Cécile Gateau
email: cecile.gateau@aphp.fr

Stéphanie Deboscker

Jeanne Couturier

Thomas Vogel

Elise Schmitt

Joris Muller

Céline Ménard

Béatrice Turcan

Rabab Syed Zaidi

Anlyata Youssouf

Thierry Lavigne

Frédéric Barbut

Local outbreak of Clostridioides difficile PCR-Ribotype 018 investigated by multi locus variable number tandem repeat analysis, whole genome multi locus sequence typing and core genome single nucleotide polymorphism typing

Keywords: C. difficile, Outbreak, PCR-ribotyping, MLVA, wgMLST, cgSNP

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

Introduction

Clostridioides (formerly Clostridium) difficile (C. difficile) is an anaerobic Gram-positive sporeforming bacillus responsible for a wide spectrum of clinical symptoms ranging from mild selflimited diarrhea to pseudomembranous colitis (PMC), toxic megacolon, septic shock and possible death [START_REF] Leffler | Clostridium difficile Infection[END_REF][START_REF] Magill | Multistate Point-Prevalence Survey of Health Care-Associated Infections[END_REF]. Previous antibiotic treatment, hospitalization, advanced age and severe underlying diseases represent major risk factors for C. difficile infection (CDI) [START_REF] Rupnik | Clostridium difficile infection: new developments in epidemiology and pathogenesis[END_REF]. Recent data have indicated that C. difficile is the leading cause of healthcare-associated infections with more than 453,000 CDI-cases per year, leading to 29,600 deaths in the U.S.A [START_REF] Lessa | Burden of Clostridium difficile Infection in the United States[END_REF]. In Europe, the estimated number of healthcare-associated CDI cases in 2013 was 126,000 per year with a 3% attributable mortality [START_REF]Point prevalence survey of healthcare associated infections and antimicrobial use in European acute care hospitals[END_REF]. The incidence of CDI has been increasing worldwide partly due to the emergence of an epidemic strain (NAP1/027/BI) responsible for large outbreaks of severe CDI in the last few years [START_REF] Planche | Differences in outcome according to Clostridium difficile testing method: a prospective multicentre diagnostic validation study of Clostridium difficile infection[END_REF][START_REF] Gerding | Clostridium difficile 30 years on: what has, or has not, changed and why?[END_REF]. Recent European surveillance has shown that NAP1/027/BI is still predominant and represents 19% of all isolates but a wide variation of ribotype distribution is observed across countries [START_REF] Davies | Diversity of Clostridium difficile PCR ribotypes in Europe: results from the European, multicentre, prospective, biannual, point-prevalence study of Clostridium difficile infection in hospitalised patients with diarrhoea (EUCLID), 2012 and 2013[END_REF]. For example, prevalence of PCRribotype (RT) 018 is high in Italy (22%), as opposed to other European countries (< 3%). C. difficile RT 018 has been reported as an emerging RT associated to severe CDI and outbreaks in Italy and more recently in Germany, Korea and Japan [START_REF] Barbanti | Characterization of Clostridium difficile PCR-ribotype 018: A problematic emerging type[END_REF][START_REF] Baldan | Clostridium difficile PCR Ribotype 018, a Successful Epidemic Genotype. Onderdonk AB[END_REF][START_REF] Senoh | Predominance of PCRribotypes, 018 (smz) and 369 (trf) of Clostridium difficile in Japan: a potential relationship with other global circulating strains?[END_REF][START_REF] Berger | Hospital outbreak due to Clostridium difficile ribotype 018 (RT018) in Southern Germany[END_REF]. A study comparing RT 018, RT 126 and RT 078 indicated that RT 018 isolates produce higher levels of toxins and have an increased adhesion to cells [START_REF] Barbanti | Characterization of Clostridium difficile PCR-ribotype 018: A problematic emerging type[END_REF]. Moreover, all RT 018 strains were multidrug-resistant (resistance to erythromycin, clindamycin and moxifloxacin). These results suggest that RT 018 strains have phenotypic traits conferring an adaptive advantage and are able to spread widely.

Materials and methods

Setting

The outbreak took place in 2 geriatric wards from a single university-affiliated hospital in Strasbourg, France. The first ward is composed of 3 different units (A, B, C). The second ward is composed of 1 unit (D). These different geriatric units (GU) (A, B, C, D) are located on 4 floors of the same building. Each unit has between 24-28 beds in single or double rooms. In 2017, the first ward performed 4,774 admissions with a total of 32,900 patient-days (PD) and the second ward performed 816 admissions with a total of 8,622 patient-days. The incidence increased from 3.24/10000 PD in 2016 to 9.15/10000 PD in 2017.

CDI diagnosis

A CDI case was defined as a patient with a clinical diarrhea (≥ 3 diarrheal stools/day) and a positive test for C. difficile in stools. CDI testing was performed upon physician's request and systematically in case of nosocomial diarrhea. CDI testing was based on a two-step algorithm using glutamate dehydrogenase (GDH) detection by enzyme immunoassay (EIA) (C. Diff Quik Chek, Alere) as a screening method followed by PCR targeting toxin genes (GenXpert C. difficile, Cepheid). In addition, for all CDI cases, stools were cultured on ChromID plates (bioMérieux, Marcy l'Etoile, France) and C. difficile isolates were sent to the National Reference Laboratory for C. difficile (Paris) for molecular typing. CDI onset after 3 days of hospitalization or community-onset CDI in a patient with an overnight hospital stay within the previous 12 weeks was classified as hospital-associated CDI; others were communityassociated CDI [START_REF] Mcdonald | Recommendations for Surveillance of Clostridium difficile-Associated Disease[END_REF]. Patient information and medical history of all CDI cases during this outbreak were collected from the electronic medical records.

Strain characterization

Antibiotic susceptibility

Susceptibility to erythromycin (ERY), clindamycin (CM) moxifloxacin (MXF), metronidazole (MZ), vancomycin (VA) and tetracycline (TET) was determined using the disk diffusion method according to the CA-SFM 2013 (Comité de l'Antibiogramme de la Société Française de Microbiologie) [14]. Briefly, strain dilution (10 8 CFU/ml) was inoculated on Brucella Agar (Becton Dickinson) supplemented with vitamin K1 (1mg/ml) (Emprove, Merck), hemin (5 mg/l) (Applichem) and defibrinated horse blood (5%) (Oxoid). Plates were incubated 48h at 35-37°C and diameters were interpreted according to the criteria for anaerobic bacteria given by the CA-SFM 2013. Resistance breakpoints were set as follows: ERY (15 UI) < 15 mm; CM (2 UI) < 15 mm; MXF (5 μg) < 18 mm; MZ (5 μg) < 21 mm; VA (30 μg) < 17 mm; TET (30 μg) < 19 mm. C. difficile ATCC 700057 was used as quality control.

PCR-ribotyping (European CDI Surveillance Network: ECDIS-net protocol)

C. difficile isolates were characterized by high-resolution capillary gel-based electrophoresis PCR-Ribotyping [START_REF] Fawley | Development and validation of an internationally-standardized, high-resolution capillary gelbased electrophoresis PCR-ribotyping protocol for Clostridium difficile[END_REF] on the ABI 3500 sequencer (Applied Biosystems), using primers described by Bidet et al. [START_REF] Bidet | Development of a new PCRribotyping method for Clostridium difficile based on ribosomal RNA gene sequencing[END_REF]. The PCR ribotypes (RT) were determined using the freely available WEBRIBO database (https://webribo.ages.at/) [START_REF] Krutova | Molecular characterisation of Czech Clostridium difficile isolates collected in 2013-2015[END_REF].

Toxin gene profiles

Genes encoding toxin A (tcdA), toxin B (tcdB), and the two components of binary toxin (cdtA and cdtB) were detected from colonies by multiplex PCR, as previously described [START_REF] Barbut | Toxigenic Clostridium difficile carriage in general practice: results of a laboratory-based cohort study[END_REF] .

MLST (Multi locus sequence typing)

MLST was performed according to Griffith 's method [START_REF] Griffiths | Multi locus Sequence Typing of Clostridium difficile[END_REF]. Seven housekeeping genes (i.e. adk, atpA, dxr, glyA, recA, sodA and tpi) were amplified by PCR and sequenced as previously described [START_REF] Griffiths | Multi locus Sequence Typing of Clostridium difficile[END_REF]. Finally, the amplified sequences were used to query the database (http://pubmlst.org/cdifficile/) to obtain the sequence type (ST).

MLVA [Multi locus VNTR (variable number-tandem repeat) analysis] typing

Seven VNTR (A6, B7, C6, E7, F3, G8, H9) were amplified for MLVA analysis, using forward primers labeled at the 5' end with a fluorochrome, as described elsewhere [START_REF] Couturier | Spatio-temporal variability of the epidemic 027 Clostridium difficile strains in France based on MLVA typing[END_REF]. The number of tandem repeats was determined for each strain by capillary gel-based electrophoresis and concatenated to generate a MLVA type, using BioNumerics software (Applied Maths NV, Sint-Martens-Latem, Belgium). The genetic distance between two strains was determined by calculating the STRD (summed tandem repeat difference), corresponding to the differences in total number of repetitions for all loci. Genomic diversity was represented by the minimum spanning tree (MST) using the Manhattan coefficient. Isolates with an STRD ≤ 10 were defined as genetically related and clonal complexes were defined by an STRD ≤ 2 [START_REF] Marchh | Multi locus Variable-Number Tandem-Repeat Analysis for Investigation of Clostridium difficile Transmission in Hospitals[END_REF].

