Morgane Salines

Mathieu Andraud

Marie Pellerin

Cécilia Bernard

Béatrice Grasland

Nicole Pavio

Nicolas Rose
email: nicolas.rose@anses.fr

Impact of porcine circovirus type 2 (PCV2) infection on hepatitis E virus (HEV) infection and transmission under experimental conditions

Keywords: Co-infection, PCV2, HEV, infection kinetic, transmission experiment

Hepatitis E virus is a zoonotic pathogen for which pigs have been identified as the main reservoir in industrialised countries. HEV infection dynamics in pig herds and pigs are influenced by several factors, including herd practices and possibly co-infection with immunomodulating viruses. This study therefore investigates the impact of porcine circovirus type 2 (PCV2) on HEV infection and transmission through experimental HEV/PCV2 co-infection of specific-pathogen-free pigs. No statistical difference between HEV-only and HEV/PCV2-infected animals was found for either the infectious period or the quantity of HEV shed in faeces. The HEV latency period was shorter for HEV/PCV2 co-infected pigs than for HEV-only infected pigs (11.6 versus 12.3 days). Its direct

Introduction

Hepatitis E virus is a non-enveloped single-stranded RNA virus that can cause acute hepatitis in humans. Chronic cases have also been described, mainly in immunocompromised patients (Lhomme et al., 2016). Genotypes 3 and 4 affect both humans and other animal species, and are responsible for sporadic autochthonous cases of hepatitis in humans in industrialised countries [START_REF] Doceul | Zoonotic Hepatitis E Virus: Classification, Animal Reservoirs and Transmission Routes[END_REF]. In particular, genotype 3 is widespread in pig populations [START_REF] Salines | From the epidemiology of hepatitis E virus (HEV) within the swine reservoir to public health risk mitigation strategies: a comprehensive review[END_REF] and a number of autochthonous cases have been linked to the consumption of undercooked pork meat, especially liver products [START_REF] Colson | Autochthonous infections with hepatitis E virus genotype 4, France[END_REF][START_REF] Guillois | High Proportion of Asymptomatic Infections in an Outbreak of Hepatitis E Associated With a Spit-Roasted Piglet, France[END_REF]. In order to limit the risk of contaminated products entering the food chain, it is crucial to understand the factors influencing HEV transmission and persistence in pig herds. High variability in HEV infection dynamics has previously been described [START_REF] Salines | From the epidemiology of hepatitis E virus (HEV) within the swine reservoir to public health risk mitigation strategies: a comprehensive review[END_REF] and may be related to husbandry practices in terms of hygiene, biosecurity and rearing conditions [START_REF] Walachowski | Risk factors associated with the presence of hepatitis E virus in livers and seroprevalence in slaughter-age pigs: a retrospective study of 90 swine farms in France[END_REF][START_REF] Lopez-Lopez | Risk factors associated with hepatitis E virus in pigs from different production systems[END_REF] or to individual characteristics such as protection conferred by maternallyderived antibodies [START_REF] Andraud | Early-Life Hepatitis E Infection in Pigs: The Importance of Maternally-Derived Antibodies[END_REF]. Various factors affecting swine immune response may also influence the course of HEV infection. Notably, in a previous study, we have shown that pigs experimentally co-infected with porcine reproductive and respiratory syndrome virus (PRRSV) exhibited chronic HEV infection with extended latency and infectious periods, increased faecal shedding and transmission, as well as an increased risk of HEV-positive livers at slaughter [START_REF] Salines | Hepatitis E virus chronic infection of swine co-infected with Porcine Reproductive and Respiratory Syndrome Virus[END_REF]. Porcine circovirus type 2 (PCV2) also has immunomodulating characteristics for instance by inhibiting IFN-α production and by increasing the expression of IL-10, an antiinflammatory cytokine [START_REF] Darwich | Immunology of porcine circovirus type 2 (PCV2)[END_REF]. PCV2 may therefore impact HEV infection dynamics. Moreover, as the primary causative agent of post-weaning multisystemic wasting syndrome (PMWS) and other porcine circovirus-associated diseases (PCVADs), it can sometimes induce hepatitis in pigs [START_REF] Rosell | Hepatitis and staging of hepatic damage in pigs naturally infected with porcine circovirus type 2[END_REF]. However, to date, only few data report on HEV/PCV2 co-infection [START_REF] Martin | Association of hepatitis E virus (HEV) and postweaning multisystemic wasting syndrome (PMWS) with lesions of hepatitis in pigs[END_REF][START_REF] Hosmillo | Molecular detection of genotype 3 porcine hepatitis E virus in aborted fetuses and their sows[END_REF][START_REF] Savic | Detection rates of the swine torque teno viruses (TTVs), porcine circovirus type 2 (PCV2) and hepatitis E virus (HEV) in the livers of pigs with hepatitis[END_REF][START_REF] Yang | Fatal disease associated with Swine Hepatitis E virus and Porcine circovirus 2 co-infection in four weaned pigs in China[END_REF][START_REF] Jackel | Coinfection of pigs with Hepatitis E and porcine circovirus 2, Saxony 2016[END_REF]. In these studies, PCV2 and HEV were simultaneously detected in pigs but the impact of co-infections on HEV dynamics was not investigated.

Given the lack of data on this specific issue, the present study was designed to investigate how PCV2 infection impacts HEV infection dynamics (in terms of viral shedding duration and quantity, transmission and humoral immune response). A transmission experiment was therefore carried out, with specific-pathogen-free (SPF) pigs infected with HEV or co-infected with HEV and PCV2 at the same time.

Material and methods

2.1.Experimental design

The trial was conducted at ANSES's air-filtered level-3 biosecurity facilities. The 44 five-week-old SPF Large White piglets included in the study were HEV-and PCV2-free and with no maternal antibodies against these two viruses at the beginning of the study. These piglets were randomly allocated into eight groups, housed in six rooms (Figure 1). Two negative control pigs were housed in Room 1. The four piglets housed in Room 2 were only orally inoculated with a PCV2-b genogroup suspension (GenBank accession number AF201311), titrating 10 5 TCID 50 /mL in a volume of 5 mL. In Rooms 5 and 6 (groups 4, 5, 6), three piglets per group were orally inoculated with 10 7 HEV RNA copies of a genotype 3 HEV suspension (strain FR-SHEV3f, GenBank accession number JQ953666) in a volume of 10 mL. In Rooms 3 and 4 (groups 1, 2, 3), three piglets per group were orally inoculated with both HEV and PCV2, following the same inoculation protocols as for the other groups. In each of the six groups (HEV-only and HEV/PCV2), the three inoculated piglets were in contact with three pen mates (contact piglets) from day 1. Individual faecal samples were collected three days before inoculation and three times a week until the end of the experiment at 49 days post inoculation (dpi). Blood samples were collected before inoculation and once a week until the end of the experiment. Clinical examination was also performed (clinical signs, rectal temperature, faeces consistence, weight, food consumption and trough cleanliness were recorded daily). After euthanasia, necropsies were performed and organ and fluid samples collected, among them liver and bile samples. The experiment was performed in accordance with EU and French regulations on animal welfare in experiments. The protocol was approved (referral 17-022) by the ANSES/ENVA/UPEC ethical committee registered under number #16.

2.2.Sample analyses

After performing manual total RNA extraction, HEV RNA in faecal samples was quantified using real-time quantitative RT-PCR as described in [START_REF] Barnaud | Thermal Inactivation of Infectious Hepatitis E Virus in Experimentally Contaminated Food[END_REF]. Results were expressed in HEV RNA copy number per gram of faeces (RNA copies/g). Since HEV shedding in faeces and presence in serum have been shown to be correlated [START_REF] Salines | Persistent viremia and presence of hepatitis E virus RNA in pig muscle meat after experimental co-infection with porcine reproductive and respiratory syndrome virus[END_REF], HEV RT-PCR was performed on serum samples of 49 day-old pigs only if their faeces were positive at 46 and/or 49 dpi. Similarly, and as bile is considered as a relevant proxy of liver status [START_REF] De Deus | Hepatitis E virus infection dynamics and organic distribution in naturally infected pigs in a farrow-to-finish farm[END_REF][START_REF] Bouwknegt | The course of hepatitis E virus infection in pigs after contactinfection and intravenous inoculation[END_REF], bile samples of 49-day old pigs having positive faecal samples at 46 and/or 49 dpi were analysed. Anti-HEV antibodies were detected using the HEV ELISA 4.0V kit (MP Diagnostics, Illkirch, France) according to the manufacturer's instructions, apart from the serum quantity used (10 µL instead of the recommended 20 µL). Samples were considered to be positive when their optical density (OD) at a wavelength of 450 nm was higher than the threshold, which was defined as the mean optical density of negative control pig samples +0.3. PCV2 DNA was extracted and quantified from the serum using real-time PCR based on TaqMan technology as described in [START_REF] Grasland | Reproduction of PMWS in immunostimulated SPF piglets transfected with infectious cloned genomic DNA of type 2 porcine circovirus[END_REF]. Results were expressed in genomic equivalent DNA copies/mL of serum. PCV2-antibodies were detected by PCV2 specific ELISA as already described with a positive cut-off for OD ratios higher than 1.5 [START_REF] Fablet | Estimation of the diagnostic performance of two ELISAs to detect PCV2 antibodies in pig sera using a Bayesian method[END_REF].

