Didier Techer

Sylvain Milla

Pascal Fontaine

Sandrine Viot

Marielle Thomas

Influence of waterborne gallic and pelargonic acid exposures on biochemical and reproductive parameters in the zebrafish (Danio rerio)

Keywords: Zebrafish, chronic toxicity, phytochemicals, gallic acid, pelargonic acid

Gallic and pelargonic acids are biologically derived substances receiving a growing interest as ecofriendly biocides with potential applications in freshwater system management. However, some data gaps remain to address their chronic ecotoxicity issue, particularly for fish. This work aimed at investigating the sublethal effects of a long-term waterborne exposure of zebrafish to these compounds. Mature fish were exposed to gallic or pelargonic acid at the concentrations of 0, 0.05, 0.5 and 5 mg/L during one month under semi-static conditions. Fecundity, hatching rate and median hatching time were regularly evaluated. Circulating sex hormone levels (11 ketotestosterone 211 KT, 17 bestradiol -E2-), plasma vitellogenin (Vtg), and gonad histology were monitored in males and females after exposure. Lactate dehydrogenase (LDH), total glutathione peroxydase (GPx) and glutathione-S transferase (GST) activities were assessed as enzymatic biomarkers of exposure in fish liver. Significant increases of GPx activity were reported in females exposed to both type of chemicals regardless the contamination level. Moreover, 5 mg/L gallic acid induced a decrease in 11-KT levels for males. For fish exposed to pelargonic acid, decreases in circulating hormone levels were reported respectively at 0.05 and 5 mg/L for 11-KT in males, and at 0.5 mg/L for E2 in females. However, no histological alteration in gonads neither significant variation in reproductive performances were detected following zebrafish exposure to gallic or pelargonic acid. Additional investigations concerning the mode of application and the environmental fate of these substances may warrant their further use in freshwater systems at concentrations compatible with bioci-dal/allelochemical effects.

Introduction __________________________________________________________________

Biologically active plant-derived compounds such as gallic or pelargonic acid [Fig. 1(A,B)] have experienced an increased interest over the last three decades as a consequence of their presumed health benefits and properties for environmental safety [START_REF] Balandrin | Natural plant chemicals: Sources of industrial and medicinal materials[END_REF][START_REF] Ow | Gallic acid and gallic acid derivatives: Effects on drug metabolizing enzymes[END_REF][START_REF] Jones | Effects of odd-numbered medium-chain fatty acids on the accumulation of long-chain 3-hydroxy-fatty acids in long-chain L-3hydroxyacyl CoA dehydrogenase and mitochondrial trifunctional protein deficient skin fibroblasts[END_REF][START_REF] Lederer | Phytotoxic activity of middle-chain fatty acids II: Peroxidation and membrane effects[END_REF]. Gallic acid (3,4,5 trihydroxybenzoic acid) is actually one of the most publicized polyphenol ingredient in functional foods [START_REF] Ow | Gallic acid and gallic acid derivatives: Effects on drug metabolizing enzymes[END_REF][START_REF] Sethiya | The total antioxidant content and radical scavenging investigation on 17 phytochemical from dietary plant sources used globally as functional food[END_REF]. Traditionally recognized as a medicinally important component in ayurve-dic herbs and formulations, this water-soluble polyphenol has been thoroughly studied for its free-radical scavenging activity that notably leads to anti-inflammatory and cytoprotective effects in mammal models [START_REF] Ow | Gallic acid and gallic acid derivatives: Effects on drug metabolizing enzymes[END_REF][START_REF] Abdelwahed | Study of antimutagenic and antioxidant activities of Gallic acid and 1,2,3,4,6 pentagalloylglucose from Pistacia lentiscus: Confirmation by microarray expression profiling[END_REF][START_REF] Sethiya | The total antioxidant content and radical scavenging investigation on 17 phytochemical from dietary plant sources used globally as functional food[END_REF].

Pelargonic acid, also known as nonanoic acid due to its nine carbon-saturated linear aliphatic chain, has been approved by the United States Food and Drug Administration as well as the Council of Europe for food use, mainly as an artificial flavoring substance and food preservative (FCT, 1978;US EPA, 2000).

Relying on their molecular structures [Fig. 1(A,B)], both gallic and pelargonic acids can demonstrate antimicrobial, as well as, algicidal or herbicidal properties [START_REF] Breeuwer | Nonanoic acid, a fungal self-inhibitor, prevents germination of Rhizopus oligosporus sporangiospores by dissipation of the pH gradient[END_REF][START_REF] Nakai | Myriophyllum spicatum-released allelopathic polyphenols inhibiting growth of blue-green algae Microcystis aeruginosa[END_REF][START_REF] Ow | Gallic acid and gallic acid derivatives: Effects on drug metabolizing enzymes[END_REF][START_REF] Lederer | Phytotoxic activity of middle-chain fatty acids II: Peroxidation and membrane effects[END_REF][START_REF] Nakai | Anti-cyanobacterial fatty acids released from Myriophyllum spicatum[END_REF][START_REF] Abdelwahed | Study of antimutagenic and antioxidant activities of Gallic acid and 1,2,3,4,6 pentagalloylglucose from Pistacia lentiscus: Confirmation by microarray expression profiling[END_REF][START_REF] Mmwd | Marin Municipal Water District Vegetation Management Plan, Herbicide Risk Assessment[END_REF][START_REF] Wang | Relationship between the allelopathic activity and molecular structure of hydroxyl derivatives of benzoic acids and their effects on cyanobacterium Microcystis aeruginosa[END_REF][START_REF] Rai | Natural Antimicrobials in Food Safety and Quality, C.A.B. International[END_REF][START_REF] Borges | Antibacterial activity and mode of action of ferulic and gallic acids against pathogenic bacteria[END_REF]. Consequently, the growing need for ecocompatible technology development has enlarged their scope of applications over these last years [START_REF] Shao | Potential for control of harmful cyanobacterial blooms using biologically derived substances: Problems and prospects[END_REF][START_REF] Sun | The antimicrobial, mechanical, physical and structural properties of chitosan-gallic acid films[END_REF]. As an illustration, the increased demand for more environmentally friendly agrochemicals, safer for human and animal health, has expanded the use of pelar-gonic acid as an adjuvant for "green" pesticide formulations [START_REF] Coleman | Organic acid enhancement of pelar-gonic acid[END_REF]. Indeed, this representative of odd numbered medium-chain fatty acid has been shown to possess nonspecific herbicidal properties toward terrestrial weeds. Its mode of action would mainly rely on its capacity to intercalate into thylakoid and plasma membranes, enhancing ultimately reactive oxygen species (ROS) formation and lipid-membrane peroxidation prior to plant cell death [START_REF] Lederer | Phytotoxic activity of middle-chain fatty acids II: Peroxidation and membrane effects[END_REF]. More recently, laboratory tests for appropriate use of pelargonic acid in order to control invasive tendencies of floating aquatic plants like duckweeds (Lemna spp.) has yielded promising results, and commercial formulations are in progress [START_REF] Webber | Pelargonic acid-a potential organic aquatic herbicide for duckweed management[END_REF][START_REF] Webber | Adjuvants affect duckweed (Lemna minor) control with pelargonic acid[END_REF]. Besides, several researchers highlighted its critical role as a bioactive phytochemical exhibiting allelopathic activity on the harmful algal bloom (HAB) forming cyanobacteria Microcystis aeruginosa, with a 50% growth inhibitory concentration (IC50) as low as 0.5 mg/L of substance [START_REF] Nakai | Anti-cyanobacterial fatty acids released from Myriophyllum spicatum[END_REF]. Concerning gallic acid, this secondary plant metabolite with its three adjacent aromatic phenoxyl groups has been also shown to act as a potent anticyanobac-terial allelochemical (toward M. aeruginosa), at an average IC50 ranging from 1 to ~5 mg/L [START_REF] Nakai | Myriophyllum spicatum-released allelopathic polyphenols inhibiting growth of blue-green algae Microcystis aeruginosa[END_REF][START_REF] Wang | Relationship between the allelopathic activity and molecular structure of hydroxyl derivatives of benzoic acids and their effects on cyanobacterium Microcystis aeruginosa[END_REF]. With this in mind, gallic and pelargonic acids show great potential for the development of novel eco-friendly and efficient water treatment process, with regards to the control of microalgae blooms or aquatic weed proliferations in freshwater systems. However, implementing integrated freshwater system management that would involve the use of biologically active phytochemicals would also bring up presumably enhanced environmental exposure issues for other nontarget aquatic organisms [START_REF] Shao | Potential for control of harmful cyanobacterial blooms using biologically derived substances: Problems and prospects[END_REF]. Currently, the information available related to aquatic ecotoxicity risks of gallic or pelargonic acid exposure is very scarce, particularly for fish (European Food Safety Authority, 2013;[START_REF] Melo | Radiolytic degradation of gallic acid and its derivatives in aqueous solution[END_REF]. As a matter of fact, only three studies have been listed in the ECOTOX Database (US EPA) relating the acute toxicity of pelargonic acid (in the range of 91 to >105 mg/L) toward three fish species, i.e., the bluegill (Lepomis macrochirus), the rainbow trout (Onco-rhynchus mykiss), and the fathead minnow (Pimephales promelas) (Brooke et al., 1984;US EPA, 2013), whereas none has been referenced for gallic acid. However, several authors raised the ecotoxicity issue for freshwater organisms by reporting cellular morphological alterations and decrease in bivalve digestive gland cell viability after 48 h in vivo exposure to gallic acid and other tannic acid derivatives [START_REF] Labieniec | Antioxidant and pro-oxidant effects of tannins in digestive cells of the freshwater mussel Unio tumidus[END_REF][START_REF] Labieniec | Response of digestive gland cells of freshwater mussel Unio tumidus to phenolic compound exposure in vivo[END_REF]. Still, fish are aquatic vertebrates also directly exposed to water enriched in such molecules and they possess an antioxidant system highly sensitive to a great variety of contaminants. More recently, [START_REF] Techer | Acute toxicity and sublethal effects of gallic and pelargonic acids on the zebrafish (Danio rerio)[END_REF] determined the 96 h lethal concentration (LC50) of pelargonic and gallic acids being respectively of 81.2 and >100 mg/L of substance using the zebrafish (Danio rerio). In this study, modulation of sublethal endpoints through antioxidant enzyme activity induction was also reported which raised the question of long-term toxicity issue for both compounds.

