⁶ Statistical analyses were selected according to the normal distribution and number of groups using Prism (Dotmatics, Boston, Massachusetts, USA).

## Additional materials and methods

Methods and reagents for lipid analysis, CRISPR-Cas9 *PTPRD* KD, RT-qPCR, western blot analysis, recombinant glutathione-S-transferase (GST)-fusion proteins and PTPRD substrate-trapping are provided in the online supplemental data.

## RESULTS

### Hepatic *PTPRD* expression is associated with signalling pathways regulating glucose and lipid metabolism

With the aim to identify signalling pathways associated with the action of PTPRD in the human liver, we employed GSEA and studied the effect of normal variations of hepatic *PTPRD* expression in a cohort of individuals without CLD (n=38), who underwent biopsy to exclude liver malignancies.¹³ Our analyses showed that in the human liver, signalling pathways were associated with low *PTPRD* expression fall mainly into four categories: upregulation of inflammatory responses (including STAT3 signalling), downregulation of gene sets associated with peroxisomal function and a perturbation of pathways regulating glucose and lipid metabolism

(figure 1a). This is highly consistent with our previous findings showing that in HCV-infected livers, low *PTPRD* expression is associated with an increased transcriptional activity of the STAT3 pathway, which in turn perturbs peroxisomal lipid metabolism.⁴,⁸ In order to validate that these pathway alterations were indeed associated with hepatic PTPRD levels, we employed a *Ptprd*-deficient mouse model.¹¹ In these animals, genetic deletion of the region coding for the first phosphatase domain of PTPRD (figure 1b) induces a significant (p<0.0001, U-test) down-regulation of *Ptprd* expression on the mRNA and protein levels (figure 1c,d). Importantly, systemic *Ptprd* deficiency did not impact the proportion of liver-resident cell populations or immune infiltration of the hepatic microenvironment (online supplemental figure 1). Nonetheless, we took into account that mice with homozygous *Ptprd* knockout (*Ptprd*−/−) exhibit a perturbed brain development, which affects feeding behaviour.¹¹ Additionally, in a diseased liver PTPRD is reduced but not completely lost.⁸ Therefore, we restricted our analysis to wild-type (*Ptprd*+/+) and *Ptprd* heterozygous (*Ptprd*+/−) mice. The analysis of liver transcriptomic data from *Ptprd*+/− mice revealed a significant (false discovery rate (FDR) <0.05) increase of transcriptional programmes associated with diabetes development and a marked downregulation of the insulin signalling pathway (figure 1e). These results further support the role of PTPRD as a regulator of metabolic pathways in the liver.

### *Ptprd* deficiency promotes a phenotype of metabolic disease

In order to investigate the role of PTPRD in a pathological context, we studied liver transcriptomic data from patients with CLD of diverse aetiologies.¹⁵ This analysis revealed that hepatic *PTPRD* expression was significantly decreased in those aetiologies associated with metabolic disorder, that is, metabolic dysfunction-associated steatotic liver disease (MASLD, p<0.05, Kruskal-Wallis test), alcohol-related liver disease (p=0.004, T-test), chronic hepatitis C (CHC, p<0.0001, T-test) and patients with PBC and PSC (figure 2a, online supplemental figure 2). As expected, *PTPRD* downregulation was most pronounced in individuals with CHC due to an additional virus-specific mechanism involving *PTPRD* mRNA suppression by microRNA.⁸ Interestingly, hepatic *PTPRD* expression was not impaired in a cohort of patients with chronic hepatitis B (CHB) (figure 2b).¹⁶ These observations are in line with previous reports describing an increased prevalence of liver steatosis in MASLD and CHC compared with CHB.²⁷ Moreover, single-cell/nuclei RNA-seq analysis of liver tissues derived from mice under HFHFD (figure 2c, online supplemental figure 3) and from patients with PSC/PBC (online supplemental figure 2), showed that hepatocytes present a marked decrease of *Ptprd* expression (figure 2d). These data suggest a specific association of *PTPRD* with the mechanisms implicated in the physiopathology of MASLD.

