Quentin Devalloir
email: quentin.devalloir@univ-fcomte.fr

Clémentine Fritsch

Yara Alchammas

Francis Raoul

Vincent Driget

Caroline Amiot

Shinji Ozaki

Nico Van Den Brink

Renaud Scheifler

Environmental pollution and nutritional quality modulate immune response of the wood mouse (Apodemus sylvaticus) through hormonal disturbances

Keywords: Immunomodulation, trace metals, selenium, glucocorticoids, mammals

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

Graphical abstract

Highlights

• Cadmium, lead and selenium modulate immune response of wild and captive wood mice • Animals from highly polluted site exhibited high inflammatory response • Captive animals from lowly polluted site fed standard food showed high response • Captive animals fed Se-deficient food exhibited low response • Inflammatory responses were related to Se and might be mediated by corticosterone

Introduction

Severe changes have occurred in many habitats of free ranging mammal populations (e.g. fragmentation, habitat loss, pollution, reduction of food availability and/or quality…) turning their living area into a stressful environment. Wild rodents are also constantly exposed to infectious diseases [START_REF] Strandin | Reviewing the effects of food provisioning on wildlife immunity[END_REF][START_REF] Viney | The Immunology of Wild Rodents: Current Status and Future Prospects[END_REF], making the immune system integrity and efficiency crucial to their health and fitness. Pollution and poor nutritional quality have been shown to have severe implication on both immune and endocrine systems [START_REF] Acevedo-Whitehouse | Effects of environmental change on wildlife health[END_REF]. The role of the immune system is to eliminate pathogens while the endocrine system ensures peripheral organ functioning to maintain homeostasis [START_REF] Wensveen | Beauty and the beast' in infection: How immune-endocrine interactions regulate systemic metabolism in the context of infection[END_REF]. However, both maintain an equilibrium between pro-(immune) and anti-(endocrine) inflammatory responses to a stress, in order to avoid autoimmune damages or an increasing susceptibility to infections [START_REF] Wensveen | Beauty and the beast' in infection: How immune-endocrine interactions regulate systemic metabolism in the context of infection[END_REF][START_REF] Martin | Stress and immunity in wild vertebrates: Timing is everything[END_REF]. In the purpose of the conservation of free ranging mammals, it is primordial to characterise the impact of both pollution and nutritional quality on the ability of organisms to respond to an infection, also known as immunocompetence. Wildlife diseases can be affected by pollutants by increasing pathogen prevalence and by decreasing immune function of the host [START_REF] Acevedo-Whitehouse | Effects of environmental change on wildlife health[END_REF].

Ensuring an efficient immune response against pathogens is central to fitness as it may reduce energetic cost allocated to reproduction for instance [START_REF] Lochmiller | Trade-offs in evolutionary immunology: just what is the cost of immunity?[END_REF]. The part of the energy allocated to immunocompetence belongs to the metabolic demand found in diet by organisms, which requires a sufficient amount of macro-and micro-nutrients [START_REF] Downs | A primer in ecoimmunology and immunology for wildlife research and management[END_REF]. Macronutrients (water, fibres, carbohydrates, lipids and proteins) maintain metabolic processes in providing vital elements (carbon, hydrogen, nitrogen…) and releasing energy. Micronutrients are idiosyncratic components of the immune system whose role varies from homeostatic regulators to constitutive elements of immunity [START_REF] Allen | Relationships among nutrition and reproduction and relevance for wild animals[END_REF][START_REF] Gombart | A Review of Micronutrients and the Immune System-Working in Harmony to Reduce the Risk of Infection[END_REF]. Among them, dietary selenium (Se) was found to enhance immune cells functioning, proliferation and activity [START_REF] Huang | The Role of Selenium in Inflammation and Immunity: From Molecular Mechanisms to Therapeutic Opportunities[END_REF]. Selenium is also a major antioxidant involved in reactive oxygen species (ROS) scavenging via selenoproteins and Se-dependent processes [START_REF] Schweizer | Selenoprotein P and Selenium Distribution in Mammals[END_REF]. In a previous study, we found that wood mice (Apodemus sylvaticus) free-ranging populations exposed to elevated levels of soil pollution by cadmium (Cd) and lead (Pb) had higher hepatic Se concentrations than control populations. We also found positive relationships between Se concentrations and haematological parameters [START_REF] Powolny | Effects of chronic exposure to toxic metals on haematological parameters in free-ranging small mammals[END_REF], suggesting that Se might play a protective role against Cd and/or Pb toxicity. In laboratory controlled conditions, Se has been shown to counteract immunosuppression as well as hepatic and renal oxidative damage induced by cadmium in orally exposed rats [START_REF] El-Boshy | Protective effects of selenium against cadmium induced hematological disturbances, immunosuppressive, oxidative stress and hepatorenal damage in rats[END_REF].

Toxic metals like Pb and Cd are known to be particularly immunotoxic to mammals [START_REF] Gera | Immunomodulation by Environmental Chemicals: Insights into Mammalian Immune Responses to Arsenic, Cadmium, and Lead[END_REF]. Complete blood cell counts have commonly been used to assess health condition of individuals exposed to various pollution types including toxic metals [START_REF] Luster | Development of a testing battery to assess chemical-induced immunotoxicity: National Toxicology Program's guidelines for immunotoxicity evaluation in mice[END_REF].

Leukocytes and derived white blood cells (i.e. monocytes, neutrophils, granulocytes) can help to interpret infectious status or to give a disease diagnosis, and have also been studied in a metal pollution context in the field [START_REF] Rogival | Metal blood levels and hematological characteristics in wood mice (Apodemus sylvaticus L.) along a metal pollution gradient[END_REF] or under controlled-condition exposure in the laboratory. Laboratory studies on mice and rats exposed to Cd or Pb showed that these metals dampened immune cell functioning, e.g. a decrease of lymphocyte proliferation or an increase of inflammatory markers [START_REF] Gera | Immunomodulation by Environmental Chemicals: Insights into Mammalian Immune Responses to Arsenic, Cadmium, and Lead[END_REF]. Both Cd and Pb can directly impair immune cell functioning by decreasing cell (e.g. T cells, NK cells) proliferation and activity [START_REF] Gera | Immunomodulation by Environmental Chemicals: Insights into Mammalian Immune Responses to Arsenic, Cadmium, and Lead[END_REF][START_REF] Wang | Effects of Cadmium Exposure on the Immune System and Immunoregulation[END_REF]. Cadmium and Pb exposure can enhance ROS production which then, can indirectly affect nuclear factor-κ B pathway in promoting an increase of (pro-)inflammatory cytokines (i.e. tumour necrosis factor alpha (TNF-α), interleukin 1 Beta (IL-1β), interleukin 6 (IL-6)) [START_REF] Hossein-Khannazer | The effects of cadmium exposure in the induction of inflammation[END_REF][START_REF] Liu | Protective role of quercetin against leadinduced inflammatory response in rat kidney through the ROS-mediated MAPKs and NF-κB pathway[END_REF]. Toxic metals can enhance cell reactivity to self (autoimmunity) and modulate immune response against pathogenic agents [START_REF] Kabata-Pendias | Interleukin-12 Promotes Enhanced Resistance to Listeria monocytogenes infection of Lead-Exposed Mice[END_REF].