This study was designed to investigate the effects of chronic waterborne exposure to the current-use phytochemi-cals gallic and pelargonic acids on zebrafish (Danio rerio). This work was achieved in order to address data gaps related to their longterm aquatic ecotoxicity risks before any large-scale field application trial in a framework of water ecotechnology development. Danio rerio is a small freshwater fish species which is particularly suitable as an experimental model for long-term toxicity assessment [START_REF] Hill | Zebrafish as a model vertebrate for investigating chemical toxicity[END_REF]. For this purpose, mature males and females were exposed to each type of contaminants under semi-static conditions for 1 month. Since breeding success is critical for fish species sustainability, population fecundity of the exposed adult zebrafish, with subsequent hatching time and rate were regularly evaluated. Then, a panel of biochemical, physiological, and histological biomarkers was assessed. Plasma sex steroid hormonal levels (11-ketotestosterone, 11-KT; 17 ßestradiol, E2), circulating vitellogenin (Vtg) concentrations, testicular, and ovarian histology were monitored respectively in males and females at the end of experiments to investigate more thoroughly any potential impact on reproductive function. Moreover, selected liver enzyme activities were assessed as exposure biomarkers. The latter included lactate dehydrogenase (LDH) which is involved into energy production [START_REF] Diamantino | Lactate dehydrogenase activity as an effect criterion in toxicity tests with Daphnia magna straus[END_REF][START_REF] Coelho | Assessing lethal and sub-lethal effects of trichlorfon on different trophic levels[END_REF], glutathione-S-transferase (GST) which plays a key role in phase II biotransformation of endogenous and xenobiotic substances, and glutathione peroxydase (GPx) that protects cells against ROS formation [START_REF] Van Der Oost | Fish bioaccumu-lation and biomarkers in environmental risk assessment: A review[END_REF][START_REF] Hermes-Lima | Storey KB (ed) Oxygen in biology and biochemistry: role of free radicals, in functional metabolism: regulation and adaptation[END_REF].

Material and Methods

Chemicals

Stock solutions of 8 g/L gallic acid (Acros Organics, 98% purity) were prepared and renewed weekly in dechlorinated tap water. Pelargonic acid was obtained from Alfa Aesar, Johnson Matthey Company. In a liquid form (97% purity) at room temperature, it was directly used for appropriate dilution in glass container tanks. Tricaine methane sulphonate (MS-222), NaHCO3, and chemicals required for LDH assays were of analytical grade and were procured from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Regarding histology preparation of gonads, OTTIX solution and Diawax paraffin substitute were provided by DIAPATH SpA, Italy/ MM France. Hematoxylin Gill III and phloxine were provided by VWR International, Briare, France, whereas Regaud's iron hematoxylin was brought from Merck & Co., Whitehouse Station, NJ, USA, and Ponceau-Fushine of Masson and light green from Sigma-Aldrich Chemical Co., St. Louis, MO, USA. For the determination of plasma 11-KT and E2 levels, enzymelinked immunosorbent assay (ELISA) kits were bought from Cayman Chemical Company, Ann Arbor, USA, whereas Zebrafish Vitellogenin Elisa Kit (V01008402) were obtained from Biosense Laboratories AS (Bergen, Norway) for Vtg measurements. Other enzyme activity assay kits used for glutathione peroxidase and glutathione S-transferase measurements were purchased from Cayman Chemical Company (Ann Arbor, USA).

Animals

All fish treatment and procedures used in this study were in accordance with the general guidelines of the Council of European Communities (European Union, No 2010/63/CEE) and the French Animal Care Guidelines. Wild-type Tuebin-gen strain zebrafish were purchased from GIS AMAGEN (UMS 3504 CNRS/UMS 1374 INRA, Gif-sur-Yvette, France) at 3 months of age. They were housed for three additional months in the laboratory in a fish containment unit (HP Aquarium, France) filled with dechlorinated and aerated tap water (at 26 ± 1°C on a 12 h:12 h light:dark cycle). They were daily fed with newly hatched Artemia nauplii and once a week with additional commercial pellets (BioMar, France). The same rearing conditions were followed during the exposure period.

Experimental Design

Two weeks before experiments, groups of randomly selected 5 males and 5 females, based on morphological differences (i.e., shape of abdomen from gravid females vs males), were introduced into experimental tanks (15 L whole-glass containers filled with 10 L of tap water, i.e., 1 fish/L). Thereafter, fish were exposed either to gallic or pelargonic acid for 1 month under semistatic conditions, i.e., 70% daily water exchange was made using peristaltic pumps connected to appropriate stock solution reservoirs of gallic/pelargonic acid (or of noncontaminated freshwater). Debris and feces were removed daily with a siphon and additional 80% water volume was exchanged two times a week to ensure steady concentrations of test compounds in each exposure tank. For each type of test compounds, the following nominal exposure concentrations were established: 0.05, 0.5, and 5 mg/L of substance (and control groups of 0 mg/L). Such a geometric progression aimed at covering a broad range of contamination levels that may be encountered when applying pelargonic or gallic acid to inhibit microalgae growth in freshwater systems in a framework of water ecotechnology development [START_REF] Nakai | Myriophyllum spicatum-released allelopathic polyphenols inhibiting growth of blue-green algae Microcystis aeruginosa[END_REF][START_REF] Nakai | Anti-cyanobacterial fatty acids released from Myriophyllum spicatum[END_REF][START_REF] Wang | Relationship between the allelopathic activity and molecular structure of hydroxyl derivatives of benzoic acids and their effects on cyanobacterium Microcystis aeruginosa[END_REF][START_REF] Shao | Potential for control of harmful cyanobacterial blooms using biologically derived substances: Problems and prospects[END_REF]. These concentrations also encompassed the theoretical toxicological reference value (TRV) of 0.46 mg/L pelargonic acid which has been established by the "Marin Municipal Water District" (MMWD) United States government agency for water quality monitoring [START_REF] Mmwd | Marin Municipal Water District Vegetation Management Plan, Herbicide Risk Assessment[END_REF]. Treatments, i.e., exposure concentrations, were randomly assigned to glass tanks, with n = 3 replicate tanks per treatment including control group (nonexposed fish in freshwater only). During all the exposure period, temperature (25.6 ± 0.1°C), pH (8.2 ± 0.0), conductivity (223.1 ± 0.5 mS/cm), and dissolved oxygen (6.9 ± 0.1 mg/L) remained within the ranges that have been reported for D. rerio rearing [START_REF] Lawrence | The husbandry of zebrafish (Danio rerio): A review[END_REF].

Spawning, Embryonic Development, and Hatching Time

Reproductive performance of zebrafish was evaluated twice a week during the exposure period. For this purpose, a spawning box equipped with a plastic mesh to prevent egg predation was introduced into each exposure tank, so that spawning boxes contained exposure solutions or clean water only for control tanks, depending on the exposure condition previously assigned to the exposure tank. Plastic plant imitations were fixed to the mesh so as to serve as spawning substrates [START_REF] Lawrence | The husbandry of zebrafish (Danio rerio): A review[END_REF]. Males (n = 5) and females (n = 5) were placed separately (using a plastic barrier) into the spawning box in the evening. The next morning, fish were allowed to mix together just before light illumination, after which mating and spawning occurred progressively. Three hours after the light had been turned on, while fish are kept into exposure tanks, spawned eggs were collected using a 10 mL sterile Falcon pipette, rinsed, and counted. Since several males and females were grouped together into the spawning box, spawning and nonspawning females could not be distinguished when collecting eggs, and thus the total number of females per exposure tank was used for mean fecundity calculations. Consequently, population fecundity was determined in this work as the average number of eggs per female per successful breeding experiment [START_REF] Penglase | Selenium and mercury have a synergistic negative effect on fish reproduction[END_REF]. Moreover, in order to rule out any mistake on gender identification, gonad examination was conducted on all fish at the end of experiments using a dissecting microscope. Following fish spawning, coagulated eggs which were milky white were immediately discarded. The remaining eggs from a spawning box were pooled and transferred into Petri dishes (in several batches of 20 eggs each) containing clear water only.

Developing embryos were regularly examined under a dissecting microscope for potential abnormalities such as spinal deformities (including spine bending), delayed yolk sac absorption, and pericardial edema [START_REF] Westerfield | The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish Danio (Brachydanio) rerio[END_REF]. Mean malformation percentage of newly hatched fry was determined for each treatment over the reproductive test period. Newly hatched fry were recorded daily to evaluate the hatching rate and the median hatching time (expressed in days post fecundation or dpf). Hatching rate (%), median hatching time (dpf), and developmental abnormalities (%) were calculated for each exposure tank where a successful breeding experiment, i.e., egg production, was noticed. Mean values were then calculated over the entire exposure period according to the treatment modality (control group or exposure to gallic or nonanoic acid), and the total number of successful breeding experiments which have been reported for each treatment modality.

Anesthesia and Biometric Measurements

After 1 month of exposure, all fish were deeply anesthetized u NaHCO3, and total length (mm) and wet weight (mg) were recorded to calculate condition factor indices according to Fulton's formula [START_REF] Nash | The Origin of Ful-ton's Condition Factor: Setting the Record Straight[END_REF]:

Fish were then killed by exsanguination for blood collection prior to circulating hormones and vitellogenin levels determination.