2.3.Statistical analyses

The infectious period and time prior to HEV seroconversion were estimated using survival analyses.

Two parametric models were tested (lognormal and Weibull survival time distributions) and compared using the Akaike Information Criterion (AIC). Cox-proportional hazard models were used to assess the effect of PCV2 co-infection on the lengths of the infectious period and the time prior to HEV seroconversion. The distributions of individual HEV viral loads in faeces were analysed according to time since inoculation (with and without co-infection). A linear mixed model taking into account repeated measurements over time was used for this investigation in order to assess the different quantities of HEV particles shed by co-infected as opposed to HEV-only infected pigs.

The HEV infection dynamics in each group were modelled using a SEIR (Susceptible -Exposed -Infectious -Recovered) model as per the estimation process described in [START_REF] Gallien | Better horizontal transmission of a US non-InDel strain compared with a French InDel strain of porcine epidemic diarrhoea virus[END_REF].

Briefly, pigs were considered as "susceptible" during the time window from exposure (day 0 = day of inoculation) to the point at which they actually became infected ( ), progressing to the "exposed" state. The time at which individuals were considered to be "infectious" (i.e. began shedding), denoted , was considered to lie between the times of the last HEV-negative PCR sample ( ) and the first HEV-positive PCR faecal sample ( ) for each animal ( < <

). The latency period therefore corresponds to the delay between infection and shedding ( = -). Pigs were considered "recovered" as soon as they no longer produced HEVpositive PCR samples. Two transmission routes were considered to be involved in this infection process: transmission by direct contact between pen mates and oro-faecal transmission via the environmental compartment. Environmental viral load represents the accumulation of viral particles in the environment through faecal shedding by infected animals. is partially offset by its clearance rate ( = 0.3 ) and was calculated as described in [START_REF] Andraud | Direct contact and environmental contaminations are responsible for HEV transmission in pigs[END_REF] and [START_REF] Salines | Hepatitis E virus chronic infection of swine co-infected with Porcine Reproductive and Respiratory Syndrome Virus[END_REF]. Let and denote direct contact and environmental transmission rates, respectively. The force of infection exerted on a typical susceptible individual located in pen at time is defined by:

! " # $ = % " # $ & -1 + " # $ & ,
where % and respectively represent the number of infectious animals and the viral load in pen at time , & being the total number of pigs in each pen. With these notations, the probability * + of individual getting infected at time , + #+$ is given by

* + = 1 -exp 0-! " 1, + #+$ 23
while the probability of having escaped infection in time interval 40, + #+$ 4 is given by

5 + = exp 0-6 ! " #7$ 7 8 9:; #9$ < 3.
An informative gamma prior was used to analyse the duration of the latency period . Its parameters were fixed using data from previous experiments and from observations of inoculated pigs #= = 4, ? = 3$. Very wide normal distributions were initially used as prior for the logtransformed transmission rates (log# $ ~D#-2,4$ and log# $ ~D#-8,4$). The global likelihood can be written as:

GH, I , , J , %, | , , , , + , =, ?L = M N 6 O#P$ Q 9:; #9$ R SP × 01 -N O18 9:; #9$ 2 3 + ∈ V WV + V S * Y1 #+$ , =, ?2 × M N 6 O# $ Z[\H] ^_`L R S + ∈ V WV + V S × M Y1 #+$ , =, ?2 + ∈ + VabW S
The first term of the likelihood denotes the probability of detected infections occurring for an individual at time , #+$ ; the second term represents the probability of observed infection failure whenever some individual would remain susceptible throughout the experiment; and the third term gives the distribution of the latency period in seeder pigs. Bayesian inference was performed using the Metropolis-Hastings algorithm: ten independent chains of 50,000 iterations were run with a burn-in period of 10%. Initial values were randomly drawn from prior distributions. Convergence was assessed by inspecting parameter outputs visually as well as through conventional diagnostic tests (Heidelberger, Geweke and Gelman-Rubin diagnostics). The impact of PCV2 infection on the HEV latency period and the transmission parameters' distribution were then assessed using a Kruskal-Wallis test. All the analyses were performed using R software (R 3.5.1).

Results

3.1.Infection data

No clinical sign related to PCV2 or HEV infection was observed in any infected pig. All PCV2 inoculated pigs and pigs in contact were seropositive at 28 dpi except one that was found seropositive at 45 dpi (Supplementary File 1). Control pigs and HEV-only inoculated pigs remained PCV2 seronegative throughout the study. PCV2 viraemia in contact pigs started between 10 and 28 dpi and lasted until 28 to 49 dpi. Viral loads ranged between 1.10 3 and 8.10 6 genomic equivalent DNA copies/mL of serum with a viraemia peak around 17 days post-inoculation (Supplementary File 1). HEV infection data are presented in Figures 2 and3 for quantitative RT-PCR on faecal samples and serological results respectively. All but two animals (one HEV/PCV2-inoculated pig and one HEV contact pig) shed HEV during the experiment. Inoculated animals started to shed HEV between 11 and 25 dpi, and contact animals between 23 and 46 dpi. Sporadic or intermittent shedding was observed in a few animals (Figure 2). Of the 36 pigs, 20 produced anti-HEV antibodies: 14 of the 18 HEV-only infected pigs versus just six of the 18 HEV/PCV2 co-infected pigs. Seroconversion occurred between 24 and 49 dpi for inoculated animals, and between 38 and 45 dpi for contact animals (Figure 3). At the end of the experiment, four out of the 17 analysed pigs (23%) had HEV RNA in their bile and one of them was viraemic (6%), with a viral load of 4.7.10 3 RNA copies/mL (Figure 2). These positive pigs were HEV/PCV2 co-infected (both inoculated and contact pigs).

3.2.Estimated durations related to HEV infection dynamics

Latency periods were estimated at 12.

3.3.HEV shedding and environmental accumulation

The distribution of the shed HEV viral load against time (with and without co-infection) is shown in Figure 4. The linear mixed model accounting for repeated measurements did not show the PCV2 infection to have any impact on the quantity of HEV particles shed by inoculated or contact animals (p > 0.05). The viral load accumulated in the environment was modelled for each experimental pen.

The environment was HEV-free until 15 to 20 dpi, when the environmental load increased and reached 4.10 5 to 2.10 6 before dropping at the end of the trial (data not shown) when there were no remaining shedders in the pen.

3.4.HEV transmission parameters

In our experimental settings, a single HEV-only infected pig was able to infect 0.04 pigs per day by direct contact ( = 0.04 [2•10 -5 -0.24]), whereas the direct transmission rate for HEV/PCV2 coinfected pigs was estimated to be significantly higher, with a three-fold difference (0.12 [5•10 -4 -0.4]; Figures 5 and6). The environmental transmission rate can be considered as the average number of animals that a single genome equivalent is able to infect when present in the pen environment. was estimated at 1.5•10 -5 g/RNA copies/day [2•10 -6 ; 4•10 -5 ] when pigs were HEV-only infected versus 4.3•10 -6 g/RNA copies/day [7•10 -8 ; 1.3•10 -5 ] when pigs were HEV/PCV2 co-infected (Figures 5 and6). It was statistically lower in cases of HEV/PCV2 coinfection than for HEV-only infected pigs (p < 0.05).

Discussion

Understanding factors likely to influence HEV infection dynamics on pig farms is a pivotal step in the design of HEV surveillance and control programmes aiming to mitigate the risk of human exposure to HEV. Of those factors, immunomodulating pathogens are suspected to play a key role and PRRSV has previously been shown to strongly influence HEV infection dynamics [START_REF] Salines | Hepatitis E virus chronic infection of swine co-infected with Porcine Reproductive and Respiratory Syndrome Virus[END_REF]. The main aim of the present study was to investigate the potential impact of PCV2 coinfection on HEV infection dynamics under experimental conditions.