Both Cd and Pb are also known to disrupt the production/secretion of corticosterone (CORT) from the adrenal gland, CORT being the main glucocorticoid (GC) in rodents [START_REF] Spackman | Corticosterone concentrations in the mouse[END_REF]. . As steroid hormones, GC production (in standard conditions) enables to decrease immune response in down-regulating immune system activity once infections are controlled. The production of GCs can reduce circulating lymphocytes, neutrophils, granulocytes and monocytes. [START_REF] Martin | Stress and immunity in wild vertebrates: Timing is everything[END_REF] proposed a temporal framework of immune response to a stressor, mediated by stress hormones. In this framework, a first neuropeptidedominated phase tends to accelerate the processes involved in the inflammatory response (within minutes to hours), and is followed by a second phase in which GC production down regulates the inflammatory response (within hours to months). The two phases constitute key regulators of the immune system. Thus, a close interaction exists between hormonal and immune response against stressors to prevent immune self-damages. The production of mice CORT by the adrenal gland can be enhanced by the release of pro-inflammatory cytokines (IL-6, TNF-α and IL-1) from immune cells [START_REF] Silverman | Glucocorticoid regulation of inflammation and its behavioral and metabolic correlates: from HPA axis to glucocorticoid receptor dysfunction[END_REF]. Indeed, immune challenge of laboratory mice showed a combined increase of both TNF-α and CORT [START_REF] Grion | Induction of nitric oxide synthase and heme oxygenase activities by endotoxin in the rat adrenal cortex: Involvement of both signaling systems in the modulation of ACTH-dependent steroid production[END_REF]. Metabolic CORT disruption could have severe consequences on the regulation of the immune system, as low levels would maintain an up-regulation of the immune response after an episodic infection. A significant decrease of serum CORT was found in male Wistar rats exposed to chronic injection of Cd chloride, which could thus act as a neuroendocrine disruptor [START_REF] Nishiyama | Effect of cadmium on plasma aldosterone and serum corticosterone concentrations in male rats[END_REF].

This study aims at determining how toxic metals can modulate the immune response of wood mouse populations inhabiting polluted (mainly by Cd and Pb) areas near the former smelter of Metaleurop Nord (northern France). To test immunocompetence, individuals from highly and lowly polluted sites were subjected to an immune challenge enhanced by bacterial lipopolysaccharides (LPS). This experiment is hereafter referred to as Experiment 1. To test the potential role of nutritional quality, a sub-sample of the animals captured in the contrasted sites were maintained in captivity for 5 days, and fed with a standard or Se-deficient rodent food. This experiment is hereafter referred to as Experiment 2. We hypothesized that the LPS challenge in wood mice from highly polluted sites would promote higher levels of TNF-α due to neuroendocrine disturbances (as assessed by serum and faecal levels of CORT) mediated by Cd and Pb exposure. We also hypothesized that Se-deficient diet would shape discrepancies in wood mice inflammatory response to the challenge among contrasted levels of pollution.

Methods

Study site

The study was conducted in the surroundings of the former smelter Metaleurop Nord northern France), which generated high levels of environmental pollution, mainly by Cd and Pb (Fritsch et al., 2010b(Fritsch et al., , 2010a)). Two groups of two (replicate) sites (of 25 ha), contrasted by their level of soil trace metal concentrations were studied, hereafter referred to as High and Low (Table S1). Median soil Cd and Pb concentrations in soils sampled in 2006 are of 1.4 and 116 µg/g dry mass for Low site, and 59.3 and 1357 for High site, respectively [START_REF] Douay | Contamination of woody habitat soils around a former lead smelter in the North of France[END_REF]Fritsch et al., 2010a). An additional soil sampling performed in 2019 confirmed that total Cd and Pb concentrations did not change significantly between 2006 and 2019 and provided data for other elements like Se (Table S1, [START_REF] Scheifler | Dynamique Spatiale et temporelle des transferts et des impacts des éléments traces métalliques sur le site de Metaleurop Nord[END_REF].

Animal sampling and selection

From the 28 th of September to the 13 th of October 2020, 70 traplines of ten nonlethal INRA traps with dormitory boxes each spaced three meters apart were set for three to five consecutive nights. Dormitory boxes were filled with apple, hay, sunflower seeds, cat food and peanut butter to ensure survival of the animals for 24 h. Traps were checked every morning.

The wood mouse (Apodemus sylvaticus) was retained as the animal model for this study considering its relevance for bioaccumulation of metals from soil and/or trophic transfer, its ubiquity and its ability to cope with captivity. Among the individuals trapped, individuals from other species than A. sylvaticus were immediately released, after checking their health status (no injury, not wet, apparent normal behaviour). Pregnant females and females exhibiting suckling, as well as young individuals (i.e. below 15g, [START_REF] Gelling | Impact of trapping and handling on leukocyte coping capacity in bank voles (Clethrionomys glareolus) and wood mice (Apodemus sylvaticus)[END_REF])) of A. sylvaticus species were also released. Adult males and non-pregnant females were kept for the experiment. A total of 91 wood mice were trapped on which 46 of the 60 males and 13 of the 31 females caught were used for the experiment (Table 1).

Experimental design

The experiment design, sample size and names given to the various groups are summarized in Table 1 and Figure S1.

In experiment 1, a group of 19 individuals (6 from High site, and 13 from Low site) was immediately treated on the day they were captured. Individuals were immune-challenged on the day they were captured and then blood was sampled before animals were sacrificed.

Corresponding haematological and biochemical data will serve as reference values for wildcaught non-captive challenged individuals from High (H-LPS) and Low (L-LPS) polluted sites.

In experiment 2, a group of 36 individuals (23 captured in sites High, 13 from sites Low) was maintained in captivity for five days post-capture. Animals were blood sampled on the day they were captured (day 0, D0) without any treatment. Corresponding haematological and biochemical data will serve as reference values for wild-caught non-captive unchallenged individuals High (H) and Low (L) polluted sites, respectively (Table 1). Animals were immunechallenged at the end of the captivity period (day 5, D5) and were then sampled again for blood before sacrifice.

Animals held captive were fed with pellet corn starch food for rodents (SAFE® SAS -basis formula AIN93G, see content in Table S2). A first group of animals coming from High (H-LPS-Se+) and Low (L-LPS-Se+) polluted sites were fed the standard food while a second group coming from High (H-LPS-Se-) and Low (L-LPS-Se-) polluted sites were fed a Se-deficient food (Table S2). Vitamin E was also deprived from Se-deficient diet as vitamin E can compensate for the antioxidant activity and immune cell protective effect of Se (Noaman et al., 2002).

We acknowledge that laboratory studies investigating the effects of Se on the immune response of rodents have been done under a longer period (weeks)(e.g. [START_REF] El-Boshy | Protective effects of selenium against cadmium induced hematological disturbances, immunosuppressive, oxidative stress and hepatorenal damage in rats[END_REF].

However, a previous pilot study from our team showed that circulating (blood) levels of Se in wood mice fed standard rodent food changed after only five days of captivity (Powolny et al., In preparation). Moreover, laboratory rats fed with 0.5 mg Se/kg diet showed an increase of the glutathione peroxidase activity in only one day (Knight and Sunde, 1988). Furthermore, rats fed zinc-deficient diet for three weeks displayed a rapid (three to seven days) recovery of their immune function after a dietary zinc supplementation (Blewett and Taylor, 2012). A fiveday lasting experiment may thus be expected as long enough to detect the early effects of Se on immune and inflammatory responses.

Housing

Captive animals (experiment 2) were housed individually (to avoid inter-individual aggressive interactions) in transparent boxes filled with a solid hemp litter, a shelter containing natural cotton and a paper roll as enrichment (Figure S2). Boxes were placed in shelves in a room with no heating system (i.e. day and night temperatures followed roughly the natural ones and was of 18-20°C during days and of 15-18°C at night for all the duration of the experiment).

Light also followed nychthemeral regime. Animals had access to water and to food ad libitum.

Individuals were weighed at day 0, and then at each cleaning of the box (i.e. at day 2, 4 and 5). Faecal samples and food remains were collected and weighed during each cleaning day.

Faecal samples were stored at -20°C until analysis.