Blood Collection, Plasma Hormones, and Vitellogenin Level Determination

Blood was collected from each individual by cardiac punctures using 10 mL pipette tip coated with heparin (5000 U/ mL), and immediately centrifuged (10,000 x g, 10 min). Plasma was removed and stored at -808C until analysis. Measurements of circulating hormone levels (11-KT for males, E2 for females) and Vtg (in females but also in males to investigate putative feminization property of the molecules) were achieved following appropriate plasma dilutions in coating buffers according to the manufacturer's instructions. All measurements from each fish were assayed using commercial ELISA kits and results were calculated from means of duplicates for each sample.

Gonad Histology

Midpart of two males and two females per exposure tank (ntotal = 12 specimens per treatment) were fixed in Bouin-Holland solution for 1 week, washed once with water, twice with 70% ethanol, and stored in absolute ethanol (Abdulfa-tah et al., 2011). Fragments of 5 mm thick slices were cut from each individual, dehydrated in OTTIX solution and embedded in the Diawax paraffin substitute (adapted from [START_REF] Langeron | Pr ecis de Microscopie: Technique-Exp erimentation-Diagnostic[END_REF][START_REF] Ammar | Continuous lighting inhibits the onset of reproductive cycle in pikeperch males and females[END_REF]. For females, sections of 6mm were cut from the Diawax block and stained with a trichrome: hematoxylin Gill III, 0.5% phloxine B, and 0.5% light green [START_REF] Rinchard | Comparative study of reproductive biology in singleand multiple-spawner cyprinid fish. I. Morphological and histological features[END_REF]. Ovarian maturity was evaluated under light microscopy with respect to oocytes in the following four general categories [START_REF] Selman | Stages of oocyte development in the zebrafish, Brachydanio rerio[END_REF]: Primary growth (type I): protoplasmic oocytes with enlarged and vacuole-free basophilic cytoplasm [Fig. 2(A)]; Early cortical alveolus stage (type II): appearance of oil droplets or yolk vesicles, occupying 2 or 3 rings in the peripheral zone of cytoplasm [Fig. 2(B)]; Late cortical alveolus stage (type III): presence of the cortical alveoli occupying all the cytoplasm, with irregular shape, cuboidal follicle cells and differentiated cellular layers [Fig. 2(C)]; Vitellogenic follicles (type IV): include follicles in the primary, secondary or tertiary yolk stage with accumulation of crystalline yolk globules in the center of oocytes and the periphery of the cytoplasm, also includes mature follicles [Fig. 2(D)]. The number of oocytes at each developmental stage was counted using an optical microscope (Nikon Eclipse Ni-U) equipped with a DS-Fi2 digital camera associated to the software NIS BR (Nikon France, Champignysur-Marne, France), and expressed as relative percentage of total follicle number in the whole section examined. For male gonad examination, sections of 4 µm of fixed midpart of specimens in Diawax blocks were cut and also stained with a trichrome (Ben [START_REF] Ammar | Continuous lighting inhibits the onset of reproductive cycle in pikeperch males and females[END_REF]): Regaud's iron haematoxylin, 1% Ponceau-Fushine of Masson, and 0.1% light green. Testes cells were classified according to three general sperm germ cell categories (Fig. 3) in accordance with the Histology and Histopathology Atlas of the Zebrafish (http://zfishtoxpat.comoj.com) and the work of [START_REF] Huszno | The reproductive cycle in the male gonads of Danio rerio (Teleostei, Cyprinidae). Stereological analysis[END_REF]: Spermatogonia: the largest of the spermatogenic cells with relatively voluminous and irregular nucleus with poorly condensed chromatin surrounded by acidophilic cytoplasm; Spermatocytes: include early and late spermatocytes, with relatively smaller cytoplasm than spermatogonia (with no affinity for dye) and easily identified by their densely stained chromatin; Spermatids and spermatozoa: dense, dark and round nuclei, spermatozoa also recognized sometimes by their light colored hair-like tails. For estimating the relative percentage of each sperm cell category, a stereological approach was followed [START_REF] Huszno | The reproductive cycle in the male gonads of Danio rerio (Teleostei, Cyprinidae). Stereological analysis[END_REF]. Briefly, photographs of a whole gonad section were taken under the microscope objective 40X. Then, a mesh with 87 X 65 points was superimposed on each photograph using the ImageJVR software (v.1.48). The number of points lying on the cysts of a specified cell category was counted and reported to the total number of points embedded in one of the three cell types of the section examined. Results were finally expressed as relative percentage for each testis germ cell category.

Biochemical Analysis

Liver Isolation and Homogenate Preparation

Livers were collected from 6 fish per exposure tank (ntotal = 18 specimens for a treatment, including males and females), using fine forceps and a dissecting microscope. Livers were homogenized individually in 1.5 mL screw-cap microtubes using a Bullet Blender [START_REF] Hoyt | Rapid, high-throughput homogenization of embryonic or larval zebrafish (Danio rerio)[END_REF]. In each microtube, prior to agitation, a mass of silica beads equals to twofold the mass of liver was introduced with 25 µL of ice-cold homogenization buffer (100 mM Tris, 150 mM KCl, 1 mM EDTA, 0.1 mM PMSF, and 10% v/v glycerol; pH 7.5). Tubes were agitated for 1 min at maximum speed and kept for 1 min on ice. Then, 90 µL of homogenization buffer were added followed by two more minutes of agitation. The resulting homogenate was centrifuged 13,000 X g, 20 min (4°C). The supernatant, corresponding to the S9 fraction, was aliquoted into 25 µL fractions and rapidly frozen (-80°C) until further use for protein content and enzymatic activity analyses.

Total Protein Determination

The volume of recovered supernatant from each liver homogenate was determined to estimate the total liver protein content. Protein content of S9 fraction was then measured on a 10-fold dilution of the recovered supernatant, according to the method developed by [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF] and using bovine serum albumin as a standard (Thermo Scientific Pierce Protein Assay kit 23236). Absorbance was read at 595 nm (Labsystem Multiskan EX, Labsystems Inc., Franklin, MA, USA) after an incubation period of samples and reagents for 15 min (258C) in microtiter plates.

Enzyme Activity Determination

Lactate dehydrogenase, glutathione transferase as well as total glutathione peroxydase activities were determined on S9 fraction spectrophotometrically using 96-well microtiter plates (Labsystem Multiskan EX, Labsystems Inc., Franklin, MA, USA).

LDH activity (EC 1.1.1.27) was evaluated according to the protocol described by [START_REF] Jesus | Lethal and sub lethal effects of the biocide chlorhexidine on aquatic organisms[END_REF] with slight modifications. The reaction mixture contained 147 mL of NADH (0.24 mM) and 23 mL of pyruvate (10 mM) in a Tris-NaCl buffer (0.1 M, pH 7.2). The decrease in absorbance at 340 nm due to the oxidation of NADH was continuously monitoring for 6 min.

For GST activity (EC 2.5.1.18) determination, the procedure of Habig and Jakoby (1981) was followed, using 1-chloro-2,4dinitrobenzene (CDNB) as the model substrate (Sigma-Aldrich CS0410). The increase in absorbance due to the conjugation of the glutathione thiol group to CNDB was monitored at 340 nm for 6 min.

The measurement of total GPx activity (EC 1.11.1.9) was based on the standard procedure of [START_REF] Drotar | Evidence for glutathione peroxidase activities in cultured plant cells[END_REF] using cumene hydroperoxide as substrate. The oxidation of reduced glutathione (GSH) to its oxidized form (GSSG) by GPx is coupled to the recycling of GSSG to GSH. The decrease in NAPDH concentration, used as a co-factor, was monitored at 340 nm for 8 min (Cayman Chemical 703102).

All enzyme activities were determined in duplicates for each liver homogenate, using 20 mL of diluted homogenate samples per microplate well. The mean obtained from duplicate measurements for one fish liver sample was used for further calculations (ntotal 5 18 per enzyme activity per treatment). All enzyme activities were finally expressed as U/mg protein. One unit corresponded to the amount of enzyme hydrolyzing one nanomole of substrate per minute.

Statistical Treatment

All data were expressed as mean 6standard error. Median hatching time was determined using REGTOX Excel macro (v.7.0.6) based on nonlinear regression (Hill model) (Vindimian, 2012). Percentages were arcsin square root transformed prior to statistical analyses. Normality and homogeneity of variance assumptions were respectively checked using Kolmogorov-Smirnov and Levene's test. Differences between groups were then determined by one-way ANOVAs for parametric distributions or Kruskal-Wallis for nonparametric ones, followed by Student Newman-Keuls post-hoc tests (p < 0.05). In the case of inequality between the treatment group size, the Dunns' test was used. All analyses were carried out at a significance level of p < 0.05 using Statistica v.10.

Results ______________________________________________________________________

Fish Condition Index

First of all, no dead fish neither any abnormality in animal behavior (swimming, feeding) could be reported during all the exposure period. Moreover, condition index was evaluated at the end of exposure as a general marker of overall fish health status (Table I). No differences were found among control and treatment groups regardless contaminant type and organisms gender (Table I).

Liver Enzyme Activities

In order to figure out eventual biological changes concomitant to the long-term exposure to each phytochemical, several biochemical biomarkers were further determined on total liver homogenates of both control and exposed fish. As reported in Table I, for both male and female specimens exposed to gallic or pelargonic acid, no significant variations were observed in total protein content, nor in LDH and GST activities. In contrast, higher levels of total GPx activity have been measured in males and females exposed to both type of substances compared to control regardless the exposure concentration (Table I). These increases in GPx activity were significant for female zebrafish exposed to 0.05, 0.5, and 5 mg/L of gallic acid on the one hand and to 0.05 and 5 mg/L of pelargonic acid on the other hand (Table I).