PCV2 infection dynamics in our experimental settings did not differ from data in the available literature [START_REF] Andraud | Quantification of porcine circovirus type 2 (PCV-2) within-and between-pen transmission in pigs[END_REF], suggesting that HEV did not impact PCV2 dynamics. Animal follow-up showed high inter-individual variability of HEV infection dynamics, both in HEV-only and HEV/PCV2-infected pigs, with average latency periods of 12.3 and 11.6 days, and infectious periods of 11.8 and 16.6 days respectively. This high variability was already highlighted in previously-published studies on the topic, especially in cases of natural infection by the oral route [START_REF] Bouwknegt | The course of hepatitis E virus infection in pigs after contactinfection and intravenous inoculation[END_REF][START_REF] Andraud | Direct contact and environmental contaminations are responsible for HEV transmission in pigs[END_REF][START_REF] Salines | Hepatitis E virus chronic infection of swine co-infected with Porcine Reproductive and Respiratory Syndrome Virus[END_REF]. This variability was taken into account for the parameter estimation by taking uninformative or little informative prior distributions; algorithm convergence therefore allows to gain confidence in the obtained results. For the HEV-only infected group, the infection kinetics slightly vary from those described in [START_REF] Andraud | Direct contact and environmental contaminations are responsible for HEV transmission in pigs[END_REF], who reported a latency period of 6.9 days [5.8-7.9] and an infectious period of 9.7 days [8.2-11.2]. This gap may be related to the different HEV strain used for inoculation (strain FR-SHEV3e in [START_REF] Andraud | Direct contact and environmental contaminations are responsible for HEV transmission in pigs[END_REF], versus strain FR-SHEV3f in the present trial) as well as to the lower inoculation dose (10 7 genomic equivalent in the present experiment versus 10 8 in the HEV/PRRSV experiment). In the trial described by [START_REF] Bouwknegt | The course of hepatitis E virus infection in pigs after contactinfection and intravenous inoculation[END_REF], the infectious period was estimated at between 13 and 49 days, depending on the replicate block, but their pigs were intravenously inoculated (versus oral inoculation in the present experiment).

From our analyses, no statistical difference was found between HEV-only and HEV/PCV2 groups, either in the infectious period, or in the quantity of HEV shed in faeces. The latency period was found to be less than one day shorter in HEV/PCV2 co-infected pigs than in HEV-only infected pigs which, although statistically significant, is likely to have a limited biological impact on HEV infection dynamics. The direct transmission rate of HEV was found to be three times higher in cases of HEV/PCV2 co-infection than in cases of HEV-only infection (0.12 versus 0.04), meaning that one co-infected pig is likely to infect three times more pigs than a pig infected only with HEV. The environmental transmission rate of HEV was found to be lower in cases of HEV/PCV2 co-infection (4.3•10 -6 versus 1.5•10 -5 g/RNA copies/day for HEV-only infected pigs), meaning that three times more HEV particles in the environment are needed in order to infect a pig already carrying PCV2.

The lower environmental force of infection in cases of PCV2 infection may delay HEV infections.

Short time to slaughter after HEV infection seems to be a key point of liver contamination. Thus, delaying HEV infection is likely to increase the risk of pig livers containing HEV at slaughter time.

Regarding immune response, fewer HEV/PCV2-infected pigs than HEV-only infected pigs presented a humoral immune response (6/18 versus 14/18 pigs, respectively). Moreover, the time prior to HEV seroconversion was 1.9 times longer in HEV/PCV2 co-infected pigs than in HEVonly infected pigs (49.4 versus 25.6 days). This could be especially problematic if pig HEV status is screened using serological method: this long time prior to HEV seroconversion would lead to many false negative animals. Although PCV2 did not affect HEV infection dynamics as much as PRRSV did in the trial that we previously conducted [START_REF] Salines | Hepatitis E virus chronic infection of swine co-infected with Porcine Reproductive and Respiratory Syndrome Virus[END_REF], it cannot be excluded that in combination with other factors, as for PMWS, it may influence HEV infection. This is consistent with the immunomodulating effect of both PCV2 and PRRSV described in literature, where innate immunity is somewhat suppressed due to a reduction in the IFNα response, delaying the onset of the adaptive response [START_REF] Darwich | Immunology of porcine circovirus type 2 (PCV2)[END_REF][START_REF] Butler | Porcine reproductive and respiratory syndrome (PRRS): an immune dysregulatory pandemic[END_REF]. Four out of the 17 tested pigs had HEV RNA in the bile at the end of the experiment, which can be considered as a reliable proxy of the liver contamination. This late-stage positivity illustrates the increased risk of having HEV positive livers entering the food chain when animals were co-infected. Moreover, the detection of one HEV/PCV2 co-infected pig being HEV viraemic at the end of the experiment also raises the question of a potential risk linked to other pork products that is still debated in the literature [START_REF] Salines | Persistent viremia and presence of hepatitis E virus RNA in pig muscle meat after experimental co-infection with porcine reproductive and respiratory syndrome virus[END_REF]. Further analyses would be necessary to assess the level of contamination of pig muscles in cases of PCV2 infection, especially as correlations between HEV RNA levels in muscles, liver and faeces have been shown [START_REF] Salines | Persistent viremia and presence of hepatitis E virus RNA in pig muscle meat after experimental co-infection with porcine reproductive and respiratory syndrome virus[END_REF]. Such analyses could inform on the risk for public health linked to the consumption of undercooked or raw pig meat or other pork products that do not contain liver. Our present results could also be used to feed dynamic models representing HEV spread and persistence on farms in which PCV2 may circulate. Our data, obtained under controlled conditions, can also add supplementary explanations to the previously published field studies in which HEV and PCV2 were detected simultaneously in pigs and in which causal relationship was suspected but not demonstrated [START_REF] Martin | Association of hepatitis E virus (HEV) and postweaning multisystemic wasting syndrome (PMWS) with lesions of hepatitis in pigs[END_REF][START_REF] Hosmillo | Molecular detection of genotype 3 porcine hepatitis E virus in aborted fetuses and their sows[END_REF][START_REF] Savic | Detection rates of the swine torque teno viruses (TTVs), porcine circovirus type 2 (PCV2) and hepatitis E virus (HEV) in the livers of pigs with hepatitis[END_REF][START_REF] Yang | Fatal disease associated with Swine Hepatitis E virus and Porcine circovirus 2 co-infection in four weaned pigs in China[END_REF][START_REF] Jackel | Coinfection of pigs with Hepatitis E and porcine circovirus 2, Saxony 2016[END_REF]. Further work is needed to investigate whether there are other underlying immune mechanisms specific to co-infecting viruses. Moreover, it should be noted that the pigs in the present experiment were simultaneously inoculated with HEV and PCV2; the same kind of study could be reproduced with different inoculation time sequences (e.g. pigs inoculated with PCV2 a week before HEV) and probably with more pigs included to reduce the impact of inter-individual variability in infection dynamics.

To our knowledge, this study is the first to focus on the impact of HEV/PCV2 experimental coinfection on HEV infection and transmission in pigs. Our results show that, in experimental settings, PCV2 co-infection increases the direct transmission of HEV and impairs the humoral immune response towards it. The effect observed in this PCV2/HEV co-infection trial was less marked than previously observed when PRRSV was involved, however, and failed to explain the long-term HEV shedding that has been observed in the field at an individual level. A combination of PCV2 co-infection with other factors may lead to chronic HEV infection. Additional studies (e.g.

on-farm intervention studies, other co-infection trials, dynamic modelling approaches) should therefore be conducted to explore the potential synergistic effects of multiple co-infections and devise effective control strategies that would include measures targeting intercurrent pathogens (vaccination, eradication programme).

Figures

  3 days [4.4-25.5] in] in HEV/PCV2 co-infected pigs. The latency period was significantly shorter in HEV/PCV2 coinfected pigs than in HEV-only infected pigs (p < 0.05).Survival analysis of the infectious period (lognormal distribution) gave a mean duration of 11.8 days[8.3-16.7] for HEV-only infected animals and 16.6 days [10.7-25.9] for HEV/PCV2 coinfected animals. No statistical difference was found between HEV-only and HEV/PCV2-infected pigs (HR = 0.6 [0.3-1.4], p > 0.05).Survival analysis of the time prior to HEV seroconversion (using the Weibull distribution) gave a mean duration of25.6 days [19.3-33.8] for for HEV/PCV2 co-infection. The time prior to HEV seroconversion was statistically longer in HEV/PCV2-than in HEV-only infected pigs (HR = 0.3 [0.1-0.8], p < 0.05).

Figure 1 .

 1 Figure 1. Experimental design of the HEV/PCV2 co-infection trial.

Figure 2 .

 2 Figure 2. HEV RNA quantification in faecal, bile and serum samples from HEV-only and

Figure 3 .

 3 Figure 3. Kinetic of HEV seroconversion. Results for individual sera samples (in different colours

Figure 4 .

 4 Figure 4. Distribution of the number of HEV genome equivalents (log RNA copies/g faeces) shed

Figure 5 .

 5 Figure 5. Running average of transmission parameter estimates from ten independent Monte-Carlo

Figure 6 .

 6 Figure 6. Distribution of direct and environmental HEV transmission parameters estimated from

Supplementary Files

Supplementary File 1. PCV2 DNA quantification in serum (a) and PCV2 antibodies detection (b) from HEV-only and HEV/PCV2 infected pigs (inoculated and contact animals, n=36).