Whole genome sequencing (WGS)

We performed WGS on 31 strains. C. difficile isolates were subcultured on Columbia agar (bioMérieux) for 48 h in anaerobic atmosphere. DNA was extracted using UltraClean Microbial DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA). Then, the DNA library was performed using Nextera XT DNA library preparation and Nextera XT index kits (Illumina, San Diego, CA, USA). Finally, we generated 2x150 base paired-end reads using MiSeq Reagent kit v3 on a MiSeq instrument (Illumina). The quality of reads was evaluated with BioNumerics 7.6.3 (Applied Maths NV, Sint-Martens-Latem, Belgium): isolates with average quality below 30 and estimated coverage below 30 were excluded from subsequent analysis.

Whole genome multi locus sequence typing (wgMLST) analyses

wgMLST analyses were performed using BioNumerics 7.6.3. Briefly, alleles were identified by the combination of a kmer frequency approach using raw reads and assembly-based BLAST approach using SPAdes [START_REF] Bankevich | SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing[END_REF] assembled genomes against a pan-genome locus scheme of C. difficile developed by Applied Maths. This wgMLST scheme contains 8745 coding loci, representing a pan-genome of C. difficile identified from 259 previously published genomes [START_REF] Kiekens | H2?u=https%3A%2F%2Fww w.escmid.org%2Fescmid_publications%2Fescmid_elibrary%2Fmaterial%2F%3Fmid%3D6289 1[END_REF]. Once all alleles were assigned to each genome, a minimum spanning tree (MST) was constructed. Loci with no allele calls and loci with less than 80% identity were ignored in the pairwise comparison during the tree construction. WgMLST scheme is more detailed in the supplementary data. The genetic relationship between two isolates was assessed by calculating the number of different alleles for wgMLST. We defined 2 isolates as genetically related or belonging to the same complex clonal (CC) when they had an allelic difference ≤ 200 or ≤ 20, respectively.

Core-genome Single-Nucleotide polymorphism typing (cgSNP) analyses

Once the genomes were assembled (described above), one outbreak isolate was selected based on the quality of the assembly (lowest number of contigs, highest N50). This full genome was used as the reference genome for mapping and SNP calls after removal of contigs smaller than 1000 bp. Mapping was performed with Bowtie2 [START_REF] Langdon | Performance of genetic programming optimised Bowtie2 on genome comparison and analytic testing (GCAT) benchmarks[END_REF]. The genetic relationship between two isolates was assessed by calculating the number of SNP. SNPs were kept for analysis if the region was mapped in all genomes, if the coverage was sufficient (5 reads in total, at least 1 read forward and 1 read reverse), if none of the genomes contained ambiguous bases or gaps at this position and if no other SNP occurred within 12 bp. SNPs were visualized on the reference genome to identify hotspots of SNPs that could be the result of recombination and no evidence for recombination events were seen.

We defined 2 isolates as genetically related when they had a SNP difference ≤ 100 and in the same clonal complex ≤ 10.

Statistical analysis

Statistical analyses were performed using the R software, version 3.3.3 (R Core Team -2017 R Foundation for Statistical Computing, Vienna, Austria). Patient demographic characteristics were described by their mean and standard deviation for quantitative variables and by the frequency for categorical variables. Quantitative and categorical variables were compared between patients infected with RT 018 and those infected with another RT 018 by Mann-Whitney-Wilcoxon and Fisher exact tests. A p value of < 0.05 was considered significant.

Results and discussion

Fig. 1 shows an increase in the number of CDI cases in 2017 (38) compared to 2016 (13) and 2018 [START_REF] Langdon | Performance of genetic programming optimised Bowtie2 on genome comparison and analytic testing (GCAT) benchmarks[END_REF]. (Fig. 2) Most of the patients with CDI (18/38, 47%) were located in Unit B on the 4th floor (Fig. 2).

The peak of the outbreak occurred during the first 4 months of 2017 (22/38; 58%). Among the 38 CDI cases, 33 strains (isolated from 32 patients) were sent to the national reference laboratory for characterization. Nineteen strains (57.6%) belong to RT 018 whereas the remaining 14 belong to RT 020 (9.1%), RT 106 (9.1%), RT 015 (6.1%), RT 014 They all belong to ST 17, MLST clade 1.

We compared the clinical features of patients infected with RT 018 (n = 19), to those infected with other RT (n = 13) (Table 1). No significant difference was observed for age, gender, leukocyte count > 15,000 cells/mm 3 , incontinence, antibiotic treatment in the previous 90 days, and origin of CDI. Nevertheless, patients infected with RT 018 strains had a higher 90-day all-cause mortality rate compared to those infected by other RT. These data are consistent with the higher virulence of RT 018 which has been associated with a higher toxin production, increased adhesion to cells and an enhanced sporulation rate [START_REF] Barbanti | Characterization of Clostridium difficile PCR-ribotype 018: A problematic emerging type[END_REF]. These microbiological features could also in part explain the rapid local spreading and interpatient transmissibility observed for strains RT 018 in the hospital environment.

Table 2 compares antimicrobial susceptibility testing of 19 RT 018 strains and 14 other RT strains. All 33 strains were susceptible to metronidazole and vancomycin but resistant to clindamycin. However, resistance rate to erythromycin and moxifloxacin was significantly higher for RT 018 compared to the other RT (p < 0.001). Erythromycin and moxifloxacin resistance has been described in RT 018 outbreak strains from Korea, Germany and Japan [START_REF] Berger | Hospital outbreak due to Clostridium difficile ribotype 018 (RT018) in Southern Germany[END_REF][START_REF] Kim | Association between PCR ribotypes and antimicrobial susceptibility among Clostridium difficile isolates from healthcare-associated infections in South Korea[END_REF][START_REF] Wakamatsu | Predominance of PCR-ribotypes, 018 (smz) and 369 (trf) of Clostridium difficile in Japan: a potential relationship with other global circulating strains?[END_REF]. In Italy, an analysis was performed on a large number of strains and showed that 100% of RT 018 were erythromycin, moxifloxacin and clindamycin resistant compared to only 75% for RT 027 [START_REF] Spigaglia | Diagnostic testing for Clostridium difficile in Italian microbiological laboratories[END_REF]. The high-level of fluoroquinolone resistance is a very common trait for epidemic RT and may have conferred a selective advantage to strains RT 018 in patients treated by antibiotics [START_REF] He | Emergence and global spread of epidemic healthcare-associated <i>Clostridium difficile</i[END_REF].

Recent studies have shown that WGS has a superior discriminatory power compared to other molecular methods [START_REF] Parkhill | Bacterial epidemiology and biology -lessons from genome sequencing[END_REF][START_REF] Pallen | Are diagnostic and public health bacteriology ready to become branches of genomic medicine?[END_REF]. The accuracy of WGS allows different approaches to assess genomic variations: nucleotide polymorphisms and allelic profiles (wgMLST or cgMLST) [START_REF] Chen | Whole Genome and Core Genome Multi locus Sequence Typing and Single Nucleotide Polymorphism Analyses of Listeria monocytogenes Isolates Associated with an Outbreak Linked to Cheese, United States[END_REF]. Recently, a study comparing a set of different C. difficile strains showed that the cgMLST was as discriminatory as the SNP analysis [START_REF] Bletz | Defining and Evaluating a Core Genome Multi locus Sequence Typing Scheme for Genome-Wide Typing of Clostridium difficile. Diekema DJ[END_REF]. In the present study, we determined the genetic relationship between RT 018 by cgSNP typing and wgMLST method and we compared the results with MLVA. Indeed, MLVA has been previously used to successfully subtype strains from the same PCR-ribotype [START_REF] Couturier | Spatio-temporal variability of the epidemic 027 Clostridium difficile strains in France based on MLVA typing[END_REF].

We performed MLVA and WGS on 31 strains: 17 RT 018 strains (CD17-039, CD17-041, CD17-042, CD17-043, CD17-124, CD17-125, CD17-126, CD17-127, CD17-128, CD17-132, CD17-133, CD17-134, CD17-136, CD17-244, CD17-245, CD17-248, CD17-313) isolated from the GU in 2017 and 2 RT 018 strains isolated in the same GU in 2015 (CD15-230, CD15-235). We also included 12 strains isolated outside of the hospital in Strasbourg: 9 epidemiologically unrelated RT 018 strains isolated in Paris (SA11-109, SA11-123, Lu15-003), Caen (Lu14-078), Montélimar (CD14-251), Cholet (CD17-161), Toulon (CD15-058), and unknown origin (Co2566), and 3 RT 018 strains isolated from a local HCF in Strasbourg (CD17-179, CD17-180, CD17-200). Among these 3 strains, 2 strains (CD17-179, CD17-200) were isolated from recurrences in a patient who presented a first episode of CDI (CD17-041) at a Strasbourg hospital 6 months before.

The minimum spanning tree analysis by MLVA is presented in Fig. 3. The analysis indicated that among the 31 RT 018 strains, 19 were included in 2 close CC separated by 5 STRD on 2 loci. The first one (cluster 1) comprised 9 strains (53%), all isolated in patients from the GU and the second one (cluster 2) included 10 strains (6 strains (35%) from the GU and 4 strains (36%) from other HCF (CD15-058, CD17-179, CD17-180, CD17-200). Four strains (21%) of the GU did not belong to the CC including two epidemic strains isolated in 2017 and 2 strains isolated in 2015. These results suggest that MLVA can successfully subtype strains belonging to the same RT, as previously shown for RT027, RT0021 and RT076 [START_REF] Couturier | Spatio-temporal variability of the epidemic 027 Clostridium difficile strains in France based on MLVA typing[END_REF][START_REF] Krutova | Czech Clostridium difficile study group. Clostridium difficile PCR ribotypes 001 and 176 -the common denominator of C. difficile infection epidemiology in the Czech Republic, 2014[END_REF].