The evidence described above prompted us to explore the direct effect of *Ptprd* deficiency during metabolic liver
<figure><img src="image_3.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>e</figcaption></figure>

<figure><img src="image_7.png" /></figure>

**Figure 1** Hepatic *PTPRD* expression is associated with signalling pathways regulating glucose and lipid metabolism. (a) GSEA showing significantly enriched (false discovery rate (FDR) <0.05) signalling pathways associated with low hepatic *PTPRD* expression in humans without CLD (n=38, GSE61260). (b) *Ptprd*-deficient mouse model presents a 200 bp deletion in the genomic region coding for the first phosphatase domain of PTPRD. Genotyping by PCR of liver samples from *Ptprd+/+* mice (n=3) compared with *Ptprd−/−* animals (n=3). (c) Genomic deletion of *Ptprd* induces a significant (U-test) downregulation of its expression at the mRNA level when comparing *Ptprd+/+* (n=6), *Ptprd+/−* (n=4) and *Ptprd−/−* (n=5) mice, as assessed by RT-qPCR. Data are presented as means±SD. (d) PTPRD protein level is impaired in *Ptprd*-deficient mice, as shown in liver samples analysed by western blot. (e) *Ptprd+/−* mice exhibit a pro-diabetic transcriptional pattern in the liver microenvironment. GSEA of liver expression data from *Ptprd+/−* mice (n=3) as compared with *Ptprd+/+* animals (n=3), showing a significant upregulation of the *Reactome diabetes pathways* gene set and a downregulation of the *Kegg insulin signalling pathway* (FDR <0.05). AMPK, AMP-activated protein kinase; CLD, chronic liver disease; FN, fibronectin; GSEA, gene set enrichment analysis; IFN, interferon; IL, interleukin; mTOR, mechanistic target of rapamycin kinase; NES, normalised enrichment score; NF-κB, nuclear factor kappa B; PKB, protein kinase B; PPARα, peroxisome proliferator-activated receptor alpha; PTPRD, protein tyrosine phosphatase delta; STAT, signal transducer and activator of transcription; TNFα, tumour necrosis factor alpha. \*P<0.05, \*\*\*p<0.0001.

injury in vivo. Therefore, we induced MASLD in *Ptprd+/+* and *Ptprd+/−* animals using CDA-HFD (figure 3a). This diet-based liver disease model closely recapitulates the progression of MASLD in humans.²⁸⁻³⁰ We observed no difference in the incremental weight gain of animals on this diet (figure 3b), excluding potential feeding abnormalities. However, *Ptprd+/−* animals developed significantly increased (p=0.02, T-test) fasting blood glucose levels after 8 weeks of CDA-HFD compared with *Ptprd+/+* mice (figure 3c). Moreover, following 16 weeks of CDA-HFD, we observed a significantly (p=0.04, U-test) higher accumulation of lipids in the liver of *Ptprd+/−* mice

as compared with the *Ptprd+/+* animals (figure 3d,e), without signs of liver fibrosis or elevated immune cell infiltration (online supplemental figures 4 and 5) suggesting that the effect of impaired hepatic PTPRD levels acts predominantly on lipid metabolism while not on inflammation or fibrosis. Lipid profiling of liver samples from these animals showed a significant increase of palmitic acid (p=0.0005, U-test) in the *Ptprd+/−* mice, which was accompanied by a decrease of oleic acid (p=0.03, U-test) and γ-linoleic acid (p=0.003, U-test) (figure 3f–h). This pattern resembles the plasma free fatty acid (FFA) distribution in patients with type II diabetes, where an increase
<figure><img src="image_8.png" /><figcaption>**Figure 2** *PTPRD* expression is impaired in the context of metabolic liver disease. (a–b) *PTPRD* expression is decreased in liver disease aetiologies associated with metabolic disorders (ie, MASLD, alcohol and chronic HCV infection), but is independent of chronic HBV infection. Patients are divided according to NAS (1–6), alcohol consumption (high: ≥200g/year, low: <200g/year), HCV infection and CHB phase. *PTPRD* expression in non-tumour liver tissues of patients with HCV infection was compared with patients with non-HCV disease aetiologies. Expression data were obtained from GSE10140 and GSE230397. (c–d) Single-cell RNA-seq liver data were derived from mice under chow or HFHFD (15, 30, 34 weeks), showing a decrease of *Ptprd* expression levels in hepatocytes. Expression data were obtained from GSE166504. CHB, chronic hepatitis B; cDCs, classical dendritic cells; CVECs, central vein endothelial cells; ENCHB, HBeAg-negative chronic hepatitis B; ENCI, HBeAg-negative chronic infection; EPCHB, HBeAg-positive chronic hepatitis B; EPCI, HBeAg-positive chronic infection; HBV, hepatitis B virus; HCV, hepatitis C virus; HFHFD, high-fat high-fructose diet; HSCs, hepatic stellate cells; KCs, Kupffer cells; LSECs, liver sinusoidal endothelial cells; MASLD, metabolic dysfunction-associated steatotic liver disease; Mig.DCs, migratory dendritic cells; NAS, NAFLD activity score; NI, non-infected; NK cells, natural killer cells; n.s., not significant; pDCs, plasmacytoid dendritic cells; PTPRD, protein tyrosine phosphatase delta; PVECs, portal vein endothelial cells. \*P<0.05, \*\*p<0.01, \*\*\*p<0.001, \*\*\*\*p<0.0001.</figcaption></figure>