Authors' contributions and acknowledgements

MS and MA developed the mathematical model, analysed the data and drafted the manuscript. MP and CB analysed the HEV and PCV2 samples respectively, and interpreted the results. NP and BG supervised the HEV-and PCV2-related laboratory work respectively, and helped coordinate the study. NR conceived and coordinated the study, in addition to participating in the animal experiment and data analyses. All the co-authors revised the manuscript and approved the final submitted version. The authors would like to thank Frédéric Paboeuf, Angélique Moro, Nadège Morin, Yann Bailly and Gérald Lediguerher for their excellent technical management of the experiment.

Competing interests and funding

The authors declare that they have no competing interests. The trial was funded by INAPORC.

Morgane Salines received a PhD grant from the French Ministry for Agriculture and Food.

Room


MD. Guillaume Lassailly
email: guillaume.lassailly@chru-lille.fr

Mohamed Bou Saleh
email: mohamed.bou-saleh@inserm.fr

Natascha Leleu
email: natascha.leleu-2@univ-lille2.fr

Massih Ningarhari
email: m.ningarhari@gmail.com

Émilie Gantier

Rodolphe Carpentier
email: rodolphe.carpentier@univ-lille2.fr

Florent Artru
email: florent.artru@chru-lille.fr

Viviane Gnemmi
email: viviane.gnemmi@chru-lille.fr

Benjamin Bertin
email: benjamin.bertin-2@univ-lille2.fr

Patrice Maboudou
email: patrice.maboudou@chru-lille.fr

Natascha Leleu-Chavain

Emilie Gantier
email: emilie.gantier@inserm.fr

Didier Betbeder
email: betbederdidier985@gmail.com

Céline Gheeraert
email: celine.gheeraert@inserm.fr

François Maggiotto

Sébastien Dharancy
email: sebastien.dharancy@chru-lille.fr

Philippe Mathurin
email: philippe.mathurin@chru-lille.fr

Alexandre Louvet
email: alexandre.louvet@chru-lille.fr

Ph. D Laurent Dubuquoy
email: laurent.dubuquoy@inserm.fr

Liric -Umr995

Nucleotide-binding oligomerization domain 1 (NOD1) modulates liver ischemia reperfusion through the expression adhesion molecules

Keywords: Liver ischemia reperfusion, Innate immunity, liver trasnplantation, adhesion molecules, hepatocytes, nanoparticles antagonist,

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

Lay summary

Nucleotide-binding oligomerization domain 1 (NOD1) is as an important modulator of polymorphonuclear (PMN)-induced liver injury such as in ischemia reperfusion (IR). Here, we show that NOD1 pathway targets liver adhesion molecule expression (ICAM-1 & VCAM-1) on endothelium and on hepatocyte through p38 and ERK signaling pathways. The early increase of adhesion molecule expression after reperfusion, emphasizes the importance of adherence molecules in liver injury severity. In this study we have generated PGLAnanoparticles loaded with NOD1 antagonist. Nanoparticles have shown to reduce liver necrosis, by reducing PMN liver infiltration and adhesion molecules expression.

Abstract (262 words)

Objective: In liver transplantation, organ shortage leads to use marginal grafts that are more susceptible to ischemia reperfusion (IR). We identified nucleotide-binding oligomerization domain 1 (NOD1) as an important modulator of polymorphonuclear (PMN)-induced liver injury such as in IR. Here we aimed at elucidating its role in the liver IR, especially on the endothelium and hepatocyte.

Design: NOD1 WT and KO mice were treated with NOD1 agonists and submitted to liver IR.

Expression of adhesion molecules were analyzed in total liver, isolated hepatocytes and endothelial cells. Interaction between PMN and hepatocyte was studied in ex vivo co-culture model using electron microscopy (EM) and LDH levels. We generated NOD1 antagonistloaded nanoparticles (np ALINO).

Results: NOD1 agonist treatment increased liver injury, PMN tissue infiltration and upregulated ICAM-1 and VCAM-1 expression 20 hours after reperfusion. NOD1 agonist treatment without IR increased expression of adhesion molecules (ICAM-1, VCAM-1) in total liver and more particularly in WT hepatocytes, but not in NOD1 KO hepatocytes. This induction is dependent of p38 and ERK signaling pathways. As compared to untreated hepatocytes, NOD1 agonist markedly increased hepatocytes lysis in co-culture with PMN as shown by the increase of LDH in supernatants. Interaction between hepatocytes and PMN was confirmed by EM. In a mouse model of liver IR, treatment with np ALINO significantly reduced the area of necrosis, transaminase level and ICAM-1 expression.

Conclusion:

NOD1 regulates liver IR injury through induction of adhesion molecules and modulation of hepatocytes-PMN interaction. NOD1 antagonist-loaded nanoparticles reduced liver IR injury and open up new perspectives to prevent IR especially in the context of liver transplantation.

Introduction

In the field of liver transplantation, organ shortage is a major issue; especially in western countries where the living donor transplantation is still scare. This situation leaded transplant units to use of liver allografts following donation after cardiac death along with marginal and extended criteria donors [START_REF] Casavilla | Experience with liver and kidney allografts from non-heart-beating donors[END_REF] . These marginal grafts are known to be more susceptible to early graft dysfunction and retransplantation, increasing morbidity and mortality [START_REF] Clavien | Preservation and reperfusion injuries in liver allografts. An overview and synthesis of current studies[END_REF][START_REF] Jaeschke | Mechanisms of Liver Injury. II. Mechanisms of neutrophil-induced liver cell injury during hepatic ischemia-reperfusion and other acute inflammatory conditions[END_REF][START_REF] Lentsch | Inflammatory mechanisms and therapeutic strategies for warm hepatic ischemia/reperfusion injury[END_REF][START_REF] Serracino-Inglott | Hepatic ischemia-reperfusion injury[END_REF] mostly due to liver ischemia reperfusion (IR) injuries [START_REF] Lee | Early allograft dysfunction in liver transplantation with donation after cardiac death donors results in inferior survival[END_REF][START_REF] Merion | Donation after cardiac death as a strategy to increase deceased donor liver availability[END_REF] . Liver IR is a biphasic phenomenon in which hypoxia-induced lesions are exacerbated after oxygen delivery is restored, shear stress and polymorphonuclear neutrophils (PMN) tissue infiltration [START_REF] Peralta | Hepatic ischemia and reperfusion injury: effects on the liver sinusoidal milieu[END_REF] . This process of liver injury occurs also during hemodynamic instability and hepatic resection. It is a frequent cause of acute liver dysfunction 2-5, 9, 10 . Thus, preventing IR and its consequences remains a clinical challenge.

The pathophysiology of liver IR involves numerous cells (i.e. PMN, liver sinusoidal endothelial cells (LSEC) and hepatocytes) and is considered to be an experimental model of PMN-mediated hepatitis [START_REF] Jaeschke | Mechanisms of Liver Injury. II. Mechanisms of neutrophil-induced liver cell injury during hepatic ischemia-reperfusion and other acute inflammatory conditions[END_REF] . The process of injury occurs during the reperfusion phase with PMN infiltration. The first step of this event is the interaction between PMN and the endothelium, allowing PMN to enter into the liver parenchyma. The upregulation of adhesion molecules (i.e. ICAM-1, VCAM-1, E-Selectin) on LSEC follows reperfusion, allowing PMN recruitment with the rolling process [START_REF] Springer | Adhesion receptors of the immune system[END_REF] through the binding of neutrophil adhesion molecules L-Selectin and Mac-1, an heterodimeric integrin (CD11b/CD18) [START_REF] Steinhoff | Intercellular immune adhesion molecules in human liver transplants: overview on expression patterns of leukocyte receptor and ligand molecules[END_REF] . The dual role and interaction between endothelium and PMN is accepted to be a cornerstone of the pathophysiology of liver IR. Thus, inhibiting adhesion molecule expression (as ICAM, VCAM, E-Selectin, CD44 and PECAM) or using blocking antibodies have been largely studied to reduce liver injury [START_REF] Ala | Role of cell adhesion molecules in leukocyte recruitment in the liver and gut[END_REF] .

Contrary to the endothelium, the mechanisms driving the interaction between PMN and hepatocytes during liver IR remain to be clarified. The expression of adhesion molecules by the hepatocyte has been suggested to be important for its interaction with PMN during IR [START_REF] Benkoel | Expression of intercellular adhesion molecule-1 (ICAM-1) during ischemia-reperfusion in human liver tissue allograft: image analysis by confocal laser scanning microscopy[END_REF] . Intercellular adhesion molecule-1 (ICAM-1) has been shown to be expressed on hepatocyte plasma membrane under IR conditions [START_REF] Benkoel | Expression of intercellular adhesion molecule-1 (ICAM-1) during ischemia-reperfusion in human liver tissue allograft: image analysis by confocal laser scanning microscopy[END_REF] . However, it remains unclear whether the regulation of ICAM expression on the hepatocyte impacts the severity of IR lesions.