Blood and tissue sampling, immune challenge

Blood was sampled from the retro-orbital sinus (volume of 1% of the body mass) using a glass Pasteur pipette and was immediately transferred into EDTA-coated tubes (Microvette®, Sarstedt). A small volume of blood (~50µL) was used fresh (i.e. stored at +5°C until analysis, which was performed within 8h following sampling) for complete blood count. The rest of the blood (~150 µL) was centrifuged at 2000g for 15 minutes to collect plasma. Plasma was stored at -20°C during the field session and, when back at the laboratory, at -80°C until analyses.

Animals were challenged by intraperitoneal injection with a solution of bacterial lipopolysaccharides (LPS O111:B4, Merck) at 500 µg/kg, which is sufficient to enhance TNFα production as found in several laboratory rodent models [START_REF] Batista | Lipopolysaccharide-Induced Neuroinflammation as a Bridge to Understand Neurodegeneration[END_REF]. Blood was collected two hours after LPS challenge.

After immune challenge and blood sampling, animals were sacrificed by cervical dislocation following ethical guidelines (Sikes and the Animal Care and Use Committee of the American Society of Mammalogists, 2016). The liver and the eyes were extracted to measure trace metal concentrations in the liver and to determine age with crystalline lens mass [START_REF] Vandorpe | An age reference model for the wood mouse, Apodemus sylvaticus (Linnaeus, 1758)[END_REF]. Livers were stored at -20°C until further preparation.

Trace metal analysis

Livers were lyophilised until constant mass, then acid digested with nitric acid (HNO3 69%, ultra-pure quality for trace analyses) and then diluted with ultra-pure water (Elga, 18.2 MV/cm).

The concentrations of Cd, Pb and Se in the liver were measured by Inductively Coupled Plasma Mass Spectrometry (ICP-MS, Thermo Fisher Scientific X Series 2). The quality of measurement was assessed by regularly measuring calibration solutions and blank (ultra-pure water). Reproducibility was checked by using internal standards of the ICP-MS. Repeatability and accuracy were verified by repeated (6 times) measurements of certified reference materials (National Research Council Canada TORT-3 Lobster hepatopancreas, National

Institute of Standards and Technology SRM® 1577c Bovine liver). Values under limits of quantification (LoQ) were replaced by the LoQ divided by the square root of two [START_REF] Helsel | Detection Limits[END_REF].

The concentration of metals was calculated in µg/g dry mass in the liver. Limits of quantification (LoQ), proportion of data < LoQ, certified values and average recoveries of the certified reference materials are provided in Table S3.

Complete blood count

Complete blood count was done with a Haematology Analyser automate (Horiba ABX Micros ES60), providing leukocyte counts (in 10 3 cells/mm 3 ): white blood cells (WBC), lymphocytes (LYM), monocytes (MON), granulocytes (GRA). Repeatability is automatically assessed by the analyser by measuring 3 times each value. Moreover, each blood sample was analysed in 2 to 3 runs in the cases of non-repeatable measurements (Table S5). Reproducibility and accuracy were determined withdaily blank controls and measurements of certified reference materials (MINOTROL 16, "low", "medium" and "high", Horiba SAS).

Determination of TNF-αlpha

TNF-α plasma concentration was determined with a high sensitivity commercial Mouse TNFα quantikine ELISA kit (R&D MH, detection range from 0.781 to 50 pg/ml). Positive controls were used to evaluate the ability of the kit to detect elevated values at 50 and 100 pg/ml (average recovery 86% in both cases). As low volume of blood did not allow performing linearity or other recovery assays, only two samples were used to control inter-assay (recovery of 104% and 105%) and two others sample to control intra-assay (recovery of 91% and 80%) quality of measurements. Four samples spiked with known amounts of TNF-α were used to assess if antigen-antibody inhibitors may be present in the sample, giving a recovery rate of 110% for a spike with 100 pg/ml (n=1), 92% with 25 pg/ml (n=1), and 89 and 73% with 3.13 pg/ml (n=2). Average recovery of 91% suggested the absence of such inhibitors.

Faecal and plasma CORT metabolite determination

The determination of faecal CORT metabolite (FCM) was performed according to the method of [START_REF] Touma | Effects of sex and time of day on metabolism and excretion of corticosterone in urine and feces of mice[END_REF] as detailed in Sánchez-González et al. ( 2018) on the wood mouse.

Briefly, faecal samples were oven dried until constant mass (40°C for 10 hours) and homogenized with a mortar and a pestle. An aliquot of 0.056 ± 0.006 g of faeces was then mixed with 500 μL of 80% methanol and 500 μL of phosphate buffer, vortexed for 15 s and left 16 hours in an orbital agitator. Faecal extract of FCM was then centrifuged for 15 min at 2500 g and the supernatants were collected in microtubes and stored at -20 °C until analyses. FCM determination was done with a commercially available enzyme immunoassay rat/mouse serum/plasma kit from DEMEDITEC Diagnostics (Kiel, Germany). A serial dilution (1:32, 1:16, 1:8, 1:4, 1:2, 1:1) of faecal extract was done to evaluate parallelism of the standard curves and the diluted extracts (73±27%). Recovery assays were performed between plates to evaluate accuracy (63±24%). The CORT concentration expressed in ng/ml was then corrected by the individual faeces mass as it has been shown to have a significant influence on the levels of FCM (Navarro-Castilla and Barja, 2014). Plasma concentration of CORT was determined using the same DEMEDITEC Diagnostics kit. Assay precision was measured with intra-assay and inter-assay recovery rates, which were respectively of 115±47 % (n=5) and 90±5 % (n=4).

For accuracy, the recovery rate from spiked samples with 185 ng/ml of CORT was 99±23 % (n=2) and the one from diluted (1:2) samples was 91±26 % (n=2).

Ethics

Blood sampling, immune challenge and euthanasia were performed under gaseous anaesthesia using isoflurane (mobile anaesthesia unit UNIVENTOR 400

Results and discussion

Experiment 1: Effect of Cd and Pb on the immunocompetence of free-ranging wood mice

Variations of liver trace metal concentrations between pollution levels

Cd hepatic concentrations did not significantly differ between Low and High sites (t = 1.2, df = 14.2, p = 0.24, Table 2), while Pb concentrations were higher in mice from High sites (t = 6.1, df = 13.8, p < 0.001, Table 2). In previous studies conducted over the same area, a chronic exposure of free-living wood mice to both metals was observed but with significantly higher internal organ concentrations in animals from the most polluted sites [START_REF] Tête | Histopathology related to cadmium and lead bioaccumulation in chronically exposed wood mice, Apodemus sylvaticus, around a former smelter[END_REF]. The contrasted findings between the two metals and between results of the different studies could be explained by variations in metal bioavailability. Indeed, comparing total concentrations and partitioning of metals in soil samples collected over the sites in 2006 and in 2019, Ozaki et al.

(2022) showed changes in both exchangeable fractions and the less mobile fractions leading to an increase of Cd mobility in the less polluted site but a decreased mobility of Pb in the most contaminated sites.

Hepatic Se concentrations were higher in individuals from High than Low sites (t = 4.4, df = 8.5, p = 0.002, Table 2). A similar result was observed in a previous study over the same area [START_REF] Powolny | Effects of chronic exposure to toxic metals on haematological parameters in free-ranging small mammals[END_REF]. [START_REF] Peraza | Effects of Micronutrients on Metal Toxicity[END_REF] hypothesised that Se, which is involved in a protective role against the toxicity of metals, would be preferentially taken up and/or accumulated in animals exposed to Cd and Pb. However, Se concentrations in soils differed between Low and High sites (median = 1.8 and 4.1 µg/g, respectively; [START_REF] Scheifler | Dynamique Spatiale et temporelle des transferts et des impacts des éléments traces métalliques sur le site de Metaleurop Nord[END_REF].