of palmitic acid is often accompanied by a decrease of oleic acid levels.³¹ Moreover, a similar depletion of hepatic γ-linoleic acid has been described in MASLD progressing to MASH.³² Taken together, our results suggest that the observed transcriptomic changes in the liver of *Ptprd*-deficient mice are associated with the development of a metabolic phenotype.

## STAT3 is a PTPRD substrate in the liver and regulates peroxisomal fatty acid metabolism

To gain further insights into the mechanisms linking PTPRD with hepatic metabolism, we identified cell populations that express *PTPRD* in the human liver microenvironment.¹⁷ The analysis of single-cell/nuclei data revealed that similar to mice (figure 2d), human *PTPRD* is predominantly expressed in the epithelial

compartment of the liver, which is constituted by hepatocytes, cholangiocytes and LPCs (online supplemental figure 6). As these cells share several cellular markers and are difficult to distinguish,³³ we zoomed into epithelial cell subpopulations and applied transcriptional lineage tracing to further resolve *PTPRD* expression. Using this strategy, we revealed that *PTPRD* is highest expressed in bipotent LPCs (figure 4a), which are putative resident stem cells able to differentiate to either cholangiocytes or hepatocytes during liver injury.³⁴ Moreover, *PTPRD* levels were significantly and negatively correlated with STAT3 target gene expression at the single-cell level (p<0.001, Spearman's r=-0.46) (figure 4b), while other previously described pathways in the brain were weakly correlated with hepatic *PTPRD* (online supplemental table 2).³⁵ ³⁶
<figure><img src="image_9.png" /><figcaption>**Figure 3** *Pptrd* deficiency promotes a phenotype of metabolic disease. (a) *Pptrd*-deficient liver disease mouse model. Male *Pptrd+/+* (n=22) and *Pptrd+/-* (n=13) mice aged 8 weeks received 16 weeks of CDA-HFD, followed by 8 weeks of normal diet before sacrifice. (b) *Pptrd*±mice show a similar incremental weight gain as compared with *Pptrd+/+* mice following CDA-HFD during the 16-week period. (c) *Pptrd+/-* mice present significantly (T-test) higher levels of fasting blood glucose compared with *Pptrd+/+* mice after 8 weeks of CDA-HFD. (d-e) *Pptrd+/-* mice (n=6) present significantly (U-test) higher hepatic lipid levels following CDA-HFD, as compared with *Pptrd+/+* mice (n=10). Microscopy analysis with Oil red O staining. Bar represents 500 µm. (f-h) *Pptrd*-deficient mice present a dysregulated hepatic lipid profile. *Pptrd+/-* mice (n=6) show a significant alteration (U-test) in the intrahepatic levels of palmitic, oleic and γ-linoleic acids, as compared with *Pptrd+/+* mice (n=10) following CDA-HFD. Data are represented as µmol/L normalised to total protein concentration. CDA-HFD, choline-deficient, L-amino acid defined, high-fat diet; n.s., not significant; Pptrd, protein tyrosine phosphatase delta. \*P<0.05, \*\*p<0.01, \*\*\*p<0.001, \*\*\*\*p<0.0001.</figcaption></figure>

Consistently, genetic deletion of *Pptrd* in mice led to an allele-dependent increase of STAT3 phosphorylation in the liver (figure 4c), reflecting a PTPRD-dependent regulation of STAT3 activity.³⁷ To validate STAT3 as a PTPRD target in the liver, we conducted substrate-trapping experiments in interleukin (IL)-6-stimulated HepaRG cells (immortalised liver epithelial cell line) using a recombinant cytosolic PTPRD fragment coupled to GST as bait. In this experimental setting, only PTPRD-GST was able to pull down phosphorylated STAT3, while GST alone did not (figure 4d). Consistently, genetic knockdown (KD) of PTPRD catalytical domain D2 in the genome of HepaRG cells promoted IL-6-induced STAT3 phosphorylation

(figure 4e). These results establish PTPRD as a negative regulator of STAT3 signalling in the liver.