The minimum spanning tree analysis of wgMLST and cgSNP typing are showed in Fig. 4 and Fig. 5.

wgMLST and cgSNP typing gave consistent information. A recent study investigating

Staphylococcus aureus outbreaks has shown that these two methods provide comparable epidemiological discrimination, but that they vary in their overall suitability for epidemiologic or infection control settings [START_REF] Cunningham | Comparison of Whole-Genome Sequencing Methods for Analysis of Three Methicillin-Resistant Staphylococcus aureus Outbreaks. Diekema DJ[END_REF]. All the RT 018 isolates from the outbreak clustered in the same CC that also included 4 strains (36%) from other HCF (CD15-058, CD17-179, CD17-180, CD17-200). Among these 4 strains, 1 (CD17-180) was isolated in another HCF in Strasbourg and 1 (CD15-058) was isolated in a city far from Strasbourg. This strain was also present in one of the 2 CC generated by MLVA. We failed to collect information about these patients and their medical history but we can hypothesize that they likely had connections with the present outbreak or were exposed to a common source. The 2 other strains present in the CC (CD17-179 and CD17-200) came from the same patient who had presented a first episode of CDI (CD17-041) in a Strasbourg hospital, 6 months before.

However, the strains CD17-179 and CD17-200 were different from CD17-041 by 1 allele as showed by wgMLST. In contrast to whole genome SNP analysis, wgMLST is based on the concept of allelic variation, meaning that recombinations, deletions or insertions of multiple positions are indeed counted as a single evolutionary event. This approach might be biologically more relevant than approaches that consider only point mutations, as it both captures isolated point mutations as well as close-by point mutations or gene exchanges that occur as one event in the DNA. So, this variation suggests a slight evolution over time.

wgMLST and cgSNP typing analyses also indicated that the 2 RT 018 strains isolated in 2015 in the geriatric unit belonged to the same CC, suggesting that this clone was already present in the ward, either in the environment or in asymptomatic carriers, and emerged again in 2017. The 2 cases of CDI in 2015 appeared 2 months apart. However, in Strasbourg hospital, specific hygiene measures are implemented only when 2 cases of CDI occur in the same unit within 15 days. These 2 cases were therefore not considered linked, and no specific hygiene measure were implemented, most likely contributing to dissemination of the strain in 2017.

During the CDI outbreak in 2017, the hygiene measures were reinforced by a daily cleaning/disinfection using a sporicidal product (Oxyfloor, Anios).

Considering that the outbreak was well documented and occurred in a short period of time and in a restricted space, we were expecting a cluster of epidemic isolates. Our results showed that wgMLST and cgSNP typing gave more relevant results than MLVA from an epidemiological prospective. A recent study showed that, compared to the WGS, the absolute numbers of STRD obtained by MLVA can over-or under-estimate direct relatedness between epidemiologic isolates due to method-related reasons (low number of loci explored, high variability of the genetic targets) [START_REF] Berger | Hospital outbreak due to Clostridium difficile ribotype 018 (RT018) in Southern Germany[END_REF]. Zhou et al. compared cgSNP typing with MLST and highlighted the ability of cgSNP typing to differentiate several lineages within the same MLST clade [START_REF] Zhou | Phenotypic and Genotypic Analysis of Clostridium difficile Isolates: a Single-Center Study[END_REF].

WGS-based methods seem to be the most accurate methods for investigating outbreaks and will likely outweigh very shortly MLST and MLVA which are currently widely used in microbiological laboratories [35,36] [37].

Conclusion

This first outbreak of C. difficile RT 018 in France associated with a high 90-day all-cause mortality rate confirmed the potential of dissemination and the high virulence of this emerging ribotype. MLVA, wgMLST and cgSNP typing analysis of the isolates gave quite consistent information but the wgMLST and the cgSNP typing better separated epidemic strains from non-epidemic strains.

1

 1

Fig. 1 .Fig. 2 . 3 Fig. 3 .

 1233 Fig. 1. Evolution of the number of CDI cases per year during the years 2016, 2017 and 2018.

Fig. 4 .

 4 Fig. 4. wgMLST analysis with minimum spanning tree of PCR-ribotype 018 strains. A different color is assigned to each HCF. Each circle represents a single wgMLST-type, the size of the circle being proportional to the number of isolates included. The numbers between the circles correspond to the number of allele between the wgMLST types. wgMLST types differing on 1 to 3 loci are connected by solid lines, those differing on 4 or more loci are connected by a dashed line. The shaded areas represent clusters, grouping the genetically linked strains.

Fig. 5 .

 5 Fig. 5. cgSNP typing analysis with minimum spanning tree of PCR-ribotype 018 strains. A different color is assigned to each HCF. Each circle represents a single cgSNP type, the size of the circle being proportional to the number of isolates included. The numbers between the circles correspond to the number of SNP between the cgSNP types. cgSNP types differing on 1 to 3 loci are connected by solid lines, those differing on 4 or more loci are connected by a dashed line. The shaded areas represent clusters, grouping the genetically linked strains.

1 Table 1 .

 11 Clinical features of 32 CDI patients p values were calculated using Mann-Whitney Wilcoxon or Fisher's exact test.

	1
	Variables	RT 018	Other RT	p a
	Age (mean ± SD), years	86 ± 8	88 ± 6	0.324
	Gender (Male), n/N (%)	8/19 (42)	4/13 (31)	0.713
	Leukocyte count (> 15 000 cells/mm3), n/N (%)	6/18 (33)	2/11 (18)	0.671
	Incontinence, n/N (%)	9/16 (56)	9/12 (75)	0.434
	Antibiotic (in the previous 90 days), n/N (%)	18/19 (95)	13/13 (100)	1
	Hospital-associated CDI, n/N (%)	18/19 (95)	12/13 (92)	1
	90-days all causes mortality, n/N (%)	12/19 (63)	3/13 (23)	0.036

a

We report here the first RT 018-related outbreak in geriatric units in France that was investigated by different molecular typing techniques including MLVA (multi locus VNTR [variable number-tandem repeat] analysis), wgMLST (whole genome multi locus sequence typing) and cgSNP typing (core genome single nucleotide polymorphism).


Céline Hernandez

Etienne B Blanc

Véronique Pène

Béatrice Le-Grand

Maxime Villaret

Lynda Aoudjehane

Arnaud Carpentier

Filomena Conti

Yvon Calmus

Philippe Podevin

Michèle Garlatti

Hélène Rouach

Arielle R Rosenberg
email: arielle.rosenberg@parisdescartes.fr

Arr Contributed

Impact of hepatitis C virus and alcohol, alone and combined, on the unfolded protein response in primary human hepatocytes

Keywords: Hepatitis C virus Alcohol Unfolded protein response Primary human hepatocytes 4 HCV, hepatitis C virus, UPR, unfolded protein response, ER, endoplasmic reticulum, BiP, immunoglobulin heavy-chain-binding protein, GRP94, 94 kDa glucoseregulated protein, ERAD, ER-associated degradation, EDEM, ER degradation infection, ffu, focus-forming units, RT-qPCR, reverse-transcription quantitative PCR, RPL13A, 60S ribosomal protein L13A, gDNA, genomic DNA, XBP1, X-box binding protein 1, LDH, lactate dehydrogenase, CYP2E1, cytochrome P450 2E1, ADHs, alcohol dehydrogenases, IRE1, inositol-requiring enzyme 1

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

Introduction

Hepatitis C virus (HCV) infection and excessive alcohol consumption are leading causes of chronic liver disease which can progress from steatosis to fibrosis, cirrhosis and hepatocellular carcinoma. In addition, these two factors often coexist in the same individual, their association leading to more severe liver injury, accelerated fibrosis progression and increased risk for the development of cirrhosis and hepatocellular carcinoma [START_REF] Bhattacharya | Hepatitis C and alcohol: interactions, outcomes, and implications[END_REF][START_REF] Mueller | Alcoholic liver disease and hepatitis C: a frequently underestimated combination[END_REF][START_REF] Punzalan | Alcoholic hepatitis and HCV interactions in the modulation of liver disease[END_REF].

Accordingly, chronic liver disease caused by HCV infection and alcohol abuse alone and in combination represents a major health problem, yet the pathogenesis of hepatic injury remains largely elusive partly due to the multifactorial and intricate nature of the underlying mechanisms. Moreover, research in this field has long been hampered by the lack of a relevant experimental model to study the impact of HCV and alcohol on their primary target cell, the normal human adult hepatocyte.