Therefore, our results did not allow discriminating between a physiologically "active" uptake or a "passive" Se accumulation because of higher soil Se concentrations in soils.

Influence of Cd and Pb exposure on the inflammatory response of free-ranging wood mice

All challenged wood mice exhibited an increase of TNF-α since its level was undetectable in animals before immune challenge (Table 3). This suggests that individuals displayed a functional response to the immune challenge, which at least produced the secretion of TNF-α.

Concentrations of plasma TNF-α were found higher in H-LPS than in L-LPS individuals (W = 53, p = 0.047, Figure 1), suggesting a higher inflammatory response in animals from polluted sites (H-LPS). Challenged individuals showed decreases of WBC count in comparison with unchallenged ones (Table 3, Figure S4). In individuals from High sites (H-LPS), leukocytes showed a significant decrease of -75% for WBC (t = -5.7, df = 8.8, p < 0.001), -77% for LYM (t = -6.2, df = 10, p < 0.001), -76% for MON (t = -3.8, df = 6.7, p = 0.007), and -51% for GRA (t = -2.7, df = 8, p = 0.026). In individuals from Low sites the variation of WBCs (-45 %; t = 2.8, df = 23, p = 0.009) may be related to LYM decrease (-52%; t = 2.9, df = 22, p = 0.007) as no variation was found for MON (t = 1.7, df = 18, p = 0.09) and GRA (t = 2.1, df = 19, p = 0.05). Similarly, [START_REF] Brooks | Non-Lethal Endotoxin Injection: A Rat Model of Hypercoagulability[END_REF] found that LPS (15 mg/Kg) induced a decrease of WBCs including LYM, GRA and MON within 4 hours post-injection in Wistar rats. The differences in leukocytes decrease between High and Low sites can be attributed to the elevated levels of TNF-α found in challenged animals from High sites. Negative relationships between WBC, LYM, MON and GRA and TNF- concentrations were found (Figure S5).

Higher levels of TNF-α and a decrease of lymphocyte were evidenced in cell culture containing 100 ng/mL of LPS and rat peripheral blood polymorphonuclear leukocytes pre-treated with Cd (i.p. injection) at 1 mg/kg [START_REF] Djokic | Differential effects of cadmium administration on peripheral blood granulocytes in rats[END_REF]. Lead was also found to enhance a marked increase of TNF-α in rats after co-exposure with different concentrations of Pb and intravenous injection of LPS at 100 μg/kg [START_REF] Liu | Coexposure of lead-and lipopolysaccharideinduced liver injury in rats: involvement of nitric oxide-initiated oxidative stress and TNF-α[END_REF]. In our study, the difference of TNF-α concentrations and of circulating WBC levels between individuals from High and Low polluted sites could suggest two levels of strength of the immune response to LPS challenge, with a possible peracute immune response in animals highly exposed to pollution.

Influence of Cd and Pb exposure on the anti-inflammatory response of free-ranging wood mice

Plasma CORT concentrations differed according to exposure to pollution and varied due to immune challenge in highly exposed individuals only. Lower levels of plasma CORT were found in unchallenged individuals from High sites in comparison with challenged ones from the same site and with unchallenged ones from the Low sites (F = 5.2, df = 51, p = 0.003, Figure 1). A significant increase of plasma CORT of 23% was found between challenged and unchallenged individuals from the High sites (t = 5, df = 15, p < 0.001; Figure 1 and Table 3) while no difference was found for animals from the Low sites (t = 1, df = 22, p = 0.3).

Furthermore, WBC and LYM were negatively related to CORT but not MON and GRA (Figure S5). The absence of significant differences in GRA, MON and CORT in individuals from Low polluted sites could indicate a differential recruitment of WBCs. One explanation can be that, in highly exposed immune-challenged animals, CORT increased to avoid a hyper-inflammatory immune response. Indeed, one effect of CORT on the immune system of rodents is to control inflammation in modulating responses once infections are controlled to avoid self-damages mediated by the activated immune cells, also called autoimmunity [START_REF] Medzhitov | Origin and physiological roles of inflammation[END_REF][START_REF] Martin | Stress and immunity in wild vertebrates: Timing is everything[END_REF]. Corticosterone can have down-regulating effects on immune parameters in reducing numbers of circulating LYM (T cells > B cells and CD4+ > CD8+) and also antigen presenting cells (i.e. macrophages, neutrophils and monocytes) promoting an immune-redistribution [START_REF] Martin | Stress and immunity in wild vertebrates: Timing is everything[END_REF]. In our study, the stress induced by the LPS challenge promoted a decrease of circulating LYM, and sometimes of other leukocytes, which may be mediated by elevated CORT enhancing a redistribution of WBCs from the blood to other organs where the infection occurs (e.g. peritoneal cavity, lymph node) and clear organisms from pathogens [START_REF] Dhabhar | Stress-induced augmentation of immune function-The role of stress hormones, leukocyte trafficking, and cytokines[END_REF]. Lower levels of CORT in the plasma of free-ranging wood mice from the most polluted sites can be attributed to endocrine disruption mediated by chronic exposure to Cd and Pb.

Indeed, Cd and Pb were found to inhibit circulating CORT by altering adrenal cortex functions [START_REF] Nishiyama | Effect of cadmium on plasma aldosterone and serum corticosterone concentrations in male rats[END_REF][START_REF] Nishiyama | Effects of heavy metals on corticosteroid production in cultured rat adrenocortical cells[END_REF]. The adrenal cortex is mainly under the control of the pituitary adrenocorticotrophic hormone, which, by a negative feedback mechanism, controls plasmatic CORT levels [START_REF] Wills | Laboratory investigation of endocrine disorders[END_REF]. Lower circulating CORT could reduce anti-inflammatory levels in wood mice from polluted site. A dampening of anti-inflammatory levels would enable organisms exposed to pollution to maintain at a higher level their basal immune functions leading to an up-regulation of the immune system, as shown by the higher inflammatory response found in animals from the most polluted sites. One might hypothesize that a higher basal immune function in wood mice inhabiting polluted sites allows a shortening of the time of effectiveness to enhance inflammatory immune response and thus quickly enhances CORT release in the blood stream.

Experiment 2: Effect of a Se-deficient diet on the immunocompetence of captive wood mice

Age structure

The age structure did not differ between individuals from the experiment 1 and 2 (df = 47, F = 0.56, p = 0.57) (Table S4). In both experiments 1 and 2, the concentrations of Cd, Pb and Se in the liver were not influenced by the age of individuals (p > 0.05).

Food consumption

Neither consumption (df = 38, F = 0.32, p = 0.61) nor body weight (df = 47, F = 2.8, p = 0.07) differed between individuals fed standard or Se-deficient food (Table S4), suggesting satisfactory housing and experimental conditions.

Variation of trace metals liver concentrations

Neither Pb nor Cd concentrations in the liver of captive wood mice showed significant differences in comparison with those sampled the day they were captured (High: Pb: df = 26, F = 0.68, p = 0.51, Cd: df = 26, F = 1.6, p = 0.21; Low: Pb: df = 23, F = 2.08, p = 0.15; Cd: df = 23, F = 0.09, p = 0.91; Table 2). This absence of decrease in the hepatic Cd concentrations during captivity is in agreement with its very long half-life in rat and mouse (200-700 days) and in human (several years or decades) [START_REF] Nordberg | Cadmium[END_REF]. Five days of captivity are not supposed to be long enough to detect a decrease in liver concentrations as its half-life in soft tissues is estimated at 40 days in human (Kabata-Pendias and Szteke, 2015). Individuals from High sites fed Se-deficient diet had lower Se concentration than those fed standard diet (t = 3, df = 16, p = 0.01, Table 2), while such a difference did not occur in individuals from Low sites (t = 0.9, df = 8, p = 0.4). Five days of captivity with Se-deficient food seemed sufficient to lead to a decrease of hepatic Se concentrations in wood mice exposed to metallic pollution.