There is evidences that damage-associated molecular patterns (DAMPs) and pathogenassociated molecular patterns (PAMPs) activate PMN 16 17 leading to IR-mediated liver injury. We previously showed that activation of the nucleotide-binding oligomerization domain 1 (NOD1), a cytosolic pattern recognition receptor (PRR), was responsible for activation of PMN function and migration [START_REF] Dharancy | Neutrophil migration during liver injury is under nucleotide-binding oligomerization domain 1 control[END_REF] . Indeed, by activating mitogen-activated protein (MAP) kinases such as p38, NOD1 leads to PMN infiltration in the liver, facilitating injury in a model of liver IR. At the opposite, NOD1 knockout (KO) mice were protected against liver IR injury 18 . In addition, NOD1 is significantly expressed in the liver and in hepatocytes [START_REF] Scott | Hepatocytes express functional NOD1 and NOD2 receptors: a role for NOD1 in hepatocyte CC and CXC chemokine production[END_REF] , thus NOD1 pathway appears as promising for liver IR injury regulation.

The aim of our study was to dissect the role of the NOD1 pathway on the expression of adhesion molecules in the liver, and more specifically in endothelial cells and in hepatocytes. As IR lesions are induced by PMN liver parenchyma infiltration, we also explored the modulation of hepatocytes and PMN interaction by NOD1. Then, as a proof of concept, we have tested NOD1 as a potential therapeutic target with the use of a NOD1 inhibitor for the prevention of IR lesions in a mouse model.

Methods

Reagents

Three different NOD1 agonists were used in this study. The natural ligand iE-DAP (1 µg/ml in vitro and 5 mg/kg in vivo) and its derivative C12-iE-DAP (100 ng/ml in vitro and 3.3 mg/kg in vivo) were obtained from InvivoGen (Toulouse, France). FK565 (synthetic ligand, 10 -6 M in vitro and 1 mg/kg in vivo) was obtained from Dr. M. Chamaillard. TNFα (50 ng/ml) and IFNγ (100ng/ml) were obtained from R&D (Lille, France). We used ML-130 (Notilinib) and ALINO73 (analog of SB711 [START_REF] Rickard | Identification of selective small molecule inhibitors of the nucleotide-binding oligomerization domain 1 (NOD1) signaling pathway[END_REF] ) synthetized by Natascha Leleu-Chavain as described as NOD1 antagonists [START_REF] Rickard | Identification of selective small molecule inhibitors of the nucleotide-binding oligomerization domain 1 (NOD1) signaling pathway[END_REF][START_REF] Khan | Identification of Inhibitors of NOD1-Induced Nuclear Factor-kappaB Activation[END_REF] . The ERK inhibitor (U0126, Sigma, Saint-Quentin Fallavier, France) and p38 inhibitors (SB203580, InvivoGen, Toulouse France) were used at 10µM. Resomer RG503H was used as a PLGA (polylactic-co-glycolic acid) matrix (Evonik Industries AG,

Deutschland). (Supplementary CTAT Table)

Animals

C57BL/6J wild-type mice (Janvier Labs, Le Genest-Saint-Isle, France), Nod1 KO (C57BL/6J background) [START_REF] Dharancy | Neutrophil migration during liver injury is under nucleotide-binding oligomerization domain 1 control[END_REF] and their wild-type littermates (Nod1 WT) were used in this study.

Animals were kept in a controlled environment (12 h light/dark cycles) and fed a standard rodent pellet diet (RM1, Special Diet Service, France) ad libitum. Experiments performed on animals were approved by the local ethics committee (n° 01757.01) in accredited facilities (n°B59-35010) according to government guidelines (n°2010/63/UE).

IR injury

We used a murine model of 70% partial hepatic ischemia for 60 min. Mice were or were not treated with a NOD1 agonist 24h and 4h before surgery. Then mice were anesthetized with 10 mg/kg xylazine and 100 mg/kg ketamine, and a midline laparotomy was performed. The left lateral and median lobes of the liver were ligatured using Ethicon mersutures F2541 (Ethicon, Issy Les Moulineaux, France). After 60 min of ischemia, the ligature was removed to initiate hepatic reperfusion. Mice were sacrificed 3hours (for hepatocyte isolation of the ex vivo experiment) or at 4, 8 and 20 hours after reperfusion. Blood and liver samples were collected for analysis at each time point. Liver samples were fixed in paraformaldehyde 4% (w/v in PBS) and paraffin embedded or snap frozen.

Cell isolation and culture

PMN were isolated following sterile peritonitis as previously described [START_REF] Seglen | Preparation of isolated rat liver cells[END_REF] . Briefly, mice received an i.p. injection of 1 mL of a solution of oyster glycogen at 150µg/µL in phosphate buffer saline (PBS). Sixteen hours later mice were sacrificed and peritoneal cells were harvested using 5 mL of sterile PBS without Ca 2+ /Mg 2+ . Exudate cells were determined to be >95% neutrophils by flow cytometry.

Hepatocytes were isolated by the collagenase perfusion method [START_REF] Seglen | Preparation of isolated rat liver cells[END_REF] . Briefly, mice were anesthetized with 2% isofluran. After laparotomy, the portal vein was cannulated (24G catheter, Terumo, Guyancourt, France). The liver was then perfused for 10 to 20 minutes at 5 mL/min with washing buffer (HBSS W/O Ca 2+ Mg 2+ , EDTA 0.5 mM, HEPES 50 mM), then perfused with collagenase buffer (HBSS W/O Ca 2+ Mg 2+ , HEPES 50 mM, CaCl 2 50 mM, Collagenase type IV 0.0025% 850 CDU/mg (C5138, Sigma Aldrich, Saint-Quentin Fallavier, France) for 6 minutes at 5ml/min. Following perfusion, the liver was collected in washing buffer and the Glisson's capsule was cut. Cell suspension was filtered in a 70 µM cell strainer. Cells were washed twice with washing buffer and centrifuged at 50g for 2 minutes at room temperature to pellet murine hepatocytes. Hepatocytes were then washed with hepatocyte culture medium (DMEM Glutamax, glucose 4.5 g/L, Gentamycin 1%, FCS 10%, 10 -7 M dexamethasone) and centrifuged at 500g for 5 minutes. One million hepatocytes were plated in 6-well plate (Sarstedt, Marnay, France).

The main challenge to investigate the hepatocyte expression of adhesion molecules was to avoid any contamination of LSEC during the isolation of the mouse primary hepatocytes. To deal with this contingency, we have optimized our experimental method and check the purity of isolated hepatocyte by FACS using CD31 as a marker for endothelial cell contamination. Considered acceptable a purity > 99% (supplementary figure1). In addition a cytospin followed by cell staining were performed before in vitro and ex vivo analysis to confirm purity. Primary Human umbilical vein endothelial cells (HUVEC) were obtained from Prof.

Eric Boulanger (Lille, France) and were maintained in endothelial cell basal medium supplemented with Bullet Kit (EBM-2) on a culture flask coated with 0.1% gelatin and maintained at 37°C with humidified of 5% carbon dioxide. HUVEC cultured from passages 4 to 8 were used for the experiments.

Hepatocyte and PMN co-culture

Freshly isolated hepatocytes were plated on a 48-well plate (1.25x10 5 cell per well) and incubated at 37°C with 5% CO 2 for 36 hours. Purified peritoneal PMN (6.25x10 5 cell per well) were or were not added to the hepatocyte culture for 4 hours in HBSS medium (w/o phenol red). Supernatants were then harvested and centrifuged at 500g for 5 min to discard cells and debris. These supernatants were used to quantify lactate dehydrogenase (LDH) activity using the manufacturer's protocol (Roche, Boulogne-Billancourt, France).

Real-time RT-PCR for mRNA quantification

Real-time PCR analyses were performed for quantification of mRNA expression.

Mouse glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was used as a housekeeping gene. Sequence-specific PCR primers were designated using Primer3 Software [START_REF] Koressaar | Enhancements and modifications of primer design program Primer3[END_REF][START_REF] Untergasser | Primer3--new capabilities and interfaces[END_REF] ; see Table S1 and Supplementary CTAT Table for details). Total RNA was extracted from isolated hepatocytes or liver samples using Nucleospin®RNAII (Macherey-Nagel EURL, Hoerdt, France). First strand cDNA was synthesized from 1 µg total RNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Courtaboeuf, France). Realtime polymerase chain reaction (RT-PCR) was performed using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) in StepOne plus (Applied Biosystems, Foster City, CA, USA).