Among the cellular responses to various stresses, the unfolded protein response (UPR) has emerged as an important contributor for a number of human diseases including, in particular, hepatic steatosis [START_REF] Malhi | Endoplasmic reticulum stress in liver disease[END_REF][START_REF] Imrie | Stress management: How the unfolded protein response impacts fatty liver disease[END_REF][START_REF] Lebeaupin | Endoplasmic reticulum stress signalling and the pathogenesis of non-alcoholic fatty liver disease[END_REF], which is often the first symptom of chronic liver disease in patients with HCV infection and/or alcohol abuse. The UPR is a cellular program which is triggered upon disruption of homeostasis of the endoplasmic reticulum (ER), the cellular organelle responsible for folding and maturation of membrane-bound and secreted proteins, a situation termed ER stress [START_REF] Walter | The unfolded protein response: from stress pathway to homeostatic regulation[END_REF]. The UPR aims to restore ER homeostasis (i) by increasing ER folding capacity through induction of ER-resident molecular chaperones such as immunoglobulin heavy-chain-binding protein (BiP) and 94 kDa glucose-regulated protein (GRP94) and (ii) by activating ER-associated degradation (ERAD) of misfolded proteins through induction of ERAD components such as ER degradation-enhancing α-mannosidase-like protein (EDEM) or homocysteine-induced ER protein (HERP). Aside from these two signalling pathways specific to ER stress, another branch of the UPR is shared by the so-called integrated stress response (ISR), which can be triggered by multiple stressors including viruses and alcohol [START_REF] Magne | ATF4 and the integrated stress response are induced by ethanol and cytochrome P450 2E1 in human hepatocytes[END_REF]. This common pathway proceeds through phosphorylation of the eukaryotic initiation factor 2α (eIF2α) which leads to the attenuation of global protein synthesis and induction of activating transcription factor (ATF) 4. The latter, in turn, up-regulates growth arrest and DNA damage-inducible protein 34 (GADD34), which can activate eIF2α dephosphorylation and hence end the translational arrest; other up-regulated targets are involved in cell fate such as ATF3, cation transport regulator-like protein 1 (CHAC) and C/EBP homologous protein (CHOP), which contribute to cell cycle arrest and trigger apoptosis in cases of overwhelming stress. Thus, the UPR aims primarily at coping with ER stress and preserving cell survival, but when the severity and/or prolongation of the stress exceed a poorly defined threshold, the response can switch to a cell death commitment [START_REF] Imrie | Stress management: How the unfolded protein response impacts fatty liver disease[END_REF][START_REF] Jäger | The unfolded protein response at the crossroads of cellular life and death during endoplasmic reticulum stress[END_REF].

HCV is expected to cause ER stress because most steps of the virus infectious cycle take place in the ER or membranes derived thereof [START_REF] Chan | Unfolded protein response in hepatitis C virus infection[END_REF][START_REF] Vasallo | Cellular stress responses in hepatitis C virus infection: Mastering a two-edged sword[END_REF]. However, whether the UPR is induced by HCV has been the matter of conflicting publications, partly attributable to differences in the experimental systems used, the UPR branches and targets examined and the timeframe chosen before readout. Expression of the HCV genome in transgenic mice was either shown to activate [START_REF] Merquiol | HCV causes chronic endoplasmic reticulum stress leading to adaptation and interference with the unfolded protein response[END_REF] or not [START_REF] Lerat | Hepatitis C virus proteins induce lipogenesis and defective triglyceride secretion in transgenic mice[END_REF] the UPR. Cell-culture-based studies involving overexpression of viral proteins independently of an infection process suggested that while inducing ER stress, HCV proteins may modulate specific components of the UPR and hence alter the typical course of UPR signalling [START_REF] Chan | Unfolded protein response in hepatitis C virus infection[END_REF][START_REF] Vasallo | Cellular stress responses in hepatitis C virus infection: Mastering a two-edged sword[END_REF]. When a system enabling productive HCV infection became available using clones of the hepatocarcinoma-derived Huh-7 cell line, UPR activation was shown to occur in infected cells, with a strong induction of pro-apoptotic genes obviously coinciding with a phase of massive cell death [START_REF] Merquiol | HCV causes chronic endoplasmic reticulum stress leading to adaptation and interference with the unfolded protein response[END_REF][START_REF] Zhong | Persistent hepatitis C virus infection in vitro: coevolution of virus and host[END_REF][START_REF] Sekine-Osajima | Development of plaque assays for hepatitis C virus-JFH1 strain and isolation of mutants with enhanced cytopathogenicity and replication capacity[END_REF][START_REF] Saeed | Role of the endoplasmic reticulum-associated degradation (ERAD) pathway in degradation of hepatitis C virus envelope proteins and production of virus particles[END_REF]. However, the transformed Huh-7 cells diverge significantly from the normal, quiescent, highly differentiated hepatocytes that are the natural host cells of HCV. The only laboratory which has used the highly relevant cell-culture model of primary human adult hepatocytes (PHH) reported that the UPR was induced upon inoculation with a modified virus replicating at low levels [START_REF] Chandra | Impaired expression of type I and type II interferon receptors in HCV-associated chronic liver disease and liver cirrhosis[END_REF]. Finally, studies using liver biopsies from HCV-infected patients led to widely divergent results as to the occurrence of UPR activation [START_REF] Chandra | Impaired expression of type I and type II interferon receptors in HCV-associated chronic liver disease and liver cirrhosis[END_REF][START_REF] Asselah | In vivo hepatic endoplasmic reticulum stress in patients with chronic hepatitis C[END_REF][START_REF] Mcpherson | No evidence of the unfolded protein response in patients with chronic hepatitis C virus infection[END_REF].

An alcohol-induced UPR was first reported in the liver of mice subjected to intragastric ethanol feeding, which involved ER chaperones (BiP and GRP94) and transcriptional factors such as CHOP [START_REF] Ji | Betaine decreases hyperhomocysteinemia, endoplasmic reticulum stress, and liver injury in alcohol-fed mice[END_REF]. The occurrence of hepatocellular UPR was not systematically observed upon alcohol exposure but has been described in various animal models and in human alcoholics [START_REF] Longato | Insulin resistance, ceramide accumulation, and endoplasmic reticulum stress in human chronic alcohol-related liver disease[END_REF][START_REF] Ji | New insights into the pathogenesis of alcohol-induced ER stress and liver diseases[END_REF]. An in vitro model based on human hepatocytes is required to gain insight into the hepatocellular response induced by alcohol, but hepatocarcinoma-derived cell lines lack physiological relevance as they retain only a weak, if any, expression of ethanolmetabolizing enzymes.

We have developed a robust experimental system which enables productive HCV infection to be achieved in PHH maintained in a differentiated state for at least 2 weeks; this provides a long-awaited model which combines virological and physiological relevance [START_REF] Podevin | Production of infectious hepatitis C virus in primary cultures of human adult hepatocytes[END_REF]. Here, we have taken advantage of this unique model to reassess, systematically, the occurrence of activation of UPR target genes in human hepatocytes infected with HCV or exposed to ethanol during the long term, and to investigate for the first time the simultaneous effect of the two stressors.

Material and methods

Statement of ethical approval

Procedures for PHH isolation [START_REF] Podevin | Production of infectious hepatitis C virus in primary cultures of human adult hepatocytes[END_REF] were performed in accordance with the relevant French laws and regulations. All the liver tissue specimens were obtained in the course of routine work during surgical procedures carried out in Paris Public Hospitals (Assistance Publique-Hôpitaux de Paris (AP-HP), France). For all samples, informed consent was obtained from the patients, no-opposition statements were signed, and the specimens were anonymized. This procedure was approved by an Ethics Committee (Comité de Protection des Personnes (CPP) Ile-de-France III) and by the French Ministry of Health (References COL 2929 and COL 2930).

Isolation and primary culture of human hepatocytes

PHH were isolated from normal-appearing liver tissue obtained from adult patients undergoing hepatectomy for the therapy of metastases or benign tumours and seronegative for HCV, hepatitis B virus and human immunodeficiency virus. Hepatocytes were isolated by a two-step perfusion technique and maintained in primary culture as described previously [START_REF] Podevin | Production of infectious hepatitis C virus in primary cultures of human adult hepatocytes[END_REF].

Briefly, the liver pieces were perfused with Liver Perfusion Medium (Life Technologies) followed by collagenase-and dispase-containing Liver Digest Medium (Life Technologies) at 37 °C until full digestion. After centrifugation at 200 × g for 1 min, hepatocytes were washed and seeded at a density of 1.2-1.5 × 10 5 viable cells/cm 2 onto 6-or 12-well plates pre-coated with Bornstein and Traub type I collagen (Sigma-Aldrich). PHH were maintained at 37 °C in a humidified 5% CO2 atmosphere, in complete hepatocyte medium consisting of Leibovitz's L-15 medium (Life Technologies) supplemented with 26 mM NaHCO3, 100 IU/L insulin (Novo Nordisk) and 10% heat-inactivated foetal calf serum (PAA laboratories). The medium was replaced 16-24 h after seeding with fresh complete hepatocyte medium complemented with 1 µM hydrocortisone hemisuccinate (SERB) until HCV inoculation and/or ethanol treatment 2 days later.