According to the literature, elimination of certain forms of Se in animals through both urine and faeces is at least biphasic, with one fast elimination phase of approximately one day (dose dependent) followed by a slower phase of 30-70 days in most of species [START_REF] Alexander | Selenium[END_REF].

Influence of dietary Se on the inflammatory response of captive wood mice

After five days of captivity, all immune challenged wood mice displayed an increase of TNF-α since no detectable concentration were found in unchallenged individuals (Figure 2). As suggested earlier, the increase in the production of TNF-α might be interpreted as a functional response to LPS challenge promoting an inflammatory response [START_REF] Brooks | Non-Lethal Endotoxin Injection: A Rat Model of Hypercoagulability[END_REF]. Dietary Se induced differences in the pollution-mediated variations of TNF-α in challenged individuals.

While no significant difference was found in TNF-α concentrations between individuals from High and Low sites fed a Se-deficient diet (p > 0.05), TNF-α concentrations were lower in individuals from High than Low sites fed a standard diet (W = 14, p = 0.03, Figure 2). The concentrations of TNF-α did not differ between individuals from Low sites fed a standard versus a Se-deficient diet (p > 0.05). Individuals highly exposed to metals but fed with sufficient amount of Se (i.e. standard diet) may exhibit a lower inflammatory in response to LPS challenge than their counterparts lacking Se or not being stressed by toxic metals. However, the concentration of TNF-α did not display differences between individuals from High sites fed a Se-deficient diet in comparison with those fed a standard diet. Mammals' homeostasis as well as reproducing performances have been shown to be connected to individual micronutrient resources [START_REF] Allen | Relationships among nutrition and reproduction and relevance for wild animals[END_REF]). Among them, Se is a key antioxidant which deficiency could impact cardiac muscles (e.g. mulberry heart disease) and immune cells (i.e. leukocytes) functioning [START_REF] Gombart | A Review of Micronutrients and the Immune System-Working in Harmony to Reduce the Risk of Infection[END_REF]. Selenium occurs in several mechanisms, including selenoproteins (e.g. GPX) and adducts (e.g. Cd-Se), which are involved in the detoxification of Cd and Pb [START_REF] Fu | Effect of selenium antagonist lead-induced damage on Th1/Th2 imbalance in the peripheral blood lymphocytes of chickens[END_REF][START_REF] Vuksic | The effect of dietary selenium addition on the concentrations of heavy metals in the tissues of fallow deer (Dama dama L.) in Croatia[END_REF]. The critical role of Se in ROS scavenging activity was found to prevent immune cell damages [START_REF] Avery | Selenium, Selenoproteins, and Immunity[END_REF].

Though pollution levels might have an effect on inflammatory response to immune challenge in animals fed a standard diet no difference was found when the TNF-α concentrations was compared between the 4 groups (p > 0.05, Table 3). Indeed, five days of captivity might be a short time to determine the effects of dietary Se-deficient diet on the inflammatory response to LPS in the wood mice.

At day 5, challenged wood mice from High sites fed a Se-deficient diet showed a significant decrease of LYM (-56%), while no significant changes was found in WBC, MON and GRA counts (Table 3, Figure S4). In contrast, challenged animals from High site but fed a standard diet displayed a significant decrease in WBC (-85%), LYM (-86%), MON (-87%) and no variation in GRA. Though no variation in TNF-α was found between H-LPS-Se+ and H-LPS-Se-the lower variation in leukocytes found in individuals fed a Se-deficient diet (i.e. only LYM decrease) might be interpreted as a lower inflammatory response in H-LPS-Se-. Similar contrasts between standard and Se-deficient diets were found for the Low sites since L-LPS-Se+ individuals showed a decrease in WBC (-53%), LYM (-56%), MON (-46%) and GRA (-71%) while only a reduction of WBC probably attributed to LYM decrease (-75%) was found in L-LPS-Se-(Table 3, Figure S4). These results might relate a potential influence of dietary Se on the inflammatory response as lower leukocyte variation was found in individuals fed a Sedeficient diet whilst no variation of the TNF-α was attributed to the diet (Table 3, Figure S4).

Nonetheless, WBC and LYM were negatively related to TNF- concentrations in individuals from High sites fed either a standard or deficient food (Figures 4 andS6, r²adj. = 0.39, F = 4.8, df = 15, p = 0.015). A significant interaction was found, , showing a steeper negative relationships between LYM and TNF- in individuals fed a standard diet than in those fed a Se-deficient diet (slope= -0.3 vs -0.02). In addition, such a pattern was not detected in animals from the Low sites. Altogether, these results might indicate that dietary Se has a positive influence on the immune response on wood mice exposed to high levels of Cd and Pb. [START_REF] El-Boshy | Protective effects of selenium against cadmium induced hematological disturbances, immunosuppressive, oxidative stress and hepatorenal damage in rats[END_REF] found in laboratory rats that dietary Se could reduce the inflammatory response (i.e; lower decrease of LYM and lower increase of TNF-α) induced by cadmium exposure. However, the functional role of dietary Se might be mitigated by inflammatory and anti-inflammatory processes which may last over months [START_REF] Martin | Stress and immunity in wild vertebrates: Timing is everything[END_REF]. These interpretations would therefore require further investigation.

Table 3 Variation of total leukocytes counts and corticosterone levels between unchallenged and challenged wood mice (for exper iment 1: H vs H-LPS, L vs L-LPS and for experiment 2: D0 vs D5), and median (and [min-max]) concentrations of plasma TNF-. NS stands for non-significant difference, *, **, *** for p < 0.05, 0.01, 0.001, respectively. Different letters stand for significan t differences between H-LPS and L-LPS. 1 Δ stands for variations of measured parameters (value of e.g. H-LPS -value of H) 2 < LD: under limits of detection of 0.295 pg/ml.

Influence of Se-deficient diet on the anti-inflammatory response of captive wood mice

Plasma CORT concentrations did not differ significantly between day 0 and day 5 in individuals from High and Low sites fed a Se-deficient diet (Table 3, Figure 2). Conversely, plasma CORT increased significantly by 26% and 22% between day 0 and day 5 in individuals from High and Low sites, respectively, fed a standard food (Table 3, Figure 2). FCM concentrations did not differ between individuals fed a standard or a Se-deficient diet (F= 1.31, p = 0.26, Figure 3) but varied according to pollution levels. FCM levels increased over the 5 days of captivity in individuals from High sites towards levels equivalent to the ones in animals from Low polluted sites (Figure 3). Lower FCM levels were found in captive individuals from High in comparison with Low sites after two days of captivity (F= 17.1, p < 0.001) while the concentrations did not differ at days 4 and 5. An increase of FCM in individuals from High sites from day 2 to 4 and from day 2 to 5, but not between days 4 to 5, was observed. No significant variations were found between days 2, 4 and 5 in individuals from Low polluted sites. The mean FCM levels in individuals from High sites reaches the ones from Low at day 4. Interestingly, these results about FCM echoed and reinforced the results described above about lower plasma CORT concentrations in individuals from High than from Low sites. These findings might be interpreted as a functional response of CORT to dietary Se where individuals fed with sufficient amount of Se (i.e. standard diet) produced higher antiinflammatory CORT levels. As discussed previously, the increase of CORT could promote a redistribution of WBCs from the blood to the sites of infection [START_REF] Dhabhar | Stress-induced augmentation of immune function-The role of stress hormones, leukocyte trafficking, and cytokines[END_REF].