As PTPRD deficiency is associated with impaired peroxisomal function in the human liver (figure 1a) and STAT3 has been described to regulate gene expression involved in peroxisomal lipid metabolism,⁴ we validated the functional link between PTPRD and STAT3 on metabolic signalling. Therefore, we transfected dHepaRG cells with siRNA against *PTPRD* and performed a transcriptomic characterisation by RNA-seq. GSEA allowed us to observe a positive enrichment of transcriptional programmes induced by STAT3 signalling, in parallel to the suppression of genes involved in peroxisomal fatty
<figure><img src="image_10.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>e</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>f</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>b</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>d</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>g</figcaption></figure>

<figure><img src="image_16.png" /></figure>

**Figure 4** STAT3 is a PTPRD substrate in the liver and regulates peroxisomal fatty acid metabolism. (a) *PTPRD* and *STAT3* target gene expression along the cholangiocyte-LPC-hepatocyte pseudotime trajectory (GSE185477). (b) *PTPRD* expression inversely correlates (Spearman's *r*=-0.46) with *STAT3* target gene expression in the human liver. (c) Genetic deletion of *Pptrd* is associated with a significantly (Kruskal-Wallis test) increased *STAT3* Y705 phosphorylation (pSTAT3) in the liver of C57BL/6 mice as assessed by western blot analysis and quantified by band densitometry. Data are presented as means±SD. (d) pSTAT3 is a binding partner of PTPRD in hepatic cells. Co-immunoprecipitation of 5 µg recombinant GST-PTPRD fusion protein (rPTPRD) or GST protein alone was incubated with 1 mg IL-6-stimulated HepaRG lysate. Subsequent immunostaining on the same membrane for stain-free (all proteins), anti-pSTAT3 (Y705) and anti-GST (after stripping). (e) Genomic *PTPRD* silencing promotes IL-6-induced STAT3 phosphorylation. PTPRD was genetically silenced in undifferentiated HepaRG cells using CRISPR-Cas9 (KD). KD and wild-type PTPRD-expressing cells (WT) were serum-starved overnight prior 30 min IL-6 stimulation and analysed subsequently by immunoblotting. Human PTPRD antibody detects a PTPRD fragment after ectodomain shedding. (f) GSEA of dHepaRG cells silenced for *PTPRD*, showing an enhanced transcriptional activity of STAT3 signalling and impaired expression of gene sets associated with fatty acid metabolism. (g) Combined silencing of *PTPRD* and *STAT3* in FFA-treated dHepaRG cells is able to reverse the gene expression alterations observed with *PTPRD* silencing alone, including peroxisomal function and fatty acid metabolism. ABCD, ATP binding cassette subfamily D; ACOX1, acyl-CoA oxidase 1; FFA, free fatty acids; GSEA, gene set enrichment analysis; GST, glutathione-S-transferase; IL, interleukin; LPC, liver progenitor cell; NES, normalised enrichment score; PEX19, peroxisomal biogenesis factor 19; PHYH, phytanoyl-CoA 2-hydroxylase; PTPRD, protein tyrosine phosphatase delta; SOCS3, suppressor of cytokine signalling 3; STAT3, signal transducer and activator of transcription 3; VLCFAs, very long chain fatty acids. \*P<0.05, \*\*p<0.001, \*\*\*p<0.0001.
a

acid metabolism (figure 4f). To recapitulate the conditions taking place during MASH, we silenced *PTPRD* and/ or *STAT3* in dHepaRG cells (online supplemental figure 7), cultured them in the presence of FFA (oleic+palmitic acids) and conducted RNA-seq. Our results show that combined silencing of *STAT3* and *PTPRD* was able to reverse the gene expression alterations observed with *PTPRD* silencing alone, including peroxisomal function (ie, *PEX19*, *PHYD*) and fatty acid metabolism (ie, *ABCD1*, *ABCD3* and *ACOX1*) (figure 4g). These findings demonstrate that the observed suppression of peroxisomal fatty acid metabolism during metabolic stress is functionally mediated by a PTPRD-STAT3 axis.

## Impaired PTPRD expression potentially blunts insulin signalling via the unfolded protein response

Given that our in vivo data also pointed towards the implication of PTPRD as a component of glucoregulatory signalling (figure 1a), we dissected this possibility at the molecular level in hepatocytes. With this aim, we took into account that one of the most important circuits regulating hepatic glucose metabolism is mediated by insulin signalling. Insulin binding to its receptor triggers a chain of phosphorylation events, including the activation of AKT that regulates the expression of glucose transporters and the activity of pathways associated with gluconeogenesis.³⁸ Therefore, we silenced *PTPRD* using siRNA in PHH and analysed AKT phosphorylation after stimulation with insulin. While insulin triggered AKT phosphorylation at serine 473 (S473) in the control condition, silencing of *PTPRD* significantly reduced insulin-induced AKT phosphorylation (p=0.02, U-test), suggesting functional relevance of PTPRD as a regulator of hepatic glucose homeostasis by maintaining insulin signalling (figure 5a, online supplemental figure 8).