HCV inoculation and treatment of PHH

High-titre stocks of HCV derived from Huh-7.5.1 cell culture (HCVcc) were prepared as described previously [START_REF] Podevin | Production of infectious hepatitis C virus in primary cultures of human adult hepatocytes[END_REF][START_REF] Beilstein | Lysophosphatidylcholine acyltransferase 1 is downregulated by hepatitis C virus: impact on production of lipo-viro-particles[END_REF]. Briefly, HCVcc preparations used in a previous study [START_REF] Podevin | Production of infectious hepatitis C virus in primary cultures of human adult hepatocytes[END_REF] were subjected to additional rounds of virus amplification by inoculation of naïve Huh-7.5.1 cells (at a multiplicity of infection (MOI) of 0.01 focus-forming units (ffu) per cell) which were then grown until infectious titres (see below for HCV titration method) reached at least 10 7 ffu/mL. Culture supernatants containing high titres of HCVcc were passed through cellulose acetate membrane filters of a 0.45-µm pore size (ClearLine R , Dutscher), then concentrated approximately 10-fold in an Amicon Ultra-15 100,000 molecular weight cutoff ultrafiltration device (Millipore). The viral stocks obtained were aliquoted and stored at -80 °C until they were used to inoculate PHH 3 days after seeding (MOI = 0.1 ffu/cell), as previously described [START_REF] Podevin | Production of infectious hepatitis C virus in primary cultures of human adult hepatocytes[END_REF][START_REF] Beilstein | Lysophosphatidylcholine acyltransferase 1 is downregulated by hepatitis C virus: impact on production of lipo-viro-particles[END_REF]. Briefly, the medium was replaced with the inoculum diluted in the smallest volume of fresh complete hepatocyte medium sufficient for covering the cells. For control experiments referred to as "mock", UV-inactivated virus was prepared as described previously [START_REF] Song | Thermal stability and inactivation of hepatitis C virus grown in cell culture[END_REF][START_REF] Aoudjehane | Infection of human liver myofibroblasts by hepatitis C virus: a direct mechanism of liver fibrosis in hepatitis C[END_REF]: the same HCVcc stock diluted in fresh complete hepatocyte medium was exposed to continuous UVC light for 2 min beneath a UVC lamp (wavelength: 253.7 nm), and used as a non-infectious "inoculum" after the residual infectivity was assessed by HCV titration assay to verify that the infectious titre had indeed fallen below the threshold of detection of 10 ffu/mL. In all cases, the inoculum was removed after a 16-h incubation at 37 °C, and cell monolayers were washed 3 times with phosphate-buffered saline. PHH were maintained in complete hepatocyte medium, half of the medium being renewed every 3 days.

Ethanol (100 mM) was added to the medium for the indicated periods of time; the loss of ethanol due to evaporation was assessed by the ECET-100 EnzyChrom TM Ethanol Assay Kit (BioAssay Systems) and compensated daily. Where indicated, thapsigargin (Sigma-Adrich) was added at 300 nM for the last 4 h of culture.

Gene expression analysis

Gene expression was analysed by reverse-transcription quantitative PCR (RT-qPCR).

RNA was extracted using RNeasy ® Mini Kit (Qiagen) and reverse transcribed with the High-Capacity cDNA Archive Kit (Applied Biosystems). Real-time PCR was performed on an ABI Prism ® 7900HT Sequence Detection System (Applied Biosystems). The relative amounts of mRNA were estimated by the delta-delta Ct method [START_REF] Livak | Analysis of relative gene expression data using real-time quantitative PCR and the 2 -∆∆CT method[END_REF] using the gene encoding 60S ribosomal protein L13A (RPL13A) as the reference gene and mock-infected cells not exposed to any stressor and harvested the same day as control cells. The primer sequences are given in the Supplementary Table S1. Of note, the forward and reverse primers for RPL13A, which was used as the reference gene in all experiments, were designed to target two different exons (exon 7 and exon 8, respectively, which are separated on genomic DNA (gDNA) by a 181-bp intron); accordingly, the qPCR species generated from reverse-transcribed RNA (i.e., cDNA) and from gDNA are different (of 124 bp and 305 bp in size, respectively). The absence of the second species in the qPCR products permitted to ascertain that the RNA samples were devoid of gDNA contamination.

Analysis of X-box binding protein 1 (XBP1) transcript splicing

PCR for XBP1 transcripts was performed using 5'-GGGAATGAAGTGAGGCCAGT-3' and 5'-TGAAGAGTCAATACCGCCAGA-3' as forward and reverse primers, respectively, and the following conditions: 40 cycles of denaturation (at 95 °C for 30 s) followed by annealing and elongation (at 60 °C for 1 min). PCR products were resolved by electrophoresis on a 2%-agarose gel, and visualized using ethidium bromide and UV trans-illumination. The expected size for the bands corresponding to the spliced (XBP1-S) and unspliced (XBP1-U) forms of XBP1 transcript was 111 and 137 bp, respectively. The additional 163-bp band corresponded to a hybrid amplicon (XBP1-H). The density of each band was quantified using ImageJ software.

HCV replication assay

To assess HCV replication, intracellular levels of negative-strand HCV RNA were quantified by a strand-specific RT-qPCR technique described previously (threshold of detection: 25 copies/reaction) [START_REF] Carrière | A novel, sensitive, and specific RT-PCR technique for quantitation of hepatitis C virus replication[END_REF].

Cytotoxicity assay

Cytotoxicity was assessed by measuring the activity of lactate dehydrogenase (LDH) released into culture supernatants using the CytoTox 96 ® Non-Radioactive Cytotoxicity Assay (Promega) as per the manufacturer's instructions. Cytotoxicity was calculated as the ratio of LDH leakage from the cells under study versus control cells not exposed to any stressor at the same day normalized to cell number.

Cell line culture

Huh-7.5.1 cells (a kind gift from F. V. Chisari, Scripps Research Institute, La Jolla, CA, USA) were grown in Dulbecco's modified Eagle's medium (Life Technologies), supplemented with 10 mM HEPES (pH 7.3), 1% non-essential amino-acids (Life Technologies), 2 mM L-glutamine (Life Technologies) and 10% foetal calf serum (PAA Laboratories) at 37 ºC in a humidified 5% CO2 atmosphere. The same HCVcc stocks used to inoculate PHH were used to inoculate Huh-7.5.1 cells at the same MOI.

HCV titration

Infectious titres in viral stocks or in filtered culture supernatants of HCVcc-infected PHH or Huh-7.5.1 cells were determined by focus-formation assay (threshold of detection: 10 ffu/mL) as described elsewhere [START_REF] Pène | Sequential processing of hepatitis C virus core protein by host cell signal peptidase and signal peptide peptidase: a reassessment[END_REF].

Western blotting

Total cell lysates were prepared using RIPA buffer (50 mM Tris-HCl pH 7.9, 1% NP40, 150 mM NaCl, 0.1% sodium dodecyl sulphate, 0.5% sodium deoxycholate) supplemented with protease and phosphatase inhibitor cocktails (Sigma-Aldrich). Cell lysates were electrophoresed in sodium dodecyl sulphate-12% polyacrylamide gels under reducing conditions, in parallel with protein molecular mass standards (PageRuler TM Prestained Protein Ladder, ThermoFisher), and, subsequently, transferred onto nitrocellulose membranes (Bio-Rad). Membranes were blocked with Odyssey Blocking buffer (LI-COR) for 1 h at room temperature, and then probed overnight at 4 °C with primary antibodies against cytochrome P450 2E1 (CYP2E1) or alcohol dehydrogenases (ADHs) diluted in the same buffer. These were a rabbit antibody against CYP2E1 (a kind gift from I. de Wazier, Inserm UMRS-1147, Paris, France), a goat antibody against pan-ADH1 (Acris, catalog number #AP16311PU-N; diluted 1:1,000) or a rabbit antibody against ADH4 (Abgent, catalog number #AP10128b; diluted 1:1,000). Membranes were reprobed with a mouse monoclonal antibody (Abcam, catalog number #ab8226; diluted 1:5,000) or a rabbit polyclonal antibody (Abcam, catalog number #ab8227; diluted 1:5,000) against β-actin. IRDye R 800CW goat anti-rabbit IgG (LI-COR, catalog number #926-32211), IRDye R 800CW donkey anti-goat IgG (LI-COR, catalog number #926-32214) and IRDye R 650 goat anti-mouse IgG (LI-COR, catalog number #926-65010) were used as secondary antibodies, all of them being diluted 1:10,000. After the final wash with phosphate-buffered saline containing 0.1% Tween-20, the signals were quantified using an Odyssey Infrared Imager (LI-COR).

Statistical analysis

The data are expressed as the means ± SEM of the indicated number of independent experiments, i.e., experiments performed with PHH originating from different donors.

Statistical analysis was carried out using R (agricolae package) software. The Kruskal-Wallis test (nonparametric comparison of k independent series) followed by the Fisher's least significant difference post-hoc method were performed to assess statistical significance (-, non-significant; *, p<0.05; **, p<0.01; ***, p<0.001).

Results

Effect of HCV infection on the hepatocellular UPR

To reassess whether HCV infection induces the hepatocellular UPR, PHH were inoculated with HCVcc at an MOI of 0.1, and transcript levels of key target genes of the different branches of the UPR were measured by RT-qPCR on different days over a period of 2 weeks after inoculation. Supplementary Fig. S1 (left and middle columns) presents the kinetics of UPR gene induction along with viral parameters (HCV genome replication and infectious titres) for two independent preparations of PHH (i.e., originating from two different donors), and Fig. 1a shows the changes in transcript levels of UPR markers as means ± SEM of at least 6 independent experiments. While the kinetics of induction of different markers could vary slightly from one PHH preparation to another (Supplementary Fig. S1a and Fig. S1b, compare the left and middle columns), the statistical analysis of independent experiments with PHH isolated from at least 6 different donors showed a significant mean increase of approximately 1.5-to 2-fold at day 3 post-inoculation for all the markers examined, including the ER chaperones BiP and GRP94, the ERAD components HERP and EDEM (Fig. 1a, left panel) and genes shared by the ISR (Fig. 1a, right panel). Similar levels of gene transcripts were maintained at day 9 post-inoculation, except for GRP94, ATF3 and CHAC the expressions of which returned to baseline. Thus, HCV infection in PHH induced a complete and moderate UPR.