Plasma Steroid Hormones and Vitellogenin (Vtg) Levels

Additional biochemical parameters were assessed in order to investigate potential impacts of each phytochemical on zebrafish reproductive physiology. Results concerning plasma sex hormones and Vtg levels after exposure to gallic acid are presented in Figure 4. Males exposed to gallic acid showed a decrease in 11-KT levels but this decrease was significant only for fish exposed to the highest concentration of 5 mg/L [Fig. 4(A)]. Despite lower amounts of plasma estrogen levels measured in females exposed to gallic acid [Fig. 4(C)], no significant differences could be observed between treatments. Additional measurements of circulating Vtg indicated plasma levels which were about four degrees of magnitude higher in females [Fig. 4(D)] than in males [Fig. 4(B)] for all gallic acid treatments (including control). Besides, no significant variations could be observed between treatments for either females or males [Fig. 4(D,B)].

Results related to pelargonic acid exposed fish are illustrated in Figure 5. Overall, similar patterns could be reported respectively for hormone [Fig. 5(A,C)] and Vtg levels [Fig. 5(B,D)] of pelargonic acid exposed organisms. A significant decrease in 11-KT was noticed for males at the exposure concentrations of 0.05 and 5 mg/L of substance [Fig. 5(A)], whereas significantly lower levels of E2 were measured for females exposed to 0.5 mg/L of compound compared to control [Fig. 5(C)]. Regarding Vtg concentrations, there was no statistical difference for plasma levels in males [Fig. 5(B)]. On the contrary, mean Vtg concentration was significantly higher for females exposed to 5 mg/L of pelargonic acid compared to control [Fig. 5(D)].

Gonad Development

Subsequent histological examination of gonad development was conducted (Figs. 2 and3) and results are summarized in Figure 6. Overall, testes and ovaries with typical organizational architecture were observed for all fish specimens from control and treatment groups. Moreover, the presence of all germ cell types could be reported in gonad sections from males and females exposed either to gallic acid [Fig. 6(A,B)] or to pelargonic acid [Fig. 6(C,D)]. Besides, a general trend has been noticed in the relative proportions of germ cells regardless contaminants' nature and concentration. Indeed, testes were mainly composed of cell types which were at their latest stage of development (spermatids and spermatozoids) and to a lesser extent in the spermatocyte stage [Fig. 6(A,C)]. The opposite pattern was observed for ovaries with a higher relative frequency of primary growth stage oocytes (type I) with typical enlarged basophilic cytoplasm [Figs. 2(A) and 6(B,D)], whereas the relative number of cortical alveolus stages (types II and III) and vitellogenic and mature follicles (grouped under "type IV" category) were lower, ranging from 10% to 15% in average. Furthermore, no significant variation was detected in relative germ cell types for both sex and contaminant exposure conditions [Fig. 6(A-D)].

Fecundity, Hatching Rate, and Embryo-Larval Development

As reported in Table II, there was no statistical difference in mean fecundity between control and treatments either for gallic or pelargonic acid exposed female zebrafish. The evaluation of overall hatching rates indicated that the lowest egg hatchability was observed for fish exposed to 0.5 mg/L of gallic acid and to 5 mg/L of pelargonic acid, with, respectively, 32.0 ± 8.2% and 36.7 ± 14.7% of total egg survival (Table II).

In addition, no significant differences were found between control and treatments regarding embryonic development malformation rate (Table II).

Discussion ___________________________________________________________________

In order to figure out any potential sublethal effects of gallic or pelargonic acid, several cellular enzyme activities were first evaluated as exposure biomarkers, as it is usually recommended to avoid misconception about cellular damages that xenobiotics could cause [START_REF] Van Der Oost | Fish bioaccumu-lation and biomarkers in environmental risk assessment: A review[END_REF][START_REF] Zorita | Assessment of biological effects of environmental pollution along the NW Mediterranean Sea using red mullets as sentinel organisms[END_REF]. Taking into account that liver is the primary organ where detoxification occurs, it is also sensitive to oxidative stress damages subsequent to the intake of toxic substances (van der [START_REF] Van Der Oost | Fish bioaccumu-lation and biomarkers in environmental risk assessment: A review[END_REF]. Thus, in this work, liver total protein content was primary determined as a form of intermediate metabolic substrate [START_REF] Sancho | Short-term exposure to sublethal tebuconazole induces physiological impairment in male zebrafish (Danio rerio)[END_REF], followed by enzyme activities such as LDH as an indicator of cytotoxicity [START_REF] Diamantino | Lactate dehydrogenase activity as an effect criterion in toxicity tests with Daphnia magna straus[END_REF][START_REF] Coelho | Assessing lethal and sub-lethal effects of trichlorfon on different trophic levels[END_REF], GST and GPx as primary antioxidant defense mechanisms involved respectively in Phase II biotransformation and in radical species neutralization [START_REF] Van Der Oost | Fish bioaccumu-lation and biomarkers in environmental risk assessment: A review[END_REF]. From all these biochemical parameters, only GPx activity was responsive to chronic exposure to gallic or pelargonic acid (Table I). GPx is a part of the primary cell defense system to cope with an excess of free radical production and subsequent oxidative damages (van der [START_REF] Van Der Oost | Fish bioaccumu-lation and biomarkers in environmental risk assessment: A review[END_REF]. In mammals, gallic acid has been shown to act as an antioxidant phenolic acid by leading to the induction of hepatic GPx, which in turn was believed to protect against adverse oxidative damages and to promote health protective effects [START_REF] Yeh | Induction of hepatic antioxidant enzymes by phenolic acids in rats is accompanied by increased levels of multidrug resistance-associated protein 3 mRNA expression[END_REF][START_REF] Hsu | Effect of gallic acid on high fat diet-induced dyslipidaemia, hepatosteatosis and oxidative stress in rats[END_REF]. Whether the same protective mechanisms could be hypothesized for fish in our experimental conditions deserves further investigations, particularly in a context of ecofriendly surface water quality management. Regarding GPx induction under pelargonic acid exposure (Table I), the mode of action of this nonspecific herbicide has been well described when sprayed onto terrestrial weeds [START_REF] Lederer | Phytotoxic activity of middle-chain fatty acids II: Peroxidation and membrane effects[END_REF]. Because of its medium chain length, this aliphatic acid would be particularly efficient to intercalate into plasma cell membrane, resulting into chlorophyll pigment dislocation and subsequent outer thylakoid ROS production along with lipid peroxidation in chloroplast [START_REF] Lederer | Phytotoxic activity of middle-chain fatty acids II: Peroxidation and membrane effects[END_REF]. Concerning exposed zebrafish, similar phenomenon may also occur at the cellular or mitochondrial level that may lead to enhanced fish hepatic ROS formation and concomitant GPx induction as to prevent cells against oxidative stress.

Besides antioxidant enzyme variation, chronic exposure to both type of phytochemicals lead to modifications into reproductive hormones and circulating Vtg levels (Figs. 4 and5). The most significant variation following gallic acid exposure was a decrease in plasma 11-KT level in males at the highest compound concentration of 5 mg/L [Fig. 4(A)]. Gallic acid autoxidation products, resulting from natural oxidation of the polyphenol in alkaline medium [START_REF] Nogales | Unravelling the gallic acid degradation pathway in bacteria: The gal cluster from Pseudomonas putida[END_REF], have been shown to possess antigonadrotropic activity in vitro [START_REF] Burczyk | Timeand Dose-Dependent Antigonadotropic Activity of Oxidation Products of Gallic Acid and Pyrogallol on Leydig Cells in vitro[END_REF]. Indeed, inhibition of basal or of LHstimulated testosterone secretion have been reported by [START_REF] Burczyk | Timeand Dose-Dependent Antigonadotropic Activity of Oxidation Products of Gallic Acid and Pyrogallol on Leydig Cells in vitro[END_REF] using cultured Leydig cells from a mouse model (Swiss strain) at exposure concentrations varying from 0.1 to 10 mg/L of oxidation products. In this study, autoxidation of gallic acid also presumably occurred taking account of the alkaline pH of 8.2 in exposure tanks (see Materials and Methods part). Thus, one may also hypothesize that similar inhibition of 11-KT synthesis took place in the long term in fish due to the presence of gal-lic acid oxidation products; however, the investigation was not designed to discriminate between the biological effects of gallic acid by itself and its autoxidation products considering that both molecular forms would occur in a field scale context. Furthermore, in spite of 11-KT plasma concentration variations in gallic acid exposed males [Fig. 4(A)], further investigation indicated circulating Vtg levels in far higher concentrations in females than in males [Fig. 4(D,B)], as itis usually reported in the literature for fish only exposed to freshwater [START_REF] Van Der Ven | Vitellogenin expression in zebrafish Danio rerio: Evaluation by histochemistry, immunohistochemistry, and in situ mRNA hybridisation[END_REF]. Moreover, since no significant variations were reported between treatments either for females or males [Fig. 4(D,B)], these results indicated thus an apparent absence of estrogenlike activity of gallic acid.

On the other hand, waterborne exposure to pelargonic acid was related to a decrease in 11-KT and E2 levels, respectively, in males and females [Fig. 5(A,C)], along with a dosedependent increase in Vtg levels in exposed females [Fig. 5(D)]. Pelargonic acid is a fatty acid naturally occurring in several plants and animals (FCT, 1978;US EPA, 2000). It is usually not assumed to possess any estrogen-like activity or endocrine disrupting potency (Federal Register, 2003;[START_REF] Mmwd | Marin Municipal Water District Vegetation Management Plan, Herbicide Risk Assessment[END_REF]. Indeed, this compound can be metabolized into a fatty acid-CoA complex and then enters the Krebs's cycle for energy production in mitochondria (FCT, 1978;Federal Register, 2003). Nevertheless, as recently shown by [START_REF] Stempin | Valproic acid and its derivatives enhanced estrogenic activity but not androgenic activity in a structure dependent manner[END_REF] using in vitro experiments, derivatives of short-chain fatty acids such as 2-(2-propynyl)-nonanoic acid (a derivative of valproic acid with pelargonic acid) could activate the estrogen receptor a (ERa) in stably transfected human cell line (HEK293). Moreover, there may be several ways in which xenobiotics, including nonsteroido-genic chemicals, could interact with the endocrine and reproductive systems of vertebrates, i.e., not only through direct interactions with nuclear receptors but also through modulation of metabolizing enzyme activities, or interferences with hormone binding serum proteins and other nongenomic pathways [START_REF] Foradori | Non-genomic actions of androgens[END_REF]De Coster and van Lare-beke, 2012). Further investigations would be required to better assess the mode of action of pelargonic acid regarding the modulation of steroidal hormone and Vtg secretion as observed in the chronically exposed zebrafish [Fig. 5(A,C,D)].