To gain further insights into the mechanism linking PTPRD to insulin signalling, we performed a differential expression analysis comparing the transcriptomes of *Ptprd*-deficient and wild-type mice. This allowed us to identify 30 upregulated and 29 downregulated genes (FDR <0.05, Wald test) that were differentially expressed in the liver of *Ptprd+/−* mice (figure 5b). To focus on genes relevant to human liver physiology, we correlated the human homologs of the differentially expressed genes identified in *Ptprd+/−* mice with *PTPRD* expression in the liver of healthy patients. This approach highlighted 16 genes that significantly correlated (p<0.05, Spearman's correlation) negatively (n=9) or positively (n=7) with hepatic *PTPRD* expression (figure 5c). Finally, to identify genes implicated in glucose homeostasis, we evaluated the association of each one of these factors with established gene sets involved in diabetes and insulin signalling pathways.²⁴ This allowed us to identify four genes (mesencephalic astrocyte-derived neurotrophic factor (*MANF*); stromal cell derived factor 2 like 1 (*SDF2L1*); heat shock protein family A Hsp70 member 5 (*HSPA5*); dnaJ heat shock protein family Hsp40 member B11 (*DNAJB11*)) that were positively correlated and one gene (acetyl-CoA carboxylase

beta (*ACACB*)) that were negatively correlated with diabetes signalling. Furthermore, the very same genes correlate inversely with insulin signalling (figure 5d). Of note, *MANF*, *SDF2L1*, *HSPA5* and *DNAJB11* are induced by the cellular unfolded protein response (UPR) during endoplasmic reticulum (ER) stress,³⁹⁻⁴¹ suggesting that PTPRD may regulate insulin signalling via the UPR. This is consistent with an upregulation of UPR transcriptional signatures in the liver of *Ptprd+/−* mice (online supplemental figure 9), which is further supported by previous studies linking hepatic UPR to the development of IR and diabetes.⁴² ⁴³ In this context, our results suggest a potential role of PTPRD as a regulator of liver metabolism by suppressing the hepatic UPR.

## Impaired hepatic PTPRD expression is associated with clinical manifestations of metabolic syndrome in patients

Aiming to validate the clinical relevance of PTPRD as a regulator of metabolic pathways in the liver, we studied *PTPRD* expression in a patient cohort with metabolic disease by integrating liver transcriptomic and clinical data from 737 patients with obesity.¹⁴ As a first step, patient samples were ranked according to hepatic *PTPRD* expression and divided into two groups, with the 20% lowest or highest *PTPRD* expression, respectively (figure 5e). Our analysis revealed that patients with low *PTPRD* expression exhibit significantly (p<0.0001, T-test) higher levels of metabolic disease markers, including fasting blood glucose, HbA1c, HOMA2 IR score, triglycerides and waist circumference (figure 5f). Similar results were observed across this dataset by correlating *PTPRD* expression levels and each clinical parameter (online supplemental figure 10). Consistent with our observations in individuals without CLD and low *PTPRD* expression, as well as in the *Ptprd*-deficient mice, we also identified an increased expression of the UPR genes *MANF*, *SDF2L1* (p<0.0001, T-test) and *DNAJB11* (p<0.05, T-test) in the liver transcriptome of these patients with obesity, in addition to decreased expression of the lipid regulator *ACACB* (p=0.0003, T-test) (figure 5g, online supplemental figure 11). Taken together, our results support the role of hepatic PTPRD in the regulation of glucose/lipid homeostasis, and how its perturbed expression is associated with the clinical manifestations of hepatic metabolic disease.