Because this pattern seemed widely different from the cellular response described in Huh-7 sublines [START_REF] Merquiol | HCV causes chronic endoplasmic reticulum stress leading to adaptation and interference with the unfolded protein response[END_REF][START_REF] Saeed | Role of the endoplasmic reticulum-associated degradation (ERAD) pathway in degradation of hepatitis C virus envelope proteins and production of virus particles[END_REF], we compared PHH and Huh-7.5.1 cells upon inoculation with the same HCVcc stock at the same MOI. As expected in the actively dividing Huh-7.5.1 cells [START_REF] Podevin | Production of infectious hepatitis C virus in primary cultures of human adult hepatocytes[END_REF], the viral parameters, i.e., intracellular amounts of negative-strand HCV RNA (a hallmark of HCV genome replication) and extracellular infectious titres, increased during the first 6 days after inoculation, and reached levels that, compared to those attained in PHH, were higher by 2-3 orders of magnitude (Supplementary Fig. S1c, compare right column with left and middle columns). In Huh-7.5.1 cells no induction was observed for any of the ER chaperones at any day post-inoculation (Supplementary Fig. S1a, right column, and Supplementary Fig. S2a), in agreement with published data [START_REF] Saeed | Role of the endoplasmic reticulum-associated degradation (ERAD) pathway in degradation of hepatitis C virus envelope proteins and production of virus particles[END_REF]. Expression of ERAD components was not induced at day 3 but increased significantly at day 9 post-inoculation and, most strikingly, a marked induction of ISR genes was observed at this time (Supplementary Fig. S1b, right column, and Supplementary Fig. S2a), consistent with previous observations [START_REF] Merquiol | HCV causes chronic endoplasmic reticulum stress leading to adaptation and interference with the unfolded protein response[END_REF]. Interestingly, this marked induction of pro-apoptotic genes without concomitant induction of ER chaperones coincided with a phase of extensive cell death visible by microscopic examination of the cultures from day 6 to day 12 post-inoculation (Supplementary Fig. S2b) and documented by a marked decline in cell count (Supplementary Fig. S1d, right column) and a massive leakage of LDH into the culture supernatant (Supplementary Fig. S2c). In contrast, daily examination of PHH monolayers showed no sign of cytotoxicity attributable to HCV infection at any time point of the 2-week kinetics post-inoculation (Fig. 2a), and no decrease in cell count (Supplementary Fig. S1d, left and middle columns) or significant increase in LDH release (Fig. 2b) was observed in HCV-infected compared to mock-infected PHH. Collectively, the results show that the levels of HCV infection, the nature and magnitude of UPR activation and the resulting cell fate were completely different in normal, quiescent PHH compared to transformed, rapidly-dividing Huh-7.5.1 cells.

Effect of alcohol on the hepatocellular UPR

Ethanol is metabolized in hepatocytes by ADHs 1 (class I) and 4 (class II) and CYP2E1.

We previously showed that the expression of the CYP2E1 transcript was maintained in PHH throughout a 2-week culture period under our conditions, at levels that largely exceeded those found in Huh-7.5.1 cells [START_REF] Podevin | Production of infectious hepatitis C virus in primary cultures of human adult hepatocytes[END_REF]. Here, we further verified the expression of both ADHs and CYP2E1 at the protein level. Indeed, whereas these enzymes were expressed only weakly or not at all in Huh-7.5.1 cells at any time point of a 9-day time course, even upon ethanol exposure, they were readily detected in PHH at day 3 (Supplementary Fig. S3a) and their expression was maintained at similar levels at day 9 (Supplementary Fig. S3b). Moreover, ethanol further increased the levels of CYP2E1 in PHH at any time point (Supplementary Fig. S3b), which confirms the maintenance of the inducibility of this enzyme throughout the culture period. PHH thus represent a suitable model for studying the hepatocellular impact of alcohol.

To reassess whether alcohol activates the hepatocellular UPR, PHH were exposed to 100 mM ethanol during a prolonged period of 9 days. For all the UPR markers tested, an approximate 2-to 4-fold increase in gene expression was observed at day 3 and the transcript levels did not return to baseline at day 9 of the kinetics (Fig. 1b). This complete UPR elicited by ethanol in PHH was not accompanied by any significant cytotoxicity throughout the exposure period (Fig. 2). We concluded that prolonged exposure to ethanol induced a chronic UPR in physiologically relevant PHH.

Effect of HCV infection or ethanol on the hepatocellular UPR induced by a strong ER stressor

To further characterize and compare the impact of HCV and ethanol on the hepatocellular UPR, we tested them in combination with thapsigargin, an established potent ER stress inducer which acts by depleting the ER calcium stores. Thapsigargin, or dimethylsulfoxide as the carrier control, was added for the last 4 h of culture to the medium of PHH at day 1 or day 3 after inoculation with HCVcc or continued exposure to ethanol. We verified that a 4-h treatment with thapsigargin induced a massive activation of all the UPR genes in PHH that were neither infected with HCV nor exposed to ethanol (Fig. 3a and Fig. 3b, white bars).

In PHH pre-exposed to ethanol for 1 day as compared to non-exposed PHH, the only significant change was a lesser induction of BiP upon thapsigargin treatment (Fig. 3a).

However, pre-exposure to ethanol for 3 days further potentiated the massive induction of HERP, EDEM, ATF4, CHOP and CHAC which was triggered by thapsigargin alone. In particular, in PHH treated with ethanol plus thapsigargin versus thapsigargin alone, expression of the pro-apoptotic genes CHOP and CHAC was increased 53.9±5.6-fold versus 30.6±1.2-fold and 36.7±5.6-fold versus 13.9±0.6-fold, respectively. Unlike ethanol exposure, HCV infection did not enhance the thapsigargin-triggered induction of UPR genes at any time point (Fig. 3b). On the contrary, treatment with thapsigargin 1 day post-inoculation produced a significantly lesser induction of BiP, ATF4 and CHOP in infected PHH versus mock-infected PHH: 25.9±1.2-fold versus 32.9±2.3-fold increase, respectively, in the expression of the pro-apoptotic gene CHOP, for example. Thus, HCV not only did not potentiate but, on the contrary, appeared to restrain the massive UPR triggered by thapsigargin. As such a massive UPR might be detrimental to viral replication [START_REF] Chan | Unfolded protein response in hepatitis C virus infection[END_REF][START_REF] Vasallo | Cellular stress responses in hepatitis C virus infection: Mastering a two-edged sword[END_REF], we assessed the consequence of thapsigargin treatment on HCV genome replication using quantitation of the intracellular amounts of negative-strand HCV RNA by a sensitive and strand-specific technique [START_REF] Carrière | A novel, sensitive, and specific RT-PCR technique for quantitation of hepatitis C virus replication[END_REF]. These were reduced by about half upon thapsigargin treatment both at 1 day and 3 days post-inoculation (Fig. 4a). Thus, HCV replication was compromised under the unresolvable ER stress induced by thapsigargin.

The UPR can be initiated by three sensor proteins, which detect the occurrence of ER stress [START_REF] Walter | The unfolded protein response: from stress pathway to homeostatic regulation[END_REF]. The most conserved and well-known of the three signalling pathways activated by these proximal ER stress sensors is the one which is mediated by inositol-requiring enzyme 1 (IRE1) and proceeds through an unconventional splicing of XBP1 transcript. As an approach to exploring a possible mechanism for the observed HCV-mediated attenuation of the thapsigargin-triggered UPR, we assessed XBP1 transcript splicing by semi-quantitative RT-PCR analysis (Fig. 3c). The ratio of spliced to unspliced XBP1 transcripts was not or only slightly increased by either HCV or ethanol alone at day 1, whereas a 4-h treatment with thapsigargin was sufficient to induce a massive increase in XBP1 transcript splicing, as expected for this strong ER stressor. Most interestingly, this massive induction of XBP1 transcript splicing triggered by thapsigargin was comparable in PHH pre-exposed to ethanol for 1 day versus non-exposed PHH, whereas it was greatly reduced in HCV-infected versus mock-infected PHH at 1 day post-inoculation. These results confirm that HCV, unlike ethanol, tended to curb the UPR induction triggered by thapsigargin, and further pointed to the IRE1-XPB1 signalling pathway as one possible target manipulated by HCV for this attenuation.

Effect of HCV infection and ethanol in combination on the hepatocellular UPR

To investigate the hepatocellular impact of HCV infection and ethanol in combination, PHH inoculated with HCVcc at an MOI of 0.1 were exposed continuously to 100 mM ethanol over a prolonged period of 9 days and the resulting UPR was compared to that elicited by each stressor separately. In PHH subjected to both stressors versus neither of them, a significant activation of about 2-to 4-fold was observed for all UPR genes both at day 3 and day 9 (Fig. 5, black bars). In HCV-infected PHH, ethanol exposure further increased the levels of all ER-stress-specific markers both at day 3 and day 9 and also significantly increased the expression of a number of ISR genes (ATF4 and ATF3 at both time points and CHAC at day 3) (Fig. 5, black versus white bars). Conversely, the gene activation levels were similar in PHH exposed to ethanol irrespective of whether they were infected or not, except for three UPR genes, namely BiP, EDEM and GADD34, the expressions of which were significantly enhanced by HCV at day 9 post-inoculation (Fig. 5, black versus grey bars).