Analyses of physiological endpoints were completed with gonad histological examination in both sexes in order to get more insights into potential reproductive consequences of the hormone and Vtg level modulation. Overall, all testes and ovaries developmental stages could be observed in control and exposed specimens, thereby confirming ongoing gametogenesis in fish which were all sexually mature. Besides, zebrafish are asynchronous spawners able to reproduce all the year round under laboratory conditions with very short mean interspawning intervals of 1.5 days [START_REF] Spence | Mating preference of female zebrafish, Danio rerio, in relation to male dominance[END_REF]. In the context of our study, regular spawns (two times a week) were conducted during 1 month and all fish were sacrificed the day after the last spawning event. The naturally high fecundity of zebrafish in conjunction with our experimental design relying on the maintenance of reproductively active females may explain the observed oogenesis patterns characterized by a predominance of oogonia [Fig. 6(B,D)].

Finally both male and female gonad development patterns as reported in this study were in accordance with previous work assessing reproductive performance of this fish species [START_REF] Daouk | Long-term food-exposure of zebrafish to PCB mixtures mimicking some environmental situations induces ovary pathology and impairs reproduction ability[END_REF], therefore, indicating that exposure to gallic and pelargonic acids did not impact the progress of gametogenesis.

According to [START_REF] Penglase | Selenium and mercury have a synergistic negative effect on fish reproduction[END_REF], the alternate method of fecundity determination in the present study may be used to estimate population fecundity relevant for the assessment of ecological impacts of contaminant exposure. Great variations in the spawning behavior considered on replicate tanks scale could be noticed during breeding experiments regardless the exposure condition (personal observations), possibly explaining discrepancies observed between treatments concerning fecundity data (Table II). Indeed, mated fish did not spawn systematically and egg production could differ substantially from one spawning event to another for the same spawning box. Nevertheless, such observations were in accordance with the literature when groups of male and female zebrafish are used for mating [START_REF] Sanden | Zebrafish (Danio rerio) as a model for investigating dietary toxic effects of deoxynivalenol contamination in aquaculture feeds[END_REF][START_REF] Penglase | Selenium and mercury have a synergistic negative effect on fish reproduction[END_REF]. Our primary objective was to evaluate the influence of phytochemicals on the reproductive performances of D. rerio as realistically as possible in accordance with native fish ecology [START_REF] Lawrence | The husbandry of zebrafish (Danio rerio): A review[END_REF]. Increasing the exposure period may help to mitigate the differences in spawning success and egg production from one replicate tank to another [START_REF] Lawrence | Generation time of zebrafish (Danio rerio) and medakas (Oryzias lat-ipes) housed in the same aquaculture facility[END_REF], and to better address the eventual effects of contaminants on fecundity as well. However, regardless of the reproductive performances, there was no difference in median-hatching time and developmental abnormality rates of larvae (Table II), indicating that eggs which developed until hatching were overall of similar "good" quality.

Conclusion __________________________________________________________________

In this study, we showed that long-term waterborne exposure to gallic or pelargonic acid can lead to antioxidant enzyme activity induction at the cellular level, with significant increases in GPx activity which were generally reported for females exposed at concentrations as low as 0.05 to 5 mg/L for both compounds. At the physiological level, waterborne gallic acid exposure leads to a marked decrease of male steroid hormone (11-KT) at the highest tested concentration of 5 mg/L, whereas no variation could be observed in female estrogen (E2) plasma level. Similar results were obtained with fish exposed to pelargonic acid, with slightly more pronounced decreases in male and female hormone levels, respectively, at 0.05 and 5 mg/L on one hand, and 0.5 mg/L of the middle-chain fatty acid on the other hand. To our knowledge, this is the first report that gallic and pelargonic acids could influence some reproductive parameters in zebrafish. However, these results had to be mitigated since histological examination indicated that all fish were reproductively mature, and without any alteration in relative germ cell types. Nevertheless, the possibility for gamete production and egg viability to get affected for longer exposure period than 1 month cannot be completely ruled out.

To sum up, slight detrimental biological effects could be reported depending on gallic or pelargonic acid exposure at concentration ranges which were compatible with potential water treatment ecotechnology development based on the direct use of these compounds. In order to warrant more environmental safety, we suggest to take into account the selected mode of application and environmental fate of phytochemi-cals (e.g., application rate, sequestration or adsorption phenomena onto organic matter, etc.) prior to any large-field-scale trial. 13.7 ± 8.0 3.6 ± 0.8 10.0 ± 3.5 5.3 ± 1.7 1.0 ± 0.5 12.1 ± 4.5 8.7 ± 3.5 également insérer ce contrôle autour de lignes d'un tableau pour répéter des parties de ce dernier.

Figure 1 .

 1 Figure 1. Molecular structures of (A) gallic acid (CAS 149-91-7) and (B) pelargonic acid (CAS 112-05-0).

Figure 2 .

 2 Figure 2. Typical germ cell types observed during the oogenesis in zebrafish. (A) Primary growth stage (type I). (B) Early cortical alveolus stage (type II). (C) Late cortical alveolus stage (type III). (D) Vitellogenic follicle (type IV). Bar =10 µm in A, B, C, and D. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 3 .

 3 Figure 3. Typical germ cell types observed during the sperma-togenesis in zebrafish. SPG, spermatogonia; SPC, sperma¬tocytes; SPZ, spermatids and spermatozoa. Bar = 50 µm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 4 .Figure 5 .Figure 6 .

 456 Figure 4. Plasma levels of (A) 11 kT and (B) Vtg in male zebrafish; (C) E2 and (D) Vtg in female zebrafish after 1 month exposure to gallic acid in the concentration range of 0, 0.05, 0.5, and 5 mg/L. Data are expressed as mean 6 SE. Asterisks indicate significant differences against control (p < 0.05). [Color figure can be viewed in the online issue, which is available at wileyonline-library.com.]

Table 1 . Fish condition index, liver total protein content, and enzyme activities of male (M) and female (F) zebrafish after 1 month exposure to gallic or pel-argonic acid, and the control group (0 mg/L of test substance)

 1

		Control	Gallic acid			Pelargonic acid
		0 mg/L	0.05 mg/L 0.5 mg/L	5 mg/L	0.05 mg/L 0.5 mg/L	5 mg/L
	Fish condition index	M 0.80 ± 0.03 0.73 ± 0.03 0.79 ± 0.02 0.80 ± 0.03 0.76 ± 0.02 0.81 ± 0.03	0.78 ± 0.02
		F 0.82 ± 0.02 0.88 ± 0.02 0.83 ± 0.03 0.90 ± 0.06 0.85 ± 0.03 0.82 ± 0.02	0.91 ± 0.02
	Liver total protein content (mg/g	M 77.5 ± 16.8 88.7 ± 13.6 96.3 ± 6.4	84.0 ± 14.3 93.6 ± 4.9	106.0 ± 11.0 82.0 ± 5.1
	fresh weight)	F 86.2 ± 8.5	62.8 ± 13.6 77.2 ± 11.6 49.2 ± 7.8	88.5 ± 5.6	77.1 ± 4.4	80.1 ± 9.3
	LDH activity (U/mg protein)	M 1839 ± 416 1322 ± 135 1922 ± 182 2116 ± 143 2448 ± 185 3269 ± 653	2517 ± 272
		F 512 ± 100	661 ± 57	446 ± 40	516 ± 75	893 ± 252	516 ± 62	578 ± 142
	GST activity (U/mg protein)	M 359 ± 50	225 ± 16	286 ± 29	332 ± 31	368 ± 31	369 ± 34	446 ± 56
		F 168 ± 11	223 ± 27	169 ± 19	175 ± 16	211 ± 31	174 ± 12	139 ± 15
	GPX activity (U/mg protein)	M 446 ± 111	507 ± 58	593 ± 97	668 ± 101	667 ± 94	608 ± 61	639 ± 98
		F 39 ± 12	214 ± 87*	171 ± 56 *	214 ± 61 *	277 ± 73 *	110 ± 31	234 ± 78 *

Table 2 . Evaluation of reproductive parameters in zebrafish over 1 month exposure to gallic or pelargonic acid in the concentration range of 0, 0.05, 0.5, and 5 mg/L of test substance

 2

				Control	Gallic acid			Pelargonic acid
				0 mg/L	0.05 mg/L	0.5 mg/L 5 mg/L	0.05 mg/L 0.5 mg/L	5 mg/L
	Fecundity	(mean	egg	12.1 ± 3.3	8.0 ± 1.5	16.5 ± 4.5 24.4 ± 4.9 16.3 ± 6.4	35.9 ± 8.1	26.0 ± 8.3
	number/female/spawning event)
	Median hatching time (dpf)		3 ± 0.3	4.1 ± 0.3	4.5 ± 0.3	4.4 ± 0.3	3.7 ± 0.3	3.6 ± 0.3	4.1 ± 0.1
	Hatching rate (%)			49.7 ± 18	65.9 ± 17	32.0 ± 8.2 59.4 ± 10 61.1 ± 10.4 47.2 ± 13.1 36.7 ± 14.7
	Developmental abnormalities (%)