## DISCUSSION

CLD is a major and fast rising public health burden causing millions of deaths per year worldwide.¹ A detailed understanding of the underlying mechanisms driving liver disease progression is essential for the development of novel therapeutic strategies, which could support measures that remove the underlying cause of CLD (eg, antiviral therapy and/or lifestyle changes). Aiming to identify regulatory circuits involved in liver homeostasis and CLD, our study of PTPRD uncovered a novel role for this phosphatase as a regulator of the UPR and liver metabolism, and tied together several important previous observations. First, single-nucleotide
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**Figure 5** Impaired *PTPRD* expression blunts insulin signalling via the unfolded protein response. (a) Silencing of *PTPRD* in PHH leads to a significantly impaired (U-test) AKT phosphorylation (S473) following insulin stimulation, as assessed by western blot analysis (n=4). (b) Identification of 30 upregulated and 29 downregulated genes (false discovery rate (FDR) <0.05. Wald test) in liver samples from *Ptprd+/−* mice (n=3) compared with *Ptprd+/+* mice (n=3) by RNA-seq. (c) Human homologs of the differentially expressed genes identified in the *Ptprd+/−* mice were correlated with *PTPRD* expression in liver samples from healthy individuals (n=38, GSE61260, Spearman's correlation). (d) Correlation of *PTPRD*-associated genes (*MANF*, *SDF2L1*, *HSPA5*, *DNAJB11* and *ACACB*) with transcriptional programmes implicated in diabetes development and insulin signalling (n=38, GSE61260, Spearman's correlation). (e) Ranking of patients with obesity according to hepatic *PTPRD* expression into high-expressing (n=147, grey) and low-expressing (n=147, green) groups. Liver transcriptomic data obtained from GSE130991. (f) Analysis of clinical data from patients with obesity ranked according to *PTPRD* expression shows a significant association (T-test) between low hepatic *PTPRD* expression and increased fasting blood glucose, HbA1c, HOMA2 IR score, blood triglycerides and waist circumference. (g) Patients with low *PTPRD* expression display a significant (T-test) upregulation of *MANF* and *SDF2L1* and a downregulation of *ACACB*, as compared with patients with high *PTPRD* expression. ACACB, acetyl-CoA carboxylase beta; AKT, AKT serine/threonine kinase; DNAJB11, dnaJ heat shock protein family (Hsp40) member B11; HbA1c, haemoglobin A1c; HOMA2, homeostasis model assessment 2; HSPA5, heat shock protein family A (Hsp70) Member 5; IR, insulin resistance; MANF, mesencephalic astrocyte-derived neurotrophic factor; PHH, primary human hepatocytes; PTPRD, protein tyrosine phosphatase delta; SDF2L1, stromal cell derived factor 2 like 1. \*P<0.05, \*\*p<0.01, \*\*\*p<0.001, \*\*\*\*p<0.0001.
polymorphisms (SNPs) in the *PTPRD* gene have been asso-
ciated with the development of type II diabetes and the
treatment response in these patients.⁴⁴⁻⁴⁶ Moreover, recent
genome-wide association studies identified *PTPRD* SNPs
to be strongly associated with low-density lipoprotein and
triglyceride levels, as well as body mass index in patients
with MASLD.⁴⁷ ⁴⁸ Second, the notion that dysregulated
hepatic signalling, that is, epidermal growth factor receptor
or STAT3, drives the development and progression of liver
diseases independently of their underlying aetiology.²² ⁴⁹
Indeed, we have previously reported an HCV-induced down-
regulation of PTPRD as a molecular driver for the develop-
ment of HCC.⁸ These findings are of clinical relevance, as
diabetes, MASLD and CHC are well-established risk factors
for the development of HCC.¹

The molecular mechanism linking PTPRD with meta-
bolic signalling pathways suggests that impaired *PTPRD*
expression attenuates insulin-induced phosphorylation
of AKT (figure 5a). Interestingly, PTPRD has been previ-
ously shown to interact with components of this pathway,
such as insulin receptor substrate 1 (IRS1).35 However,
the functional significance of this interaction remains
unknown. Based on our results, a direct PTPRD-mediated
dephosphorylation of tyrosines on the insulin receptor
or IRS1 would not match the observed phenotype since
both proteins are activated by tyrosine phosphorylation.38
Therefore, we propose an indirect action of PTPRD on
the regulation of insulin signalling by a potential inter-
play between PTPRD and other known activators and
regulators of AKT and insulin signalling, as for example
receptor tyrosine kinases⁵⁰ or the UPR, which is the most
evident transcriptional change observed in the liver of
*Ptprd*/− animals. Among these genes, several, including
*HSPA5*, *DNAJB11* and *SDF2L1* have been associated with
the development of IR and type II diabetes.⁵¹⁻⁵³ Interest-
ingly, a similar situation has been reported in a mouse
model deficient for protein tyrosine phosphatase 1B
(*PTP1B*).⁵⁴ In this work, hepatic expression of *PTP1B*
plays a central role in the regulation of glucose and lipid
metabolism. Moreover, the phenotype of this mouse
model was independent of adiposity and was also medi-
ated by ER stress. However, the detailed mechanism about
how PTPRD influences UPR remains to be elucidated in
future studies.