These three key components of the UPR are known to assist protein folding in the ER, to target misfolded proteins to the ERAD pathway and to mediate a negative feedback loop to end the translational arrest induced as part of the ISR, respectively [START_REF] Walter | The unfolded protein response: from stress pathway to homeostatic regulation[END_REF]. This suggests that their selective induction by HCV in PHH exposed to ethanol may contribute to coping with this increased stress. Importantly, ethanol exposure of HCV-infected PHH neither caused cytotoxicity (Fig. 2), nor affected the levels of negative-strand HCV RNA (Fig. 4b). This indicates that both cell survival and HCV replication were, indeed, maintained in spite of the additional stress caused by ethanol.

Discussion

Even though HCV and alcohol are generally considered to provoke an ER stress in hepatocytes, the existence of conflicting publications has left unclear whether the downstream response itself, i.e., the UPR, is actually activated and, if so, which of its branches are involved. In this work we first wanted to reassess the occurrence of UPR induction in PHH, the closest in vitro model of normal human adult hepatocytes, over a 9-day period of productive infection with HCV or continuous exposure to ethanol. We focused on the genes of the response itself, comprising genes that are specific to ER stress and those shared by the ISR, because it is the pattern of activated target genes which eventually determines the outcome ranging along a spectrum that goes from homeostasis restoration to cell death commitment and includes cell dysfunction [START_REF] Imrie | Stress management: How the unfolded protein response impacts fatty liver disease[END_REF]. Each of the two stressors was found, indeed, to activate the UPR in PHH. However, the response differed both quantitatively and qualitatively. Indeed, the degree of gene induction appeared only modest upon HCV infection as compared to ethanol exposure. Moreover, when combined with thapsigargin used as a classical ER stress inducer, ethanol amplified the UPR, with an exacerbated induction of proapoptotic genes (CHOP, CHAC), whereas HCV not only did not intensify but instead appeared to restrain the activation of key UPR genes. The second goal of our study was to investigate the impact of the combination of HCV and alcohol in PHH. Interestingly, we found that the resulting UPR did not simply represent the sum of the responses induced by each stressor separately; instead, HCV appeared to enhance selectively the expressions of three pro-survival genes (BiP, EDEM and GADD34) and HCV replication was spared in spite of the added stress due to ethanol. Collectively, the results suggest that whereas alcohol activates indiscriminately all the branches of the hepatocellular UPR, HCV apparently manipulates UPR signalling towards the maintenance of its host cell survival. This seems to be a viral strategy to permit maintenance of its own replication.

HCV generally is believed to activate the hepatocellular UPR as a response to ER stress, yet most publications supporting this view have used ectopic expression of part or all of the viral genome in cells that significantly differ from normal human hepatocytes [START_REF] Chan | Unfolded protein response in hepatitis C virus infection[END_REF][START_REF] Vasallo | Cellular stress responses in hepatitis C virus infection: Mastering a two-edged sword[END_REF]. In fact, the evidence is even less clear when considering only those studies involving a bona fide infection process in physiologically relevant human hepatocytes. Indeed, the few publications that used chimeric mice with humanized livers reported increased expression of either BiP and CHOP [START_REF] Mishima | Cell culture and in vivo analyses of cytopathic hepatitis C virus mutants[END_REF] or BiP but not CHOP [START_REF] Joyce | HCV induces oxidative and ER stress, and sensitizes infected cells to apoptosis in SCID/Alb-uPA mice[END_REF] in HCV-infected hepatocytes. Moreover, the comparison of liver biopsies from HCV-infected patients versus normal liver tissues showed no evidence of UPR [START_REF] Mcpherson | No evidence of the unfolded protein response in patients with chronic hepatitis C virus infection[END_REF], or only activation of the UPR sensors with no induction of downstream target genes [START_REF] Asselah | In vivo hepatic endoplasmic reticulum stress in patients with chronic hepatitis C[END_REF], or else activation of UPR genes including BiP [START_REF] Chandra | Impaired expression of type I and type II interferon receptors in HCV-associated chronic liver disease and liver cirrhosis[END_REF]. Our in vitro model in PHH enabled us to study the impact of HCV infection in normal human hepatocytes while avoiding potential confounding factors inherent to in vivo studies. Despite the inter-individual variability of PHH originating from different donors, we found a significant induction of almost all the UPR target genes. These results confirm previous data obtained in a system where PHH inoculated with a modified virus supported only low levels of HCV replication [START_REF] Chandra | Impaired expression of type I and type II interferon receptors in HCV-associated chronic liver disease and liver cirrhosis[END_REF], and extend them to a situation of robust, productive HCV infection [START_REF] Podevin | Production of infectious hepatitis C virus in primary cultures of human adult hepatocytes[END_REF]. While, at first glance, the magnitude of UPR activation appears, nevertheless, to be modest, it should be emphasized that any biochemical change induced by HCV in infected cells is diluted among the majority of cells that remain uninfected whereas other stressors such as ethanol are expected to affect the whole cell population. Such a mechanism was put forward as a possible explanation for the discrepancy between the absence of detectable UPR gene induction in the liver tissue from HCV-infected patients and the detection of signs of ER stress in situ in small clusters of hepatocytes scattered in the liver parenchyma [START_REF] Asselah | In vivo hepatic endoplasmic reticulum stress in patients with chronic hepatitis C[END_REF]. However, direct evidence supporting this hypothesis would rely on single-cell analysis of viral replication and host gene expression with the use of sophisticated high-resolution techniques that are not yet widely available.

It generally is admitted that HCV is a non-cytotoxic virus, in vivo, and that the death of hepatocytes during HCV-associated hepatitis is attributable mainly to their clearance by cytotoxic T cells. With this in mind, the detection of a transient phase of massive cell death upon HCV infection of Huh-7 sublines was, in fact, unexpected [START_REF] Zhong | Persistent hepatitis C virus infection in vitro: coevolution of virus and host[END_REF][START_REF] Sekine-Osajima | Development of plaque assays for hepatitis C virus-JFH1 strain and isolation of mutants with enhanced cytopathogenicity and replication capacity[END_REF]. Here, we found that upon inoculation with the same HCV stock at the same MOI, Huh-7.5.1 cells underwent massive cell death between day 6 and day 12 post-inoculation whereas PHH showed no evidence of cytotoxicity throughout the 2-week time course. At least two facts can be put forward to explain this difference between the two sources of human hepatocytes. First, the phase of massive cell death seen in the Huh-7 system occurs when virtually all the cells in the culture are infected [START_REF] Zhong | Persistent hepatitis C virus infection in vitro: coevolution of virus and host[END_REF], i.e., when the levels of infection are far higher than those supported by PHH or which occur in the liver of patients during natural infection [START_REF] Liang | Visualizing hepatitis C virus infections in human liver by two-photon microscopy[END_REF][START_REF] Stiffler | Focal distribution of hepatitis C virus RNA in infected livers[END_REF][START_REF] Kandathil | Use of laser capture microdissection to map hepatitis C virus-positive hepatocytes in human liver[END_REF][START_REF] Wieland | Simultaneous detection of hepatitis C virus and interferon stimulated gene expression in infected human liver[END_REF]. Second, cell death coincided with a massive induction of pro-apoptotic genes of the ISR but not of chaperones characteristic of the ER-stress-specific branch of the UPR. Whereas it is established that HCV infection in Huh-7 sublines triggers activation of the apoptotic gene CHOP, discrepancies exist as to whether BiP also is induced [START_REF] Merquiol | HCV causes chronic endoplasmic reticulum stress leading to adaptation and interference with the unfolded protein response[END_REF][START_REF] Sekine-Osajima | Development of plaque assays for hepatitis C virus-JFH1 strain and isolation of mutants with enhanced cytopathogenicity and replication capacity[END_REF][START_REF] Saeed | Role of the endoplasmic reticulum-associated degradation (ERAD) pathway in degradation of hepatitis C virus envelope proteins and production of virus particles[END_REF]. In fact, such transformed cells were reported to express high levels of BiP constitutively [START_REF] Shuda | Activation of the ATF6, XBP1 and grp78 genes in human hepatocellular carcinoma: a possible involvement of the ER stress pathway in hepatocarcinogenesis[END_REF], which make them an unsuitable model for studying the occurrence of an ER-stress-specific response. In contrast to Huh-7.5.1 cells, PHH inoculated in parallel displayed only a slight, if any, induction of ISR-associated genes accompanied by an activation of ER-stress-specific genes including its master regulator BiP. Thus, HCV is not cytotoxic for PHH, consistent with these normal hepatocytes developing a complete UPR response that does not reach the threshold leading to cell death.

An increased expression of most UPR markers was found in liver biopsies from patients with chronic alcohol-related liver disease [START_REF] Longato | Insulin resistance, ceramide accumulation, and endoplasmic reticulum stress in human chronic alcohol-related liver disease[END_REF]. However, only some of the UPR target genes and pathways were reported to be induced by short-term ethanol exposure of human hepatocarcinoma-derived cell lines engineered to express ethanol-metabolizing enzymes [START_REF] Magne | ATF4 and the integrated stress response are induced by ethanol and cytochrome P450 2E1 in human hepatocytes[END_REF][START_REF] Howarth | Alcohol disrupts endoplasmic reticulum function and protein secretion in hepatocytes[END_REF], possibly due to their intrinsic inability to activate the ER-stress-specific response [START_REF] Shuda | Activation of the ATF6, XBP1 and grp78 genes in human hepatocellular carcinoma: a possible involvement of the ER stress pathway in hepatocarcinogenesis[END_REF].