Dr Guillaume Monneret
email: guillaume.monneret@chu-lyon.fr

The Journal of Immunology

IL-7 Restores Lymphocyte Functions in Septic Patients

Fabienne Venet,* , † Anne-Perrine Foray,* Astrid Villars-Me ´chin,* Christophe Malcus,* Franc ¸oise Poitevin-Later,* Alain Lepape, †, ‡ and Guillaume Monneret* , † Septic syndrome is the leading cause of mortality for critically ill patients worldwide. Patients develop lymphocyte dysfunctions associated with increased risk of death and nosocomial infections. In this study, we performed preclinical experiments testing the potential of recombinant human IL-7 (rhIL-7) as a lymphostimulating therapy in sepsis. Circulating IL-7 and soluble IL-7 receptor a-chain (soluble CD127) concentrations were measured in plasma, whereas cellular CD127 expression was evaluated on circulating CD4 + and CD8 + lymphocytes from septic shock patients and healthy volunteers. Lymphocyte proliferation, IFN-g production, STAT5 phosphorylation, and B cell lymphoma 2 induction were measured ex vivo in response to T cell stimulation in the presence or not of rhIL-7. We show that IL-7 pathway (plasmatic IL-7 concentration and cellular and soluble CD127 expressions) is not overtly altered and remains activable in septic patients. Most importantly ex vivo treatment of patients' cells with rhIL-7 significantly improves lymphocyte functionality (CD4 + and CD8 + lymphocyte proliferations, IFN-g production, STAT5 phosphorylation, and B cell lymphoma 2 induction after stimulation). To our knowledge, this constitutes the first report of rhIL-7 ability to restore normal lymphocyte functions in septic patients. These results support the rational for initiating a clinical trial testing rhIL-7 in septic shock. The Journal of Immunology, 2012, 189: 5073-5081.

S

eptic syndrome is the leading cause of mortality for critically ill patients worldwide although an enormous effort has been expended during the past 20 y to find new therapies that decrease the mortality of this syndrome [START_REF] Hotchkiss | The sepsis seesaw: tilting toward immunosuppression[END_REF]. It is now largely demonstrated that septic patients develop severe immune alterations (innate and adaptive), which intensity and duration are correlated with increased risk of death and nosocomial infections [START_REF] Hotchkiss | The sepsis seesaw: tilting toward immunosuppression[END_REF]. Thus, immune-stimulating therapies are now considered in the treatment of sepsis [START_REF] Hotchkiss | Immunotherapy for sepsis-a new approach against an ancient foe[END_REF].

Sepsis-induced lymphocyte alterations share many similarities with immune dysfunctions described in patients with chronic viral infections such as HIV [START_REF] Monneret | Monitoring immune dysfunctions in the septic patient: a new skin for the old ceremony[END_REF][START_REF] Mackall | Harnessing the biology of IL-7 for therapeutic application[END_REF][START_REF] Munier | Acutely dysregulated, chronically disabled by the enemy within: T-cell responses to HIV-1 infection[END_REF]. In particular, severe lymphopenia due to increased apoptosis, decreased T cell proliferation, and cytokine production after stimulation as well as increased percentage of CD4 + CD25 + regulatory T cells (Treg) have been described in both clinical contexts. In HIV infection, innovative immunorestorative therapies such as recombinant human IL-7 (rhIL-7) are currently tested as adjunctive treatment in phase I and II clinical trials. IL-7 is a 25-kDa glycoprotein secreted by stroma cells and other epithelial cells. IL-7 is a nonredundant cytokine for T and B cell development and function. IL-7 activity is mediated via its binding to a receptor (IL-7R) constituted of two chains: the a-chain (CD127) and the common cytokine receptor g-chain. This receptor is expressed continuously on most resting human T cells with high levels on naive and central memory cells and lower levels on Treg [START_REF] Lundstro ¨m | IL-7 in human health and disease[END_REF]. In healthy volunteers, rhIL-7 therapy induces B cell lymphoma 2 (BCL2) upregulation and sustained increased in peripheral blood CD4 + and CD8 + T cell numbers. Consequently, this T cell expansion caused a significant broadening of circulating TCR repertoire diversity independently of subject's age [START_REF] Sporte `s | Administration of rhIL-7 in humans increases in vivo TCR repertoire diversity by preferential expansion of naive T cell subsets[END_REF]. Interestingly, rhIL-7 therapy is associated with a reduction in the circulating proportion of Treg [START_REF] Sporte `s | Administration of rhIL-7 in humans increases in vivo TCR repertoire diversity by preferential expansion of naive T cell subsets[END_REF].

In chronic viral infection, rhIL-7 treatment has proven to be safe, well tolerated and to result in potent immunorestorative effects (4). In sepsis, three studies in mice have described beneficial effects of rhIL-7 treatment on mortality and sepsis-induced immune dysfunctions [START_REF] Unsinger | Interleukin-7 ameliorates immune dysfunction and improves survival in a 2-hit model of fungal sepsis[END_REF][START_REF] Kasten | Interleukin-7 (IL-7) treatment accelerates neutrophil recruitment through gdTcell IL-17 production in a murine model of sepsis[END_REF][START_REF] Unsinger | IL-7 promotes T cell viability, trafficking, and functionality and improves survival in sepsis[END_REF].

The objective of the current study was thus to perform an ex vivo preclinical study of rhIL-7 therapy in septic shock patients. We first tested whether parameters of IL-7 pathway were altered in patients in comparison with controls. In a second set of experiments, patients' lymphocyte response to rhIL-7 treatment was tested ex vivo. These experiments should provide the preliminary results necessary to the initiation of a clinical trial testing rhIL-7 in septic shock.

Materials and Methods

Patients

Septic patients belong to a global study on intensive care unit (ICU)-induced immune dysfunctions approved by our Institutional Review Board for ethics ("Comite ´de Protection des Personnes") and registered at Ministe `re Franc ¸ais de la Recherche et de l'Enseignement (no. DC-2008-509). The study group consisted in 70 septic shock patients according to the diagnostic criteria of the American College of Chest Physician/Society of Critical Care Medicine (Table I) [START_REF] Levy | SCCM/ESICM/ ACCP/ATS/SIS International Sepsis Definitions Conference[END_REF]. The onset of septic shock was defined as the beginning of vasopressor therapy. EDTA-anticoagulated blood was collected at two time points after diagnosis: day 1-2 and day 3-4. Mortality was defined as death occurring within 28 d after the onset of shock. Secondary ICU-acquired infections were defined according to European definitions of European Centre for Disease Prevention and Control.

Reference values and control samples for functional assays were obtained from a cohort of 55 healthy volunteers after informed consent was given.

Reagents

Anti-CD3-CD28-CD2 coated beads (T cell activation/expansion kit human) were purchased from Miltenyi Biotec (Auburn, CA). PE and PE-cyanine7-conjugated anti-CD3, Alexa Fluor 647-conjugated anti-phosphorylated STAT5, PE-conjugated anti-BCL2, and isotypic controls as well as protein transport inhibitor (GolgiPlug) were purchased from BD Biosciences (San Jose, CA). Allophycocyanin-conjugated anti-CD8, PE-conjugated anti-CD4, allophycocyanin-conjugated anti-CD4, PE-cyanine7-conjugated anti-CD8, PE-conjugated anti-IFN-g as well as anti-human CD127 Abs (clone R34.34) and isotypic controls were purchased from Beckman Coulter (Hialey, FL). rhIL-7 as well as goat anti-human CD127 polyclonal Abs and recombinant human CD127-Fc chimera were purchased from R&D Systems (Minneapolis, MN).

Quantification of plasma CD127 and IL-7 concentrations

A soluble CD127 (sCD127) ELISA test was designed as described previously [START_REF] Crawley | Soluble IL-7Ra (sCD127) inhibits IL-7 activity and is increased in HIV infection[END_REF]. Briefly, 96-well plates were coated overnight with mouse anti-human CD127 mAb (Beckman Coulter). Plates were blocked with BSA for 1 h, washed, and then samples were incubated for 1 h. Bound sCD127 was detected following a 1-h incubation with biotinylated goat anti-human CD127 polyclonal Abs (R&D Systems) and streptavidin-HRP. Reactions were developed using the tetramethylbenzidine microwell peroxidase substrate (BioMe ´rieux, Marcy l'e ´toile, France) and visualized with a plate reader at 450 nm. Sample concentrations were also extrapolated from standard curves of recombinant human CD127-Fc chimera (R&D Systems).

Plasmatic IL-7 concentration was measured using a multiplex ELISA (Bio-Rad, Hercules, CA) by using the manufacturer's recommendations.

Cell isolation and culture

PBMCs were isolated by Ficoll-Paque PREMIUM (GE Healthcare, Waukesha, WI) density gradient centrifugation. Cells were washed three times in sterile PBS (bioMe ´rieux) and resuspended in complete culture media. (RPMI 1640 medium with HEPES [Eurobio, Courtaboeuf, France] supplemented with 2 g/l sodium bicarbonate 7.5% [Institut de biotechnologies Jacques Boy, Reims, France], 2 mM L-glutamine [Lonza, Basel, Switzerland], 10% human AB plasma [Etablissement Franc ¸ais du Sang, Gerland, Lyon, France], 20 U/ml penicillin and 20 mg/ml streptomycin [Sigma-Aldrich, St. Louis, MO], and 2.5 mg/ml fungizone [Bristol-Myers Squibb, Rueil-Malmaison, France].)

The number of cells per well was adjusted to 1 3 10 6 cells/ml. Cells were stimulated with anti-CD2-CD3-CD28 Abs coated beads (ratio beads/ cells = 4:1) and/or with rhIL-7 (100 ng/ml).

Tritiated thymidine incorporation

T cells proliferation was analyzed by detecting 3 H-labeled methyl-thymidine incorporation (20 mCi/well) (PerkinElmer, Boston, MA). 3 H-labeled methylthymidine was added 24 h before harvesting cells on a fiberglass filter using an automated cell harvester (PerkinElmer). Incorporated radioactivity was measured in a direct beta counter (PerkinElmer). Assays were carried out in triplicate.