Among the non-UPR genes responsive to PTPRD, we
found *ACACB* to be significantly downregulated. ACACB is
a key rate-limiting enzyme in mitochondrial fatty acid oxida-
tion and its impaired expression or SNPs in this locus have
been associated with IR and MASLD.⁵⁵ Considering that long-
chain fatty acids are metabolised in both the peroxisome and
the mitochondria, in addition to our transcriptomic analysis
showing that *ACACB* expression and the peroxisomal func-
tion are impaired (figures 1a, 4f and 5b), suggesting that
low *PTPRD* expression may alter the metabolism of long-
chain fatty acids and thus promote fat deposition in the liver.
Indeed, this is consistent with our observation of spiked intra-
hepatic palmitic acid levels and an accelerated accumulation
of lipid droplets in the liver of *Ptprd*/− mice, which had been

fed with a high-fat diet rich in palmitic acid (figure 3e). More-
over, we have previously identified STAT3 as a negative regu-
lator of peroxisomal function in hepatocytes.⁴ Considering
that we identified PTPRD as a STAT3 phosphatase in the liver
(figure 4) leading to elevated activity of this pathway in *Ptprd*-
deficient animals (figure 4c), this strongly suggests STAT3
being the key player implicated. This is further supported by
the observed partial rescue of peroxisomal genes suppressed
by *PTPRD* silencing in HepaRG cells when combined with
*STAT3* silencing (figure 4g). A more in-depth fatty acid
profiling of mouse livers also revealed a significant drop of
oleic and γ-linoleic acid in *Ptprd*/− mice compared with
control animals fed with a high-fat diet (figure 3f,h). This
pattern is consistent with the fatty acid profile of patients with
MASH.³² Importantly, an altered hepatic palmitic acid/oleic
acid ratio has a key impact on glucoregulatory signalling, as
the increase of palmitic acid and the decrease of oleic acid
favour the development of IR.³¹ Thus, this lipidic imbalance
together with the UPR represents two indirect mechanisms
of how *PTPRD* deficiency hampers insulin signalling. The
consequence of the largely perturbed lipid and glucose
homeostasis in the *Ptprd*deficient mice is also evident by
the absence of increased body weight of *Ptprd*/− animals,
despite the accelerated hepatic fat accumulations and
elevated blood glucose levels (figure 3c–h). This suggests that
PTPRD is required for efficient lipid/glucose homeostasis
in the liver. The clinical relevance of the above-described
observations was validated by the analysis of patients with
obesity with low hepatic *PTPRD* expression, who displayed
significantly higher metabolic disease markers (figure 5f),
highlighting the role of hepatic PTPRD as a regulator of
glucose homeostasis in a pathological context. Although we
observed impaired levels of hepatic *PTPRD* in those aetiolo-
gies associated with metabolic dysfunction, the mechanistic
drivers of this suppression remain unknown but potentially
involve STAT3 feedback mechanisms during chronic injury
or an indirect effect of fatty acid excess on PTPRD expres-
sion and its epigenetic regulation during chronicity. Indeed,
a recent study suggested hepatic oxidation of protein tyrosine
phosphatases by a high-fat diet as a mechanism leading to
their inactivation during MASLD.⁴⁹ Moreover, we previously
identified impaired PTPRD levels being associated with an
epigenetic footprint persisting in the livers of patients with
CHC postviral cure.⁹

STRENGTH AND LIMITATION

Our data highlight an important regulatory role of
hepatic PTPRD in maintaining hepatic lipid and
glucose homeostasis and how its impaired expression
is associated with the clinical manifestations of meta-
bolic disease. Therapeutic strategies either rescuing
PTPRD function or targeting PTPRD-sensitive signal-
ling pathways will therefore represent interesting
options to accompany measures aiming to eliminate
the underlying liver disease aetiology and restore meta-
bolic liver homeostasis. A limitation of our study is the
use of a CDA-HFD model, which is well established to
induce a MASH-like phenotype,²⁹ ³⁰ but not optimal to study glucose metabolism and diabetes. Future validation using a conventional high-fat diet model, potentially combined with a liver-specific knockout and/or overexpression of *Ptprd* could further strengthen our findings and validate its regulatory role. Moreover, our analysis of single-cell transcriptomic data suggests that *PTPRD* is expressed mainly in the epithelial compartment, but additional experiments are needed to validate the hepatic cellular distribution of PTPRD protein expression. So far, the potential of PTPRD as a putative minimally invasive biomarker remains to be evaluated, as we did not assess whether potentially shed components of the membrane protein PTPRD are secreted into the blood and associated with CLD. These points will be addressed in dedicated follow-up studies.