More unexpectedly, PHH also showed no significant activation of ER chaperones when exposed to ethanol over the short term (24 or 30 h) [START_REF] Magne | ATF4 and the integrated stress response are induced by ethanol and cytochrome P450 2E1 in human hepatocytes[END_REF][START_REF] Kao | Human immunodeficiency virus protease inhibitors modulate Ca2+ homeostasis and potentiate alcoholic stress and injury in mice and primary mouse and human hepatocytes[END_REF]. Importantly, the UPR should be viewed as a dynamic process. Therefore, an undetected marker at early time points does not necessarily rule out its activation upon prolonged exposure to the stressor and time course studies are needed to reveal how the pattern of activated markers varies with the duration of the stress. Here, we subjected PHH to prolonged ethanol exposure and we observed a significant activation of all the UPR genes at day 3 that did not return to baseline at day 9, which is indicative of a chronic UPR. Of note, we observed that none of the UPR genes was induced in Huh-7.5.1 cells after 3 days of ethanol exposure. This suggests that the metabolism of ethanol probably contributes to the UPR activation seen in PHH, which, unlike Huh-7.5.1 cells, were demonstrated to have sustained expression of the alcohol-metabolizing enzymes. Indeed, alcohol-induced ER stress may be related to the production of acetaldehyde and reactive oxygen species generated from ethanol-induced CYP2E1 and to perturbations in calcium homeostasis and in homocysteine metabolism [START_REF] Magne | ATF4 and the integrated stress response are induced by ethanol and cytochrome P450 2E1 in human hepatocytes[END_REF][START_REF] Ji | New insights into the pathogenesis of alcohol-induced ER stress and liver diseases[END_REF].

Ethanol was reported to potentiate the response to ER stress caused by other factors such as human immunodeficiency virus protease inhibitors in PHH [START_REF] Kao | Human immunodeficiency virus protease inhibitors modulate Ca2+ homeostasis and potentiate alcoholic stress and injury in mice and primary mouse and human hepatocytes[END_REF], primary mouse hepatocytes or hepatic stellate cells [START_REF] Hu | Effects of combined alcohol and anti-HIV drugs on cellular stress responses in primary hepatocytes and hepatic stellate and kupffer cells[END_REF], palmitic acid in primary rat hepatocytes [START_REF] Yi | Ethanol promotes saturated fatty acid-induced hepatoxicity through endoplasmic reticulum (ER) stress response[END_REF] and tunicamycin or thapsigargin in cells of neuronal origin [START_REF] Chen | Ethanol promotes endoplasmic reticulum stress-induced neuronal death: involvement of oxidative stress[END_REF]. In our study, PHH pre-exposure to ethanol also potentiated the UPR triggered by thapsigargin, with a massive induction of the pro-apoptotic genes CHOP and CHAC. In sharp contrast, the thapsigargin-triggered induction of UPR target genes was not enhanced in HCV-infected PHH and a slight, yet significant attenuation in expression of the ISR genes ATF4 and CHOP was observed at 1 day postinoculation. Our comparison of HCV and ethanol thus highlights the striking capacity of HCV to manipulate the UPR towards an adaptive response, a property likely to be essential for a virus prone to persist in its host [START_REF] Chan | Unfolded protein response in hepatitis C virus infection[END_REF][START_REF] Vasallo | Cellular stress responses in hepatitis C virus infection: Mastering a two-edged sword[END_REF]. Interestingly, the massive induction of XBP1 transcript splicing triggered by thapsigargin was reduced in HCV-infected PHH. This is reminiscent of the impact of murine cytomegalovirus on the UPR [START_REF] Stahl | Cytomegalovirus downregulates IRE1 to repress the unfolded protein response[END_REF]. Indeed, this virus was reported to induce only a slight and transient increase in XBP1 transcript splicing and to suppress its induction by tunicamycin via IRE1 downregulation. Although additional studies would be required to unravel the exact mechanisms by which HCV tends to curb the UPR, these data suggest that manipulation of the IRE1-XBP1 signalling pathway may be a strategy shared by different viruses.

An increased serum and intra-hepatic viral load was reported in HCV-infected patients with excessive alcohol consumption, suggesting that ethanol might promote HCV replication [START_REF] Romero-Gómez | Intrahepatic hepatitis C virus replication is increased in patients with regular alcohol consumption[END_REF][START_REF] Carrière | Low density lipoprotein receptor transcripts correlate with liver hepatitis C virus RNA in patients with alcohol consumption[END_REF]. However, studies using Huh-7 sublines as cellular models gave conflicting data as to whether ethanol decreases or increases HCV genome replication and a variety of mechanisms have been incriminated to explain the observed effects [START_REF] Zhang | Alcohol potentiates hepatitis C virus replicon expression[END_REF][START_REF] Plumlee | Effect of ethanol on innate antiviral pathways and HCV replication in human liver cells[END_REF][START_REF] Mccartney | Alcohol metabolism increases the replication of hepatitis C virus and attenuates the antiviral action of interferon[END_REF][START_REF] Seronello | Ethanol enhances hepatitis C virus replication through lipid metabolism and elevated NADH/NAD+[END_REF][START_REF] Ye | Alcohol impairs interferon signaling and enhances full cycle hepatitis C virus JFH-1 infection of human hepatocytes[END_REF][START_REF] Bukong | Ethanol facilitates hepatitis C virus replication via up-regulation of GW182 and heat shock protein 90 in human hepatoma cells[END_REF][START_REF] Hou | Alcohol facilitates HCV RNA replication via up-regulation of miR-122 expression and inhibition of cyclin G1 in human hepatoma cells[END_REF]. Theoretically, a decrease in HCV replication would be expected to occur as a result of the cellular stress induced by ethanol, as illustrated here under conditions of overwhelming ER stress caused by thapsigargin. In our model of HCV infection in physiological PHH, the addition of ethanol did not affect significantly the level of HCV genome replication as evaluated, directly and rigorously, by the specific quantitation of negative-strand HCV RNA. Interestingly, the UPR resulting from the combination of ethanol and HCV did not simply represent the sum of the effects due to each stressor separately; instead, HCV appeared to increase specifically the expression of BiP, EDEM and GADD34 all of which can contribute to coping with the ER stress added by ethanol. Future studies are needed to unravel the mechanism of the alcoholinduced increase in viral load in the absence of any effect on the step of HCV genome replication. One hypothesis is that alcohol might affect the step of viral particle morphogenesis, which is expected to be promoted under conditions that increase the capacity of folding of the viral envelope glycoproteins in the ER.

In conclusion, the hepatocellular UPR has been incriminated in the pathogenesis of a variety of liver injuries and diseases, most notably steatosis for which a causal relationship has been supported by an increasing body of experimental evidence [START_REF] Malhi | Endoplasmic reticulum stress in liver disease[END_REF][START_REF] Imrie | Stress management: How the unfolded protein response impacts fatty liver disease[END_REF][START_REF] Lebeaupin | Endoplasmic reticulum stress signalling and the pathogenesis of non-alcoholic fatty liver disease[END_REF]. However, the UPR certainly should be viewed as a multitude of responses which differ in the patterns of activated markers and subsequent outcomes, not all of them being pathologic [START_REF] Imrie | Stress management: How the unfolded protein response impacts fatty liver disease[END_REF][START_REF] Lebeaupin | Endoplasmic reticulum stress signalling and the pathogenesis of non-alcoholic fatty liver disease[END_REF]. Here, we found different patterns of UPR activation depending upon the duration of the stress and the nature of the stressor, HCV alone, alcohol alone, or HCV and alcohol in combination. The latter pattern could not be predicted simply by the addition of the effects of the two stressors taken separately, owing to their complex interplay and the peculiar ability of HCV to subvert UPR signalling. It is all the more important to characterize in detail the different types of UPR which result from different situations as new therapeutic avenues targeting specific UPR signalling pathways are being developed with the aim of switching the UPR from a diseasecausing response to an adaptive, beneficial response [START_REF] Lebeaupin | Endoplasmic reticulum stress signalling and the pathogenesis of non-alcoholic fatty liver disease[END_REF][START_REF] Cao | Targeting endoplasmic reticulum stress in metabolic disease[END_REF].

Figure legends

Fig. 1 .

 1 Fig. 1. Effect of HCV infection and ethanol on the UPR in PHH. PHH were (a) inoculated

Fig. 2 .

 2 Fig. 2. Absence of cytotoxicity of HCV infection and ethanol, alone and combined, in PHH.

Fig. 3 .

 3 Fig. 3. Effect of HCV infection and ethanol on the UPR induced by thapsigargin in PHH.

Fig. 4 .

 4 Fig. 4. Effect of thapsigargin and ethanol on HCV replication in PHH. PHH were inoculated

Fig. 5 .

 5 Fig. 5. Combined effect of HCV infection and ethanol on the UPR in PHH. PHH were

Acknowledgements

This work was supported by the ANRS (France REcherche Nord&sud Sida-hiv Hépatites); the Inserm ("PNR alcool"); and the Ireb (Institut de recherches scientifiques sur les boissons).

CH and MV received a doctoral fellowship from the ANRS. VP was the recipient of a postdoctoral fellowship from the ANRS.

Author contributions

PP, MG, HR and ARR conceived and designed the study. CH, EBB, VP, BL, MV, LA and AC performed the experiments. CH, EBB, VP, BL, MV and AC analysed and interpreted the data. FC and YC provided resources. CH, EBB, VP, HR and ARR drafted or wrote the manuscript. All authors approved the final version for submission.

Conflict of interest declaration

The authors declare no conflict of interests.