Flow cytometry

HLA-DR expression on circulating monocytes (mHLA-DR), percentage of Treg (CD4 + CD25 high CD127 low ) as well as CD127 expression on CD4 + and CD8 + T cells were monitored as described previously [START_REF] Monneret | Persisting low monocyte human leukocyte antigen-DR expression predicts mortality in septic shock[END_REF][START_REF] Venet | Increased circulating regulatory T cells (CD4 + CD25 + CD127 -) contribute to lymphocyte anergy in septic shock patients[END_REF]. CD4 + and CD8 + T cell proliferation was analyzed by monitoring decreased fluorescence after cell labeling with CFSE (Molecular Probes, Grand Island, NY) at a concentration of 10 pM and after cell culture during 5 d. STAT5 phosphorylation in T cells was analyzed by intracellular staining after overnight TCR stimulation and 15-min incubation with rhIL-7. BCL2 expression in T cells was analyzed by intracellular staining after 2-d incubation with TCR stimulation and rhIL-7. IFN-g production by CD8 + T cells was analyzed by intracellular staining after 4 h incubation with GolgiPlug, TCR stimulation and rhIL-7. The samples were run on a FACSAria II flow cytometer and analyzed using FACSDiva software (BD Biosciences).

Statistics

Results are presented as box-plots with individual values in Figs. 123and as means and SEM in Figs. 4567. Comparisons between groups were made using the nonparametric Mann-Whitney U test. The nonparametric Wilcoxon paired test was used to assess variations between time points in septic patients or between cell culture conditions in the ex vivo experiments. Statistical analyses were performed using Prism software (version 4.03; GraphPad Software, La Jolla, CA). A p value ,0.05 was considered as statistically significant.

Results

Plasmatic IL-7 concentration is slightly increased after septic shock Plasmatic IL-7 concentration was measured in septic shock patients at days 1-2 and 3-4 (n = 24) in comparison with healthy controls (n = 26). We observed that circulating IL-7 level was increased in septic shock patients at day 1-2 and at day 3-4 in comparison with controls (Fig. 1A). However, these levels remained relatively low (,10 pg/ml) and no significant differences were observed between survivors (n = 15) and nonsurvivors (n = 9; Fig. 1B) or between patients developing or not a nosocomial episode (n = 4 and n = 20, respectively; Fig. 1C).

This suggests that IL-7 plasmatic level may be increased in a homeostatic manner after septic shock. Because circulating IL-7 concentration is mostly regulated by its scavenging rate by T cells, such increased concentration is not surprising in markedly lymphopenic hosts such as septic shock patients (Table I) [START_REF] Mackall | Harnessing the biology of IL-7 for therapeutic application[END_REF][START_REF] Venet | Early assessment of leukocyte alterations at diagnosis of septic shock[END_REF].

CD127 expression is maintained on CD4 + and CD8 + lymphocytes after septic shock

A strong regulation of IL-7 receptor a-chain (CD127) has been described in various clinical conditions including HIV infection [START_REF] Crawley | The influence of HIV on CD127 expression and its potential implications for IL-7 therapy[END_REF]. We thus measured CD127 expression by flow cytometry on CD4 + and CD8 + T cells from 20 septic shock patients sampled at days 1-2 and 3-4 after the onset of shock in comparison with 30 healthy volunteers (Fig. 2).

No significant differences were observed between patients and controls at day 1-2 either on CD4 + cells (Fig. 2A) or on CD8 + cells (Fig. 2B). At day 3-4, a slightly decreased CD127 expression was measured on CD8 + T cells from septic patients in comparison with healthy volunteers (Fig. 2B), but no difference was observed for CD4 + cells (Fig. 2A). In paired samples, a decrease over time was observed for CD127 expression on CD4 + between days 1-2 and 3-4 but not on CD8 + cells. To note, absolute numbers of circulating CD4 + CD127 + and CD8 + CD127 + cells were calculated. As expected considering the marked lymphopenia observed after septic shock, these parameters were significantly decreased in septic patients versus healthy volunteers (data not shown).

No significant differences were observed between survivors (n = 12) and nonsurvivors (n = 8) for CD127 expression measured either on CD4 + cells (Fig. 2C) or on CD8 + lymphocytes (Fig. 2D). Similarly, no significant differences were observed between patients who will develop or not a nosocomial episode (n = 4 and n = 16, respectively; Fig. 2E,2F). Overall, these results show that CD127 expression is not overtly regulated on circulating lymphocytes after septic shock.

The plasmatic concentration of the soluble form of CD127 is increased in patients with ICU-acquired nosocomial infection after septic shock Importantly, plasmatic concentration of sCD127, known to possess in vitro antagonistic activity on IL-7 [START_REF] Crawley | Soluble IL-7Ra (sCD127) inhibits IL-7 activity and is increased in HIV infection[END_REF][START_REF] Crawley | The influence of HIV on CD127 expression and its potential implications for IL-7 therapy[END_REF] was measured in septic shock patients (n = 33) and healthy volunteers (n = 16). No significant differences were observed between patients and healthy controls neither at day 1-2 nor at day 3-4. This is in contrast with the observation made in HIV infection of a significantly increased sCD127plasmatic level [START_REF] Crawley | Soluble IL-7Ra (sCD127) inhibits IL-7 activity and is increased in HIV infection[END_REF][START_REF] Crawley | The influence of HIV on CD127 expression and its potential implications for IL-7 therapy[END_REF]. Meanwhile, a significant decrease was measured between these two time points in patients (Fig. 3A).

No differences were observed between survivors (n = 20) and nonsurvivors (n = 13), although a significant decrease over time was observed in both groups of patients (Fig. 3B). Interestingly and despite the low number of patients in each group, a significantly higher concentration was measured in patients who developed a nosocomial episode (n = 5) in comparison with patients who remained free of any secondary infectious challenge (n = 28). This difference was present both at day 1-2 and at day 3-4 (Fig. 3C).

In total, this first set of observational studies shows that, in contrast with results observed during chronic viral infections, parameters of the IL-7 pathway are not markedly modified after septic shock. This suggests that this pathway remains potentially activable during sepsis and thus that rhIL-7 could restore normal lymphocyte functions in patients. To test this hypothesis, we de- Blood samples were obtained from 70 septic shock patients after the onset of shock. For clinical parameters, categorical data are presented as number of cases and percentages respective to the total population in parentheses. Continuous data as well as biological parameters are presented as medians and interquartile ranges [Q1-Q3] in brackets. The following leukocyte subsets were analyzed at day 3-4 after the onset of shock: number of circulating CD3 + CD4 + T lymphocytes per microliter, percentage of CD4 + CD25 High CD127 Low Treg measured among CD3 + CD4 + T lymphocytes and percentage of monocytes expressing HLA-DR among total monocytes (mHLA-DR). Normal values from our laboratory for age-matched individuals are 500-1250 CD4 + T cells/ml, 5-7% Treg, and .90% mHLA-DR.

SAPS II, Simplified Acute Physiologic Score II calculated at inclusion in the protocol; SOFA score, Sequential Organ Failure Assessment score measured after 24 h of ICU stay.

veloped ex vivo functional assays with purified mononuclear cells from septic shock patients incubated with rhIL-7.

rhIL-7 restores ex vivo sepsis-induced decreased cell proliferation

Lymphocyte proliferation was measured after TCR stimulation of PBMCs extracted from septic shock patients (n = 10) and healthy volunteers (n = 10) and cultured in presence or not of rhIL-7. Proliferation was evaluated by tritiated thymidine incorporation.

Septic patients presented with a significantly decreased proliferation in response to TCR stimulation in comparison with healthy volunteers (Fig. 4A). Importantly, cell incubation with rhIL-7 significantly increased this proliferative response in patients whereas no significant effect was measured in healthy volunteers (Fig. 4A). This is illustrated by the significant difference of rhIL-7-induced fold increase in cell proliferation observed between patients and healthy controls (Fig. 4B).

rhIL-7 restores ex vivo sepsis-induced decreased CD4 + and CD8 + T cell proliferations

To decipher whether this rhIL-7-induced cell proliferation was affecting selectively CD4 + and/or CD8 + cells, PBMCs extracted from septic patients (n = 7) and healthy volunteers (n = 4) were stained with CFSE before culture in presence of TCR stimulation and rhIL-7.

We observed that both CD4 + (Fig. 5A,5C) and CD8 + (Fig. 5B) T cell proliferations were decreased in patients versus controls. This decrease was significant for CD4 + T lymphocytes (Fig. 5A). Most importantly, incubation with rhIL-7 significantly increased CD4 + lymphocyte proliferation to a level that was not different from that of healthy volunteers (Fig. 5A). A similar trend was observed for CD8 + T cells (Fig. 5B). Interestingly, the number of CD4 + cell divisions after rhIL-7 stimulation was increased in septic shock patients to a level similar to control values (Fig. 5D). Similar results were observed for CD8 + cells (data not shown). Representative examples of CFSE stainings of CD4 + T cells from one septic shock patient and one healthy control are shown in Fig. 5C. These results suggest that rhIL-7 is efficacious in restoring both CD4 + and CD8 + T cell dysfunctions after septic shock.

rhIL-7 restores ex vivo sepsis-induced decreased IFN-g lymphocyte production

Another aspect of sepsis-induced lymphocyte dysfunctions is a reduction in IFN-g production by lymphocytes. We thus measured by flow cytometry intracellular IFN-g production by CD8 + T cells in patients (n = 12) and controls (n = 10) after TCR stimulation and incubation with rhIL-7.