Western blot

For western blot studies, proteins were extracted from liver samples or isolated hepatocytes in a lysis buffer including PBS with 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS), 100mM Phenyl Methyl Sulfonyl Fluoride (PMSF) and a classical protease inhibitor cocktail (Roche Diagnostics, Penzberg, Germany) as well as phosphatase inhibitors (Sigma-Aldrich, Lyon, France). Fifty micrograms of total proteins were then separated by SDS-PAGE and electroblotted on nitrocellulose membranes.

Membranes were incubated overnight with primary antibodies (see Table S2 and Supplementary CTAT Table for details). Immunodetection was completed with a secondary peroxidase-conjugated antibody (1:1000, Dako Laboratories, Trappes, France) and chemiluminescence was performed according to the manufacturer's protocol (ECL, Amersham Pharmacia Biotech, Orsay, France). β-actin was used as a loading control. Band intensity was analyzed using the MM4000 Pro (CareStream, Noisy-le-Grand, France), quantified using Image J (NIH) and compared to β-actin. Results were expressed as relative units.

Immunohistochemistry, necrosis area quantification & liver PMN quantification

Liver samples were fixed in paraformaldehyde (PFA) 4% and embedded in paraffin.

Four-micrometer-thick sections were exposed to primary antibodies after a specific step of heat-performed antigen retrieval (see Table S2 for details) and then to a biotinylated secondary antibody for 1 h. After washing in TBS + 0.05% Tween 20, sections were incubated with strepavidin-horseradish peroxidase (Dako Laboratories, Trappes, France).

Staining was revealed using 3,3′-diaminobenzidine substrate (Dako Laboratories) for 1 to 5 minutes before the reaction was stopped in distilled water, and counterstained with hematoxylin. Negative controls were incubated with irrelevant serum or isotype-matched immunoglobulin instead of the specific antibody. Stained slides were observed and analyzed under a microscope (Leica, Bensheim, Germany). Pictures were obtained using Axio Scan.Z1 (Zeiss, Göttingen, Germany).

Quantification of necrosis was obtained on digital images of IR livers (3 per mice)

obtained from Axio Scan Z1 (Zeiss, Göttingen, Germany). The area of necrosis was defined as the ratio of (area of necrosis/ total liver area) x 100. Quantification of PMN infiltration was performed by counting PMN on 8-10 fields of necrosis and/or centrolobular area (magnification, 400) for each mouse.

Human ICAM-1 promoter analysis

The proximal human ICAM-1 promoter was amplified from human liver genomic

Nanoparticle synthesis, formulation and characterization.

Poly-Lactic co-glycolic acid (PLGA) nanoparticles (np) were prepared as previously described [START_REF] Broc-Ryckewaert | Development of innovative paclitaxel-loaded small PLGA nanoparticles: study of their antiproliferative activity and their molecular interactions on prostatic cancer cells[END_REF] . The Resomer RG503H (10mg) was dissolved in 1ml of an organic phase consisting of acetone and ethanol (85:15). After complete dissolution of the Resomer, it was injected into 10ml water by stirring for 30 minutes at room temperature (21 to 24 °C).

Organic solvents were then evaporated with a rotary vacuum evaporator at 120rpm, 28°c for 4 minutes. The resulting nanoparticles are used as a control (np). Association with the NOD1 antagonist was performed by post-loading and adding the antagonists into the nanoparticle suspension. ML130 was dissolved in DMSO at 100mg/ml and loaded with np from 0.075% to 3% (w/w) for in vitro cell delivery corresponding to treatment from 0.125 to 5µM (NP-ML130). ALINO73 was dissolved in DMSO at 20mg/ml and loaded with np from 0.2% to 4% (w/w) for in vitro cell delivery corresponding to treatment from 0.25 to 5µM (npALINO).

For in vivo delivery, ALINO73 was loaded at 7.5% (w/w NP) and 5% (w/v) of glucose was added to the final suspension to compensate for osmolarity.

The in vivo formulation of np ALINO was characterized for size (Z-Average = 92.8 nm) and zeta potential (-11.8 mV) respectively by dynamic light scattering and electrophoretic mobility analysis on a ZetaSizer NanoZS (Malvern Instrument, France). The poly dispersity index (PDI) was 0.259.

Statistics

Data were expressed as medians, means ± SEM or means ± SD. All comparisons were analyzed using the Mann & Whitney test. Statistics were calculated using GraphPad Prism version 5.0, (GraphPad Software, San Diego, CA, USA). Differences were considered to be statistically significant if p was <0.05.

Results

Induction of adhesion molecule expression in liver of mice treated with NOD1 agonists, in normal condition (without liver IR)

Twenty hours after treatment with NOD1 agonist injection in normal condition (without liver IR), mouse livers displayed significant induction of proteins ICAM-1 and VCAM-1, two important adhesion molecules (1.4 RU vs. 0.8 RU, p=0.032 and 0.25RU vs.

0.00 RU, p=0.008 respectively), compared to vehicle-treated mice. There was no effect of NOD1 agonist on E-selectin and P-selectin protein expression (figure 1A). The immunostaining of ICAM-1 showed that NOD1 agonist treatment increased ICAM-1 expression not only on endothelium but also on hepatocyte membranes compared to untreated livers which mainly displayed endothelial staining (figure 1B).

NOD1 agonist increases liver IR injury and up-regulates ICAM-1 and VCAM-1.

Twenty hours after IR, liver of mouse treated with NOD1 agonist displayed more necrosis area than those treated with vehicle (figure 2A). 2E).

Considering other adhesion molecules, there was no difference of expression after NOD1 agonist treatment in liver IR condition for E-selectin and P-selectin (p<0.05) (figure 2E).

NOD1 pathway targets ICAM-1 and VCAM-1 expression in vitro in primary endothelial cell and hepatocyte

HUVEC were cultured and exposed to NOD1 agonist at different dosage (0, 0.1 and 1 µg). The mRNA expression of Icam-1, Vcam-1 and E-selectin were induced by NOD1 agonist treatment in a dose dependent manner. However, Pecam mRNA expression was not significantly modified by NOD1 activation (figure 3A).

Mouse primary hepatocytes were cultured in vitro. Before analysis, the purification of isolated cells was confirmed (hepatocyte purity > 99%, supplementary figure 1). Primary hepatocytes were then exposed to NOD1 agonists under basal or inflammatory (TNF/IFN) conditions. As shown in figure 3B, Icam-1 mRNA was induced twice in Nod1 wild-type (WT) hepatocytes treated with the NOD1 agonist, as well as under inflammatory conditions induced by TNF/IFN. Vcam-1 mRNA was significantly induced by NOD1 activation (1.2 RU vs 3.3 RU, p=0.005, figure 3A). Induction of Icam-1 and Vcam-1 mRNA expression was synergistic with the NOD1 agonist and TNF/IFN treatments (figure 3B). These data were confirmed at the protein level in mouse primary isolated hepatocytes which showed that ICAM-1 and VCAM-1 were significantly increased by the NOD1 agonist both under basal and inflammatory conditions (figure 3D). To confirm the specificity of the NOD1 agonist, hepatocytes were isolated from Nod1 KO mice and exposed to NOD1 agonist treatment under basal and inflammatory conditions. Real-time PCR and Western-blot showed that the NOD1 agonist did not induce ICAM-1 and VCAM-1 in NOD1 KO hepatocytes but responded to TNF/IFN treatment (figure 3C,E).

NOD1 pathway targets ICAM-1 and VCAM-1 expression ex vivo in hepatocyte

In ex-vivo conditions, NOD1 agonist treatment of sham operated mice induced significant hepatocyte expression of Icam-1 and Vcam-1 mRNA (6-fold and 10-fold increase, respectively) compared to vehicle-treated animals (figure 4A). IR alone also induced Icam-1 and Vcam-1 mRNA (4 fold and 5-fold increase, respectively). NOD1 activation during IR markedly increased hepatocyte Icam-1 and Vcam-1 mRNA expression (25-fold and 20-fold increase, respectively) compared to vehicle-treated sham animals (figure 4A). At the protein level, NOD1 agonist treatment in sham-operated mice strongly induced hepatocyte expression of ICAM-1 and VCAM-1 (7.1-fold and 4.0-fold, respectively, figure 4C) compared to untreated mice. After 3 hours of reperfusion, induction of the ICAM-1 and VCAM-1 proteins also increased significantly (2.1-fold and 2.0-fold respectively, figure 4C). NOD1 agonist treated Nod1 WT mice that underwent liver IR showed massive induction of hepatocyte expression of ICAM-1 and VCAM-1 protein (37-fold and 109-fold, respectively) compared to vehicle-treated sham animals. More importantly, NOD1 agonist treatment increased ICAM-1 and VCAM-1 protein expression by 17.7 and 54.5 fold respectively upon liver IR conditions (figure 4C).