Significant variations of CORT and leukocyte counts occurred in individuals fed a standard diet but not in the ones fed a Se-deficient diet (Table 4). Negative relationships were found between LYM counts and CORT concentrations in individuals from High sites (r²adj. = 0.53, F = 7.1, df = 13, p = 0.005) with a lower slope in individuals fed a Se-deficient diet than in individuals fed a standard diet (slope= -1.1 vs -0.3, Figure 4). [START_REF] Nishiyama | Effect of cadmium on plasma aldosterone and serum corticosterone concentrations in male rats[END_REF][START_REF] Nishiyama | Effects of heavy metals on corticosteroid production in cultured rat adrenocortical cells[END_REF]. The variations of CORT levels may provide an explanation of the discrepancies found in TNF- concentrations, which may be lower in individuals from High polluted sites fed a standard diet, as higher basal CORT levels could promote a higher anti-inflammatory activity. In contrast, individuals fed a Se-deficient diet showed a lower immune response to the immune challenge as LYM could probably be less dependent to plasmatic TNF- and CORT. Lower quality of diet regarding Se would thus disturb both pro-and anti-inflammatory activity of TNF- and CORT in individuals exposed to toxic metals.

Conclusion

Our study provides new insight at the interface of immunology and ecotoxicology to highlight physiological disturbances originating from anthropogenic activities in a terrestrial small mammal. Individuals caught in polluted sites showed higher exposure to and accumulation of Pb and Se, a higher exposure to but not accumulation of Cd, and a significant decrease in hepatic concentrations during captivity for Se only. Free-ranging animal chronically exposed to Cd and Pb displayed a higher inflammatory response to immune challenge with a decrease in leukocytes (mainly mediated by lymphocytes) and a higher production of TNF-α and CORT.

The increase in the immunocompetence of individuals from the polluted site was attributed to neuroendocrine disturbance which might reduce the production of anti-inflammatory CORT.

Wild-caught animals maintained in captivity fed a Se-deficient diet showed a lower immune response to LPS challenge than ones fed a standard diet, which might be explained by a

Figure 1

 1 Figure 1 Experiment 1. Concentration of plasma TNF - (pg/ml) and of CORT (ng/ml) in response to LPS challenge in wood mice captured in contrasted levels (high and low) of soil pollution. Levels of significance of t and U (only TNF-) tests, *, ** for p < 0.05, 0.01, respectively; <LD: below limits of detection.

Figure 2

 2 Figure 2 Experiment 2. Concentration of plasma TNF - (pg/ml) and of CORT (ng/ml) in response to LPS challenge in wood mice captured in contrasted levels (high and low) of soil pollution and fed for 5 days a standard or a Se -deficient diet. Levels of significance of t and U (only TNF-) tests: ns: non-significant difference, *, *** for p < 0.05, 0.01, respectively; <LD: below limits of detection.

Figure 3

 3 Figure 3 Experiment 2. Trends in faecal corticosterone metabolite (FCM, ng/g d r y f a e c e s ) of wood mice captured in high level of soil pollution during five days of captivity. FCM was measured at day 2, 4 and 5 of the experiment in two groups of individuals fed a standard or a Se-deficient diet. Levels of significance of a pairwise paired T -tests between pollution levels: p-value > 0.05 (ns), <0.05 (*), <0.01(**), <0.001(***).

Figure 4

 4 Figure 4 Experiment 2. Relationships between lymphocyte counts and plasma TNF- and CORT after LPS challenge in captive wood mice from "High" levels of soil pollution fed a standard (H-LPS-Se+) or a Se-deficient (H-LPS-Se-) diet.

  lowered immune function in captive individuals fed a low quality diet. Altogether, our results highlight an immunomodulation of the inflammatory response indirectly mediated by Cd and/or Pb exposure and diet quality, related to a disturbance in circulating CORT levels. The role of dietary Se on the inflammatory and anti-inflammatory responses to challenge remains unclear. Further experiments on longer period of captivity considering the nutritional status of individuals might allow determining how dietary Se could display a functional role on the immunocompetence of wild animals chronically exposed to Cd and Pb.

Figure S9

 S9 Figure S9 Parameters of the linear regression models on the effect of LPS challenge in individuals from experiment 2 on LYM in function of plasma TNF-α (A) and CORT (B) concentrations in interaction with diet (models are presented in Figure 4). ................................................................ 47

Figure S1

 S1 Figure S1 Experimental design.

Figure S3

 S3 Figure S3 Cadmium (Cd), lead (Pb) and selenium (Se) concentrations (µg/g, log scaled) in the liver of captive and non-captive wood mice among contrasted levels (high and low) of pollution. Levels of significance: p > 0.05: ns, p < 0.05: *, p < 0.01: **, p < 0.001: ***.

Figure S5 Experiment 1 .

 1 Figure S5 Experiment 1. Variations of white blood cells (WBC), lymphocytes (LYM), monocytes (MON), granulocytes (GRA) counts in response to plasma TNF- and CORT after LPS challenge in individuals from "High" (red dots) and "Low" (blue dots) levels of soil pollution.

Figure S6

 S6 Figure S6Experiment 2. Variations of white blood cells (WBC), lymphocytes (LYM), monocytes (MON), granulocytes (GRA) counts in response to plasma TNF- and CORT after LPS challenge in captive wood mice from "High" levels of soil pollution either fed with standard (H-LPS-Se+) and Sedeficient (H-LPS-Se-) diets.

Figure S7

 S7 Figure S7 Correlation matrix of Spearman of the physiological variables measured in wood mice captured on contrasted levels of pollution (high and low) measured 2 hours after LPS challenge (i.p. injection).

Figure S8

 S8 Figure S8 Correlation matrix of Spearman of the physiological variables measured 2 hours after LPS challenge (i.p. injection) in wood mice captured on contrasted levels of pollution (high and low) at 5 days of captivity with standard (A) and selenium deficient laboratory rodent diet. A/Standard diet

Table 1

 1 Sampling design, acronyms and number (in parenthesis) of wood mice caught on high and low levels of soil pollution. In bold are the acronyms that are used throughout text, tables and figures to refer to the different experimental groups .

	Soil pollution levels	Study design	Type of food	Day 0 (D0)	Day 5 (D5)	Name of the tested groups	Experiment
		No captivity (6)	No food (6)	Challenged high (6)		H-LPS (6)	1
	High (29)	Captivity	Standard food (12)	Control high (H)	Challenged (12) high standard	H-LPS-Se+ (12)	2
		(23)	Se-	(23)	Challenged
			deficient food		high Se-deficient	H-LPS-Se (11)	2
			(11)		(11)
		No captivity (13)	No food (13)	Challenged low (13)		L-LPS (13)	1
	Low (23)	Captivity	Standard food (7)	Control low (L)	Challenged (7) low standard	L-LPS-Se+ (7)	2
		(13)	Se-	(13)	Challenged
			deficient food		low Se-deficient	L-LPS-Se-(6)	2
			(6)		(6)

Table 2

 2

	Experiment	Group	Cd	Pb	Se
		H-LPS	4.9 a [4.5, 0.9 -11]	0.9 a [0.9, 0.6 -1.6]	7.1 a [6.4, 5.1 -10]
	1
		L-LPS	4.8 a [1.3, 0.1 -26]	0.2 b [0.04, 0.005 -1.5]	2.8 b [2.5, 0.8 -6]
		H-LPS-Se+ 8.0 a [4.0, 0.7 -23]	1.1 a [0.6, 0.2 -3.7]	6.8 a [6.7, 5.2 -9.4]
		L-LPS-Se+	4.7 a [4.1, 0.2 -16]	0.02 b [0.007, 0.006 -0.08]	4.1 b [4.3, 3.4 -5]
	2
		H-LPS-Se-	5.1 a [0.9, 0.5 -25]	1.2 a [0.5, 0.05 -5.4]	5.4 c [5.5, 4.1 -7.1]
		L-LPS-Se-	3.1 a [2.9, 0.3 -5.8]	0.03 b [0.01, 0.006 -0.1]	3.7 b [3.5, 2.6 -5]

Mean (and [median, min -max]

) Cd, Pb and Se hepatic concentrations in the experimental groups. Different letters stand for significant differences between H -LPS and L-LPS.