### Author affiliations

X Armando Andres Roca Suarez @andresroca and Barbara Testoni @TestoniResearch

**Acknowledgements** We would like to thank Noriko Uetani (McGill University, Montréal, Quebec, Canada) for the *Ptprd*-deficient mouse model, Tania Sorg-Guss and Eve Geronimus (Animal facility ICS, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France) for breeding and genotyping the mice, Cathy Obringer (Inserm UMR-U1112, Ciliopathies Modeling and Associated Therapies Team), Isabel Aubry (McGill University, Montréal, Quebec, Canada) and Christine Thumann (Inserm UMR_S1110) for excellent technical support.

**Contributors** Conceptualisation: JL; formal analysis: AARS, FJ, JM, LM, AV, NB, SCD, MAO, ES, RM, LM-H, CB, ZB, LG, EO, CJ, DB, ND-Y, MS-P, BK and NF; funding acquisition: JL and TFB; investigation: AARS, FJ, JM, LM, AV, NB, SCD, MAO, ES, RM, LM-H, CB, ZB, LG, EO, CJ, DB, ND-Y, MS-P, BK and NF; resources: EF, PP, BT and FZ; supervision: JL and TFB; visualisation: AARS, FJ and JM; writing—original draft: AARS and JL; writing—review and editing: TFB, AM, BT, FZ, YH, MLT, RP and CS; JL acts as guarantor of this work.

**Funding** This work was supported by the European Union (ERC-AdG-2014 HEPCIR ERC POC PRELICAN 755460, ERC POC HEPCAN 862551 to TFB, EU H2020 HEPCAR 667273 to TFB and JL, HORIZON-HLTH-2021-DISEASE-04-07

D-SOLVE #101057917 to TFB and JL, IP-cure-B project #847939 to FZ, ERC-AdG-2020-101021417 to YH), the French Cancer Agency (TheraHCC2.0 IHU201901299 to TFB), the Agence Nationale de la Recherche (ANR-21-RHUS-001 to TFB), the ANRS Maladies infectieuses émergentes (ANRS-MIE) (ECTZ103701, ECTZ131760, ECTZ160436, ECTZ171594 to JL, ECTZ104017 and ECTZ75178 to TFB, ECTZ4446 and ECTZ206376 to AARS), the Fondation de l'Université de Strasbourg (HEPKIN) (TBA-DON-0002), SATT Conectus, University of Strasbourg (CANCLAU) (TFB), the Inserm Plan Cancer 2019-2023, the US National Institutes of Health (CA233794, CA255621, CA282178, CA288375 and CA283935 to YH) and the Cancer Prevention and Research Institute of Texas (RR180016, RP200554 to YH). The ÆPIC animal facility platform is financially supported by the CoRTecS network of the University of Strasbourg. This work of the Interdisciplinary Thematic Institute IMCBio, as part of the ITI 2021-2028 programme of the University of Strasbourg, CNRS and Inserm, was supported by IdEx Unistra (ANR-10-IDEX-0002), SFRI-STRAT-US project (ANR 20-SFRI-0012) and EUR IMCBio (ANR-17-EURE-0023) under the framework of the French Investments for the Future Programme, as well as state funds managed within the France 2030 programme (reference ANR-21-RHUS-0001).

**Competing interests** None declared.

**Patient and public involvement** Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.

**Patient consent for publication** Not applicable.

**Ethics approval** In vivo experiments were performed at the ÆPIC animal facility platform of Inserm UMR_S1110 according to local laws and ethics committee approval (APAFIS#9466-2017033117328355).

**Provenance and peer review** Not commissioned; externally peer reviewed.

**Data availability statement** Data are available in a public, open access repository. Transcriptomic profiles from *Ptprd*/+ (n=3) and *Ptprd*/− (n=3) mice and *PTPRD/STAT3*-silenced dHepaRG cells reported in this study are accessible at the NCBI GEO database, accession numbers GSE150848 and GSE266460.

**Supplemental material** This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.

**Open access** This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.

### ORCID iDs

Barbara Testoni http://orcid.org/0000-0001-5588-5465
Fabien Zoulin http://orcid.org/0000-0002-2245-0083
Joachim Lupberger http://orcid.org/0000-0003-3996-3907

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