In Nod1 KO mice, the NOD1-mediated induction of ICAM-1 and VCAM-1 expression was blunted both following IR or a sham-operation (figure 4B,D).

NOD1 activation in hepatocytes amplifies the interaction with PMN and subsequent lysis

Examination of the co-culture by electron microscopy revealed important adhesion of PMNs on hepatocytes which showed signs of distress such as swelling of mitochondria, cytoplasm clarification and lysosome (figure 5A).

Hepatocytes isolated from Nod1 WT mice that were or were not treated with a NOD1 agonist showed low levels of spontaneous lysis when cultured alone (figure 5B). A significant increase in LDH activity was observed when hepatocytes from untreated mice were cocultured with PMN isolated from untreated WT mice. When hepatocytes from NOD1 agonisttreated mice were co-cultured with PMN isolated from untreated WT mice, there was a significant increase in LDH activity compared to hepatocytes from vehicle-treated animals (figure 5B).

To confirm the specificity of NOD1 in relation to this event, we reproduced these experiments with hepatocytes isolated from Nod1 KO mice treated or not with a NOD1 specific agonist. As observed in figure 5C, targeting of Nod1 KO hepatocytes by PMN was reduced and NOD1 agonist-induced lysis was completely blunted.

NOD1 signaling pathways driving ICAM-1 and VCAM-1 expression in hepatocytes

A liver active form of ERK (phospho ERK) was induced from 10 to 30 min after treatment with a NOD1 agonist (figure 6A). A similar pattern was observed for p38 since active phospho p38 was induced at 10 and 30 min and then returned to the steady state 60 min after stimulation with a NOD1 agonist (figure 6B). Treatment with the ERK inhibitor (U0126) significantly limited the increase in Icam-1 and significantly blocked the Vcam-1 mRNA overexpression induced by the NOD1 agonist (figure 6C-D). The p38 inhibitor (SB203580) further blocked NOD1-induced hepatocyte Icam-1 and Vcam-1 mRNA expression. Cotreatment with both ERK and p38 inhibitors did not increase blockade of the NOD1 effect.

In vitro, we first cloned the human ICAM-1 promoter (figure 6E) and showed that NOD1 activation by an agonist resulted in a 50% increase in luciferase activity driven by this promoter compared to untreated cells (figure 6F).

NOD1 antagonist development to prevent IR-induced liver injury

ML130 and ALINO73 are two specific NOD1 inhibitors that have been identified by screening different libraries of chemical compounds for activation or inhibition of NOD receptors [START_REF] Rickard | Identification of selective small molecule inhibitors of the nucleotide-binding oligomerization domain 1 (NOD1) signaling pathway[END_REF][START_REF] Khan | Identification of Inhibitors of NOD1-Induced Nuclear Factor-kappaB Activation[END_REF] . In an in vitro model that allows evaluation of NOD1 activation, cells were treated with a NOD1 agonist and increasing doses of either ML130 or ALINO73

(supplementary figure 3A-B). Treatment with a NOD1 agonist induced significant expression of the reporter gene (SEAP, OD 655 0.165±0.014). Addition of ML130 resulted in a dosedependent decrease in NOD1 activity from 0.16µM (OD 655 0.106±0.014) to maximum inhibition at 5µM (OD 655 0.013±0.014). Treatment with ALINO73 led to dose dependent inhibition of NOD1 activity from 0.16µM (OD 655 0.197±0.020 vs 0.140±0.021) to maximum inhibition at 2.5µM (OD 655 0.030±0.014).

Because both NOD1 antagonists are not soluble in injectable vehicles used for in vivo applications, ML130 and ALINO73 were associated with PLGA nanoparticles to facilitate in vivo delivery to the liver.

The effect of antagonist-loaded nanoparticles on NOD1 activity was tested in the same in vitro system as above (supplementary figure 3C-D). As shown in supplementary figure 3C when ML130 was associated with PLGA nanoparticles, it lost its inhibitory potential on NOD1 activity, ALINO73-loaded nanoparticles (np ALINO) appeared to be at least as effective as free ALINO73 for the dose-dependent inhibition of NOD1 activity in vitro (supplementary figure 3D).

Modulating IR-induced liver injury with NOD1 antagonist nanoparticles

np ALINO was then used to test the in vivo potential of NOD1 antagonists. Mice received an intravenous injection of nanoparticles that was either empty (np Empty) or loaded with the NOD1 inhibitor (np ALINO) before undergoing one hour of ischemia. As shown in figure 7A, mice treated with np Empty displayed large areas of IR-induced necrosis.

However, when treated with the np ALINO the area of necrosis was lower in mouse livers (26.0±3.2% vs 15.6±2.9%, p=0.038) (figure 7B-C). These results were confirmed by determining serum transaminase. After treatment with np ALINO, serum ALT significantly decreased compared to treatment with empty nanoparticles (3935±493 IU/L vs 5814±615 IU/L, p=0.040) (figure 7D). This improvement was associated with a lower liver expression of KC mRNA and lower infiltration of PMN in liver parenchyma, whereas the PMN in centrolobular vessels were unchanged (figure 7E-7F). The expression of ICAM-1 in the liver was reduced after treatment with np ALINO, as observed using immunohistochemistry (figure 7G).

DISCUSSION

We have demonstrated in this study that NOD1 pathway modulates liver IR injury through the induced expression of adhesion molecules. These adhesion molecules are mainly ICAM-1 and VCAM-1 and are expressed on LSEC as well as on hepatocytes. With an exploratory experiment using NOD1 antagonist-loaded nanoparticles, we strongly suggest that targeting NOD1 in vivo is able to reduce liver IR injury in part by reducing the expression of ICAM-1 in the liver.

NOD1 has been previously shown to impact liver IR through increased CD11b expression in PMN [START_REF] Dharancy | Neutrophil migration during liver injury is under nucleotide-binding oligomerization domain 1 control[END_REF] . However, the role of NOD1 pathway in the liver, and more specifically the expression of adhesion molecules on hepatocyte and endothelium was unknown.

Numerous studies have demonstrated the clear impact of adhesion molecules, especially on endothelium for the regulation of histological lesions and PMN tissue infiltration [START_REF] Ala | Role of cell adhesion molecules in leukocyte recruitment in the liver and gut[END_REF][START_REF] Eltzschig | Ischemia and reperfusion--from mechanism to translation[END_REF][START_REF] Zhai | Ischaemia-reperfusion injury in liver transplantation--from bench to bedside[END_REF] . Our study emphasizes the role of NOD1 on adhesion molecule expression in the pathophysiology of liver IR. Indeed, ICAM-1 and VCAM-1 expression were increased upon NOD1 agonist treatment during liver IR. This up-regulation of adhesion molecules in the whole liver and hepatocyte occurred within the first hours of reperfusion, before necrosis establishment and PMN infiltration. This early expression of adhesion molecules is then probably a part of the first steps of pathophysiological mechanisms at the origin of reperfusion lesions.

At a cellular level, and more precisely in the context of in vitro culture of endothelial cells, Eselectin in addition to ICAM-1 and VCAM-1 is largely expressed and modulated after NOD1 pathway activation. However, the absence of significant induction of E-selectin expression after NOD1 activation at the whole organ level suggests that the endothelium is not the exclusive actor of expression of adhesion molecule in the liver. The ex-vivo and in vitro experiments demonstrate that hepatocytes are able to express ICAM-1 and VCAM-1 and these last are inducible upon NOD1 agonist treatment. Other adhesion molecules (CD44, Pselectin, PECAM) have been screened on hepatocytes, but with no significant modulation of their expression by NOD1 agonist (data not shown).

Although it is central, the PMN/hepatocyte interaction during IR has not been as extensively investigated as the PMN/endothelial interaction. During warm/cold ischemia, endothelial tissue is injured [START_REF] Selzner | Protective strategies against ischemic injury of the liver[END_REF] , sinusoidal endothelial cells are detached and sloughed into the sinusoid lumen [START_REF] Gao | Apoptosis of sinusoidal endothelial cells is a critical mechanism of preservation injury in rat liver transplantation[END_REF] , allowing PMN direct access to the hepatocyte [START_REF] Jaeschke | Neutrophils contribute to ischemia/reperfusion injury in rat liver in vivo[END_REF][START_REF] Imamura | Effects of extended cold preservation and transplantation on the rat liver microcirculation[END_REF] as confirmed by our analysis by electron microscopy (EM) (supplementary figure 4). We then designed a coculture experiment to model the PMN/hepatocyte interface and clarify the hepatocellular role of NOD1 activation in this interaction. EM analysis of our model confirmed the direct interaction between both cell types leading to hepatocyte death. Quantification of LDH shows that activation of NOD1 in the hepatocyte increases hepatocellular death upon PMN binding.

The results of the interaction between Nod1 KO hepatocyte and PMN suggest that blocking hepatocyte NOD1 could prevent PMN-induced hepatocyte death in vitro.