Table 4

 4 Summary of the main trends in the experiments, in terms of stressors encountered by animals and responses as measured by variation of WBCs, CORT, and TNF- and by Cd, Pb, and Se concentrations. Only statistically significant variations are represented. Decreases and increases are represented by ↘ and ↗, respectively.

	Variations of 0 to 33%, 34 to 66%, and 67 to 100% are represented by 1, 2 and 3 arrows,
	respectively. Cd, Pb, and Se concentrations are qualified of high (H) or low (L), and an
	arrow is used to represent a decrease/increase during captivity.
	Group	Stressors	WBCs LYM	MON	GRA	CORT TNF- Cd Pb Se
		High exposure
	H-LPS		↘↘↘	↘↘↘ ↘↘↘ ↘↘↘ ↗	↗↗↗	H	H	H
		Natural conditions
		Low exposure
	L-LPS		↘↘	↘↘				↗↗	H	L	L
		Natural conditions
		Past high exposure
	H-LPS-Se+	Captivity (no exposure)	↘↘	↘↘	↘↘		↗↗	↗	H	H	H
		Standard food
		Past low exposure
	L-LPS-Se+	Captivity (no exposure)	↘↘↘	↘↘↘ ↘↘↘ ↘↘↘ ↗↗	↗↗	H	L	L
		Standard food
		Past high exposure
	H-LPS-Se-	Captivity (no exposure)		↘↘				↗	H	H	H↘
		Se-deficient food
		Past low exposure
	L-LPS-Se-	Captivity (no exposure)	↘↘↘	↘↘↘				↗	H	L	L
		Se-deficient food
	Dietary Se might counteract Cd and/or Pb impacts on the immune system allowing individuals
	fed a standard diet to perform an efficient immune response. As discussed above, lower CORT
	levels in individuals from High sites might be attributed to endocrine disturbance induced by
	Cd or Pb exposure

Table S1

 S1 Characteristics of the study sites (made of two replicate sites for each level of pollution, distance from the former smelter, median, minimum and maximum soil concentrations of Cd, Pb and Se with year of sampling and analysis in brackets) near the former Metaleurop Nord smelter.

			High	Low
				3.0-11.0
	Distance from Metaleurop Nord (median, km)		0.6 (two adjacent sites)	(one at 3, the other at 11 km from the
				former smelter)
		Cd (2006)	59.3 (15.3 -236.5)	1.4 (0.7 -5.3)
	Soil metal concentrations (µg/g dry mass)	Pb (2006)	1357 (755 -6809)	116 (43 -200)
		Se (2019)	4.1 (3.1 -10.0)	1.8 (1.5 -2.3)

Table S3

 S3 Limits of quantification (LoQ) of ICP-MS analysis of metals, proportion of data < LoQ, certified values and average recoveries of the certified reference materials used in this study (the number of reference samples analyzed for each certified reference material is 6).

	Parameter	Cd	Pb	Se
	LoQ (µg/L)	0.018	0.006	0.056
	Proportion of data	High: 0%	High: 0%	High: 0%
	under LoQ (%)	Low: 0%	Low: 38%	Low: 0%
	TORT-3 certified
	values (and
	uncertainty: 95	42.3(1.8)	0.225(0.018)	10.9(1.0)
	confidence limits) in
	µg/g
	Recovery (%)	93±2	71±1	99±3
	SRM 1577c
	certified values (and
	uncertainty: 95	0.097(0.001)	0.063(0.001)	2.031(0.045)
	confidence limits) in
	µg/g
	Recovery (%)	70±5	61±7	105±3

Table S5

 S5 Raw data of blood counts given for each individual captured during the field session.

	F01 D03 O01	2 2 2	5 5 0	High High Low	1.1 67.4 10.2 7.1	0.9 42.2 7.3 5.0	0.03 9.80 2.20 0.90	0.17 15.35 0.70 1.20
	ID: individual code; EXP: experiment 1 or 2; DAY: day 0 or 5 of experiment 2, or CS: free-1.1 0.9 0.10 0.10 66.9 41.8 9.80 15.30 10.3 7.2 2.30 0.80 O01 2 5 Low 0.4 0.2 0.00 0.18
	ranging animals (not maintained in captivity); POLL: pollution level; WBC: White blood cells; 1.1 0.9 0.00 0.20 67.8 42.6 9.80 15.40 J05 2 0 High 4.0 3.1 0.67 0.30 0.4 0.3 0.00 0.10
	G01 D04 J05 LYM: Lymphocytes, MON: Monocytes; Granulocytes. 2 0 High 3.9 2.3 2 0 High 19.7 16.1 2 5 High 1.3 0.9 0.4 0.2 3.8 2.3 20.0 16.5 1.1 0.8 R01 1 CS Low 9.5 5.2	0.75 1.90 0.10 0.00 0.80 1.80 0.10 2.60	0.85 1.75 0.30 0.20 0.70 1.70 0.20 1.63
		EX	DA	POL	WBC 4.0 19.4 1.1 9.0	LYM 2.3 15.6 0.7 5.4	MON 0.70 2.00 0.10 2.40	GRA 1.00 1.80 0.30 1.20
	ID G01 D04	P 2 2	Y 5 5	L High High	(10 3 /mm 3 ) 1.7 1.2 1.6 9.3	(10 3 /mm 3 ) 0.9 1.1 4.9	(10 3 /mm 3 ) 0.10 0.10 2.70	(10 3 /mm 3 ) 0.20 0.40 1.70
	D01 G02 E02 K01	2 2 2 1	0 0 0 CS	Low High High High	9.4 19.3 4.7 1.0 10.1	8.3 17.1 3.8 0.8 5.4	0.87 1.60 0.50 0.00 2.70	0.30 0.53 0.40 0.18 2.00
	E02 S01	2 2	5 0	High Low	9.3 18.7 4.5 1.0 8.7	8.3 16.6 2.2 0.9 5.1	0.80 1.60 1.45 0.00 1.80	0.20 0.50 0.80 0.10 1.80
	E03 S01	2 2	0 5	High Low	9.5 20.5 5.0 1.0 1.4	8.4 18.3 4.6 0.8 1.1	0.80 1.60 0.30 0.00 0.20	0.30 0.60 0.15 0.20 0.13
	G02 K02	2 1	5 CS	High High	9.5 3.2 4.9 1.5 1.4	8.1 2.3 4.5 1.1 1.1	1.00 0.53 0.30 0.20 0.20	0.40 0.33 0.10 0.20 0.10
	D01 K04	2 1	5 CS	Low High	0.2 3.2 5.1 1.9 1.5	0.1 2.4 4.6 1.5 1.1	0.00 0.50 0.30 0.10 0.20	0.10 0.30 0.20 0.25 0.20
	D02 E04 S02	2 2 1	0 0 CS	Low High Low	2.5 3.3 8.7 1.7 1.3	1.8 2.3 7.3 1.4 0.9	0.40 0.60 1.00 0.10 0.20	0.30 0.40 0.40 0.20 0.20
	D02 H01 E04 S03	2 2 2 1	5 0 5 CS	Low High High Low	1.0 3.9 0.9 2.0 2.1	0.5 2.7 0.6 1.6 1.5	0.20 0.60 0.10 0.10 0.15	0.30 0.67 0.20 0.30 0.45
	F01 F01 L01	2 2 1	0 0 CS	Low High High	6.3 3.9 9.1 1.8 2.1	4.3 2.7 7.6 1.6 1.5	1.10 0.60 1.05 0.10 0.20	0.90 0.60 0.50 0.10 0.40
	F01 F01 A01	2 2 2	5 5 0	Low High Low	1.1 4.0 9.6 6.5 2.1	0.8 2.7 6.6 5.2 1.5	0.10 0.60 2.17 0.80 0.10	0.17 0.70 0.87 0.55 0.50
	I01 S04	2 1	0 CS	High Low	1.1 9.3 9.4 6.4 7.9	0.9 7.2 6.5 5.1 6.0	0.10 1.50 2.10 0.80 1.37	0.10 0.60 0.80 0.50 0.57
					1.1 9.3 10.0 6.6 7.6	0.8 7.2 6.7 5.2 5.8	0.10 1.50 2.30 0.80 1.30	0.20 0.60 1.00 0.60 0.50
	H01 I01 F02 A01	2 2 2 2	0 5 0 5	Low High High Low	3.2 1.5 2.8 1.8 8.1	2.6 1.1 2.5 1.4 6.1	0.40 0.20 0.20 0.10 1.40	0.20 0.20 0.10 0.30 0.60
	H01 J01 F02 I01 T01	2 2 2 2 1	5 0 5 0 CS	Low High High Low Low	0.2 6.3 0.9 2.9 1.3	0.1 5.1 0.6 2.2 0.8	0.00 0.80 0.10 0.40 0.10	0.10 0.40 0.20 0.25 0.40
	J01 G02 T02	2 1 1	5 CS CS	High High Low	0.2 1.3 4.2 2.9 5.8	0.1 1.0 2.8 2.3 2.9	0.00 0.20 0.93 0.40 2.17	0.10 0.10 0.53 0.20 0.80
	A01	2	0	High	0.2 10.5 4.2 2.8 6.0	0.1 8.0 2.8 2.1 3.0	0.00 1.70 0.95 0.40 2.30	0.10 0.80 0.50 0.30 0.70
	H02 A01 I01	2 2 2	0 5 5	Low High Low	3.1 10.7 4.3 2.2 5.7	2.5 8.1 2.8 1.5 2.8	0.40 1.60 0.90 0.40 2.10	0.20 1.00 0.60 0.35 0.80
	H02 C02 H01	2 2 2	5 0 0	Low High High	1.7 1.6 10.1 2.1 5.8	0.8 1.2 8.9 1.4 2.8	0.50 0.20 0.90 0.40 2.10	0.40 0.20 0.35 0.30 0.90
	J01 C02	2 2	0 5	Low High	11.6 9.8 9.8 2.2	8.4 7.8 8.7 1.5	2.30 1.10 0.80 0.40	0.90 0.90 0.30 0.40
	C03 I02	2 1	0 CS	High Low	11.5 11.3 10.4 4.8	8.4 8.5 9.0 2.8	2.30 2.07 1.00 1.00	0.80 0.73 0.40 0.95
	H01	2	5	High	11.6 11.0 2.2 4.6	8.3 8.4 1.8 2.7	2.30 2.00 0.17 1.00	1.00 0.60 0.20 0.90
	J01 H02	2 1	5 CS	Low High	2.1 11.4 1.4 4.9	1.5 8.6 1.0 2.9	0.30 2.10 0.15 1.00	0.35 0.70 0.20 1.00
	I01 I02	2 2	0 0	High Low	2.1 11.5 5.6 2.8	1.5 8.5 4.4 2.1	0.30 2.10 0.80 0.40	0.30 0.90 0.40 0.35
	C03 I01	2 2	5 5	High High	2.1 5.9 1.0 2.8	1.4 3.6 0.7 2.1	0.30 1.40 0.10 0.40	0.40 0.90 0.20 0.30
	L01 C04 I03	2 2 2	0 0 0	Low High High	8.9 3.4 3.9 2.8	6.6 3.0 3.4 2.0	1.47 0.20 0.30 0.40	0.83 0.25 0.20 0.40
	I02	2	5	Low	8.9 3.3 3.8 1.5	7.3 2.9 3.4 1.0	1.30 0.20 0.30 0.20	0.30 0.20 0.10 0.25
					9.1 3.5 3.9 1.4	7.3 3.0 3.3 1.0	1.30 0.20 0.30 0.20	0.50 0.30 0.30 0.20
	C04 I03	2 2	5 5	High High	8.6 4.9 3.1 1.5	5.1 3.0 2.6 1.0	1.80 1.00 0.30 0.20	1.70 0.85 0.17 0.30
	L01 L02	2 1	5 CS	Low Low	1.1 4.8 3.0 1.0	0.7 3.0 2.6 0.7	0.15 1.10 0.30 0.10	0.25 0.70 0.10 0.20
					1.1 4.9 3.1 1.0	0.7 3.0 2.6 0.7	0.20 0.90 0.30 0.10	0.20 1.00 0.20 0.20
	D01 J01 L05	2 2 1	0 0 CS	High High Low	1.0 8.9 6.7 5.1	0.6 7.6 5.9 3.8	0.10 1.00 0.40 0.95	0.30 0.35 0.40 0.35
	N01 J01	1 2	CS 5	Low High	2.6 8.7 4.2 5.1	2.4 7.5 3.1 3.8	0.10 0.90 0.65 1.00	0.15 0.30 0.45 0.30
					2.6 9.1 4.3 5.0	2.4 7.6 3.2 3.7	0.10 1.10 0.70 0.90	0.10 0.40 0.40 0.40
	D01 B01	2 1	5 CS	High Low	2.6 0.6 4.1 0.7	2.3 0.5 3.0 0.6	0.10 0.00 0.60 0.00	0.20 0.10 0.50 0.10
	N02 D02 J03 B02	1 2 2 1	CS 0 0 CS	Low High High Low	2.0 4.5 12.1 1.3	1.4 3.2 7.6 0.8	0.20 0.73 3.65 0.10	0.35 0.50 0.85 0.40
	D02 N01	2 2	5 5	High Low	1.9 2.0 11.8 1.0	1.4 1.7 7.4 0.7	0.20 0.10 3.60 0.10	0.30 0.20 0.80 0.20
	F01 D03 NO J03 1	2 2 2 2	0 0 5 0	High High High Low	2.0 2.9 2.8 2.9 8.8 8.6 8.9 12.4 10.1 9.5 9.7 9.3 9.6	1.4 2.1 2.0 2.1 6.8 6.7 6.8 7.8 7.1 7.5 6.7 7.4 7.6	0.20 0.55 0.60 0.50 1.15 1.10 1.20 3.70 2.30 1.40 2.40 1.40 1.40	0.40 0.25 0.20 0.30 0.85 0.80 0.90 0.90 0.57 0.70 0.50 0.60 0.60

Acknowledgments

The authors gratefully thank Nadia Crini from the PEA2t platform (Chrono-environnement, University Bourgogne Franche-Comté, UMR CNRS 6249, France) for their technical help in metal analyses. Anne-Claude Goydadin is also gratefully thanked for her technical help in sample analyses and Loïc Angonin for his help in the field.

Funding

This work was supported by the Agence De l'Environnement et de la Maîtrise de l'Energie (ADEME, programmes BIOTROPH, grant number N°1172C0030, and DYSPAT, grant number 1572C0309), the Conseil Régional de Franche-Comté (for having financially supported the purchase of the HORIBA ABX Micros ES 60 haematology analyser), the LTER Zone Atelier Arc Jurassien (for the financial support to purchase the field anaesthesia device), and the OSU THETA and the University of Franche-Comté (programmes DEMENOL, MEXCO and OREAS, which supported the costs of fieldwork and field and laboratory analyses).

Data availability

Data will be made available on request.

Supplementary Material

Table of content

Table S1 Characteristics of the study sites (made of two replicate sites for each level of pollution, distance from the former smelter, median, minimum and maximum soil concentrations of Cd, Pb and Se with year of sampling and analysis in brackets) near the former Metaleurop Nord smelter.