In relation to the intracellular pathway, the effect of NOD1 appeared mediated through ERK and p38 phosphorylation in hepatocytes. The regulatory effect of ERK and p38 inhibitors confirms the role of the MAPK pathway in NOD1-mediated ICAM-1 and VCAM-1 expression. These findings support data obtained in other cellular types such as PMN [START_REF] Dharancy | Neutrophil migration during liver injury is under nucleotide-binding oligomerization domain 1 control[END_REF][START_REF] Scott | Hepatocytes express functional NOD1 and NOD2 receptors: a role for NOD1 in hepatocyte CC and CXC chemokine production[END_REF] .

NOD1 is expressed widely in many organs and cell types [START_REF] Philpott | NOD proteins: regulators of inflammation in health and disease[END_REF] . The clinical potential of NOD1 antagonists could be extended to other organs such as renal IR pathogenesis [START_REF] Shigeoka | Nod1 and nod2 are expressed in human and murine renal tubular epithelial cells and participate in renal ischemia reperfusion injury[END_REF] or myocardium infarct [START_REF] Yang | Activation of NOD1 by DAP contributes to myocardial ischemia/reperfusion injury via multiple signaling pathways[END_REF] . In the kidney, Nod1 deficient mice are partially protected from IRinduced kidney injury. NOD1 activation by diaminopimelic acid worsens cardiac IR injury in particular through p38 MAPK. Nod1 knockdown using siRNA in cardiomyocytes attenuates cell death. The role of NOD1 in innate immune cells suggests that NOD1 antagonists should be evaluated for sepsis [START_REF] Cartwright | Selective NOD1 agonists cause shock and organ injury/dysfunction in vivo[END_REF] or PMN-related hepatitis (i.e. alcoholic hepatitis) [START_REF] Takahashi | Ultrastructural findings on polymorphonuclear leucocyte infiltration and acute hepatocellular damage in alcoholic hepatitis[END_REF][START_REF] Taieb | Polymorphonuclear neutrophils are a source of hepatocyte growth factor in patients with severe alcoholic hepatitis[END_REF][START_REF] Lazaro | Osteopontin deficiency does not prevent but promotes alcoholic neutrophilic hepatitis in mice[END_REF] . The present results can serve as a basis for future studies investigating the therapeutic potential of NOD1 antagonists.

Our results and previous studies performed in the liver [START_REF] Dharancy | Neutrophil migration during liver injury is under nucleotide-binding oligomerization domain 1 control[END_REF][START_REF] Scott | Hepatocytes express functional NOD1 and NOD2 receptors: a role for NOD1 in hepatocyte CC and CXC chemokine production[END_REF] strongly support evaluating the proof of concept for the use of NOD1 antagonists to prevent liver IR injury.

Previous screening of chemical libraries on NOD receptors 20 led us to choose two prototypic NOD1 inhibitors from 2 different chemical entities based on their optimal IC50: ML130 and ALINO73 with the greatest inhibitory effect (supplementary figure 3). ML130 and ALINO73 are insoluble in aqueous solutions and insufficiently soluble in non-toxic hydrophobic solvents. Because these low hydrophilic properties prevented their use in vivo, we used nanoparticles to transport insoluble NOD1 antagonists to the liver [START_REF] Park | Cellular distribution of injected PLGAnanoparticles in the liver[END_REF] . We choose PLGA nanoparticles because of their high biocompatibility and FDA approved safety. PLGA is currently used in several drugs such as: Sandostatin LAR© (USAN (United States Adopted Name): Octreotide) [START_REF] Zhang | Inhibition of peptide acylation in PLGA microspheres with water-soluble divalent cationic salts[END_REF] for neuroendocrine tumors, Bydureon© (USAN: Exenatide) [START_REF] Qi | Comparative studies on the influences of primary emulsion preparation on properties of uniform-sized exenatide-loaded PLGA microspheres[END_REF] in diabetes and Risperdal Consta© (USAN: Risperidone) in schizophrenia [START_REF] Rawat | Comparison of in vitro-in vivo release of Risperdal((R)) Consta((R)) microspheres[END_REF] .

The in vitro testing of PLGA nanoparticles loaded with ML130 or ALINO73 showed that np ML130 was not effective while np ALINO inhibited NOD1 activity at least as efficiently as free ALINO73 suggesting a high bioavailability despite the PLGA integration.

In mice, this NOD1 antagonist-loaded nanoparticle, np ALINO, decreased the area of necrosis induced by liver IR by about 40%. This decrease in histological lesions was associated with a 33% decrease in serum transaminase supporting npALINO as an interesting therapeutic approach to prevent liver IR. The npALINO treatment was also associated with lower KC mRNA expression in the liver, lower PMN infiltration and reduced ICAM expression. The reduction of KC expression could have reduced total amount of PMN recruited to the liver. Indeed, npALINO did not impact the number of PMN in centrolobular vessels, but reduced significantly the severity of PMN infiltration in necrosis areas. These results raise the therapeutic potential of NOD1 antagonists in preventing hepatic IR in humans. The use of NOD1 antagonists should be tested during different steps of liver transplantation. These NOD1 antagonists could be use either in the donor before the organs are harvested, or added to the preservation protocol, as well as a treatment during reperfusion in the recipient.

In conclusion, this study states that NOD1 regulates adhesion molecules expression in the

ACKNOWLEDGEMENTS:

Authors would like to thank Nathalie Jouy, Anne Loyens and Meryem Tardivel from the Flow Core Facility -BioImaging Center Lille (F-59000 Lille, France) for the access and excellent technical support. Thanks to Melanie Legrand, Cyril Degraeve, Amélie Mine, Emeline Gorecki, Justine Beauchamp, Mélanie Bollier, Perrine Six and Arnaud Dendooven for technical assistance.

C-D:

Representative western-blots for quantification of ICAM-1 (upper blot) and VCAM-1 (lower blot) protein expression that were compared to β-actin in ex vivo primary hepatocytes.

Nod1 KO mice (D) or their wild-type littermates (Nod1 WT mice, C), were treated or not with a NOD1 agonist (FK565) after sham surgery or ischemia/reperfusion (isch/reperf).

Ratios of band intensity are indicated as relative units (RU).

E

  .G., M.B.S., L.D., G.L., F.A., F.M., C.G., P.M. and M.N. generated data, L.D., G.L., S.D. and Ph.M. designed experiments; V.G. performed histological analysis; N.L.C., R.C., B.B., D.B. designed and provided essential tools; L.D., G.L., A.L. analyzed the data; L.D., G.L., N.L.C., R.C. prepared the manuscript; A.L., S.D., F.A. and Ph.M. edited the manuscript; L.D., G.L. and Ph.M. provided experimental funding.

  ) corresponding to fragments between -895 and -57 upstream from ATG. The purified fragment was cloned in the multiple cloning site of the reporter plasmid pGL4.10 [luc2] (Promega, France) using SalI and BglII (NEB, USA). This construct (Prom ICAM, figure5B) was amplified and purified before sequencing to confirm correct integration of the promoter. CV-1 cells (ATCC ® CCL-70 ™ ) were plated in 48-well plates (40000 cells/well) and transfected with empty pGL4.10 [luc2] or Prom ICAM (100 ng/well) and a human NOD1 expression vector (20 ng/well, pHA-NOD1 kind gift from Dana J. Philpott). After 20 hours, cells were washed and lysed for luminescence quantification.

  liver. NOD1 activation and blockage experiments confirm that NOD1 pathway modulates the severity of liver IR injury. These data complement the impact of NOD1 on PMN function and migration. Thus NOD1 has a dual role and targets two different actors implicated in reperfusion injury. However, the relative contribution of each actor (PMN or adhesion molecule) on liver IR injury is unclear. New experiments are needed to puzzle out the full mechanism of NOD1 pathway. This work supports the development of NOD1 inhibitors.

Figure 1 :

 1 Figure 1: NOD1 activation induced liver expression of adhesion molecules under

Figure 2 :B:C:

 2 Figure 2: NOD1 activation induced liver expression of adhesion molecules after liver IR

Figure 3 : 1 (

 31 Figure 3: NOD1 activation induces hepatocyte expression of ICAM-1 and VCAM-1.

Figure 4 :

 4 Figure 4: During ischemia reperfusion, NOD1 pathway induced hepatocyte expression

Figure 5 :Figure 6 :F:

 56 Figure 5: NOD1 pathway activation leads to hepatocyte lysis by neutrophils.

(VCAM-1), wild-type (WT). The authors declare no conflict of interest in relation to this manuscript. Financial support: The author acknowledges funding support from the National Institute on Alcohol Abuse and Alcoholism grant 1U01AA021908, AFEF, program StartAIRR ANOD1 Region Haut de France. Ethics committee number for animal experiments: n° 01757.