We observed that TCR-induced IFN-g production was significantly reduced in patients versus controls (Fig. 6). Most importantly, incubation of patients' cells with rhIL-7 significantly increased this cytokine response to a level that was not different from healthy controls. Representative examples of flow cytometry stainings are shown in Fig. 6B. These results show that rhIL-7 is efficacious in restoring CD8 + T cell dysfunctions after septic shock.

rhIL-7 efficiently activates parameters of its intracellular pathway in septic shock patients

A key signaling molecule for IL-7 is STAT5 (signal transducer and activator of transcription 5), which is phosphorylated following recruitment of CD127. Beside, one significant consequence of CD127 signaling is the maintenance of cell survival by increasing BCL2 protein expression. We thus investigated the expressions of these molecules in response to TCR stimulation and/or rhIL-7 in septic patients (n = 10) and controls (n = 10).

To our knowledge, we show for the first time that TCR stimulation was associated with a significantly decreased induction of STAT5 phosphorylation (Fig. 7A,7B) and BCL2 expression (Fig. 7C) in patients compared with healthy volunteers. Most importantly, cell incubation with rhIL-7 was able, by itself and after TCR stimulation, to significantly increase the expression of these molecules in patients and controls (Fig. 7). This shows that IL-7 intracellular pathway is mobilizable in septic patients although its activation is not adequate after TCR stimulation alone.

Discussion

Severe sepsis and septic shock represent the first cause of mortality in ICU [START_REF] Hotchkiss | The pathophysiology and treatment of sepsis[END_REF]. Septic syndromes induce alterations of the immune response that have, for long, been solely considered as an overwhelming proinflammatory syndrome. However, the failure of numerous clinical trials testing anti-inflammatory drugs to show any improvement in mortality has led to the re-evaluation of our understanding of severe sepsis and septic shock pathophysiology. In line, it is now globally accepted that, in parallel with a tremendous proinflammatory response leading to shock and organ dysfunctions, septic patients develop an immunosuppressive phase associated with immune dysfunctions [START_REF] Hotchkiss | Immunotherapy for sepsis-a new approach against an ancient foe[END_REF][START_REF] Monneret | Monitoring immune dysfunctions in the septic patient: a new skin for the old ceremony[END_REF]. Importantly, it has recently been shown that, in patients, these dysfunctions are not only observed at the systemic level but also locally in organs therefore illustrating the importance of this compensatory mechanism [START_REF] Boomer | Immunosuppression in patients who die of sepsis and multiple organ failure[END_REF]. In addition, observational clinical studies have linked the intensity and duration of these sepsis-induced immune dys-functions with increased risk of death and of secondary nosocomial infections (3). Consequently, clinical trials testing immunostimulating drugs are now initiated in septic patients [START_REF] Hotchkiss | Immunotherapy for sepsis-a new approach against an ancient foe[END_REF].

Sepsis-induced immune alterations affect both innate and adaptive immune responses [START_REF] Monneret | Monitoring immune dysfunctions in the septic patient: a new skin for the old ceremony[END_REF][START_REF] Skrupky | Advances in the management of sepsis and the understanding of key immunologic defects[END_REF]. So far, most work has been devoted to the study of the innate part of these sepsis-induced immune dysfunctions. Conversely, far less work has been dedicated to the study of lymphocyte alterations although a link between a reduced delayed-type hypersensitivity response (predominantly mediated by T cells and a hallmark of immune suppression) in ICU patients and increased risk of nosocomial infections and death has been described more than 30 y ago [START_REF] Meakins | Delayed hypersensitivity: indicator of acquired failure of host defenses in sepsis and trauma[END_REF]. Indeed, sepsis-induced lymphocyte dysfunctions make up 1) a dramatic lymphopenia affecting every lymphocyte subsets associated with major apoptosis, 2) functional alterations such as decreased proliferation and cytokine production in response to stimulation, 3) phenotypic alterations such as increased coinhibitory receptor (CTLA-4 and pro- grammed cell death-1 [PD-1]) and decreased costimulatory receptor (CD80 and CD86) and CD3 expressions, 4) decreased TCR diversity, and 5) increased percentage of circulating CD4 + CD25 + Treg [START_REF] Monneret | Monitoring immune dysfunctions in the septic patient: a new skin for the old ceremony[END_REF][START_REF] Venet | Decreased T cell repertoire diversity in sepsis: a preliminary study[END_REF]. These lymphocyte dysfunctions are likely illustrated in patients by viral reactivations normally solely occurring in immunocompromised hosts (CMV and herpes simplex virus). Importantly, they are associated with deleterious outcome after septic shock [START_REF] Heininger | Cytomegalovirus reactivation and associated outcome of critically ill patients with severe sepsis[END_REF][START_REF] Kalil | Prevalence and mortality associated with cytomegalovirus infection in nonimmunosuppressed patients in the intensive care unit[END_REF]. Therefore immunostimulating therapies able to restore these lymphocyte dysfunctions would represent an innovative therapeutic strategy in septic shock.

Interestingly, sepsis-induced lymphocyte dysfunctions share many similarities with cell alterations observed in patients with chronic viral infections. In particular, HIV-infected patients also present with a marked decrease in circulating CD4 + T cell number, functional alterations such as decreased lymphocyte proliferation after stimulation, phenotypic alterations such as increased coinhibitory receptor expression (i.e., PD-1), decreased TCR diversity and increased percentage of circulating Treg [START_REF] Munier | Acutely dysregulated, chronically disabled by the enemy within: T-cell responses to HIV-1 infection[END_REF]. Interestingly, in this clinical context, rhIL-7 is currently tested as an adjunctive therapy in phase I and II clinical trials to restore these lymphocyte dysfunctions. As of 2011, at least 15 ongoing studies of rhIL-7 therapy were underway. Investigators are seeking to use rhIL-7 to enhance viral clearance in the setting of chronic viral infections (hepatitis B virus, hepatitis C virus, and HIV). rhIL-7 is also being tested for its capacity to augment immune reconstitution following chemotherapy and/or bone marrow transplantation and in the setting of idiopathic CD4 + lymphopenia. Finally, trials have been initiated to test rhIL-7 efficacy as a vaccine adjuvant in aged populations and to determine whether this molecule will augment the efficacy of tumor vaccines and adoptive T cell ther-apy for cancer. Thus far, this agent has been well tolerated, inducing only mild constitutional symptoms and no evidence of the capillary leak syndrome observed with rhIL-2 therapy. In each of the studies, patients treated with rhIL-7 show diversification of their TCR repertoire and increases in circulating CD4 + and CD8 + T cell numbers stable for weeks to months after completion of rhIL-7 therapy. Interestingly, some of these trials showed treated patients experience substantial decreases in the relative frequency of Treg within the peripheral T cell pool [START_REF] Lundstro ¨m | IL-7 in human health and disease[END_REF].

We thus postulated that rhIL-7 could represent an innovative therapy in septic shock patients. Therefore, we first studied the regulation of parameters of IL-7 pathway in patients. In a second set of experiments, ex vivo functional testings were performed to evaluate rhIL-7 capacity to restore normal lymphocyte functions in septic patients.

We observed that parameters of the IL-7 pathway (plasmatic IL-7 concentration, CD127 expression on circulating lymphocytes and sCD127 level) were not markedly modified after septic shock. The only significant change was an increase in circulating IL-7 level in patients versus healthy controls. However, because an inverse relationship between serum IL-7 levels and lymphocyte counts has been constantly described in various settings of lymphocyte depletion (e.g., in HIV-infected patients), such increase in circulating IL-7 is not surprising in markedly lymphopenic hosts as septic shock patients. Moreover, this elevation is not of major importance since max concentrations measured in patients peaked at 13 pg/ml, whereas normal values for circulating human IL-7 range from 2 to 8 pg/ml [START_REF] Lundstro ¨m | IL-7 in human health and disease[END_REF].

Interestingly, cell surface and plasmatic concentrations of CD127 were not significantly different in septic patients versus FIGURE 6. IFN-g production by CD8 + T lymphocytes is restored by rhIL-7 in septic shock patients. Mononuclear cells were isolated from peripheral blood of septic shock patients (gray columns, n = 12) and healthy volunteers (open columns, n = 10). Cells were cultured for 4 h in the presence or not of anti-CD2/CD3/CD28 Ab-coated beads (TCR Stim, ratio of beads/cells = 4:1) and rhIL-7 (100 ng/ml). IFN-g production by CD8 + T cells was then measured by flow cytometry (intracellular staining). (A) Results are presented as mean 6 SEM of percentages of IFN-g-producing cells among total CD8 + T lymphocyte-proliferating cells. (B) One representative example of an IFN-g staining after culture in one septic shock patient and one healthy volunteer is presented. Nonparametric Wilcoxon paired test was used to evaluate evolution over time within a group. healthy controls. This is in contrast with results observed in HIV infection, as significantly decreased CD127 lymphocyte expression as well as marked increased concentration of its soluble form have been observed in patients [START_REF] Crawley | Soluble IL-7Ra (sCD127) inhibits IL-7 activity and is increased in HIV infection[END_REF][START_REF] Crawley | The influence of HIV on CD127 expression and its potential implications for IL-7 therapy[END_REF]. Interestingly and despite these alterations, rhIL-7 is effective in restoring lymphocyte dysfunctions in HIV-infected patients (4). Thus, as we did not observe such alterations of IL-7 pathway in our cohort, we postulated that rhIL-7 should be all the more effective in restoring normal lymphocyte functions after septic shock.

To test this hypothesis, we measured ex vivo the response of septic patients' lymphocytes to stimulation in the presence or absence of rhIL-7. As previously described, we observed that patients present with significantly decreased lymphocyte proliferation (both of CD4 + and CD8 + T cells) in response to TCR stimulation. In addition, IFN-g response was decreased in these patients as well. Importantly, we describe here two additional sepsisinduced lymphocyte dysfunctions. To our knowledge, we show for the first time that STAT5 phosphorylation and BCL2 induction in response to T cell stimulation are significantly altered after septic shock. This suggests that intracellular TCR activation pathway is dysfunctional in septic shock patients. Overall, our experiments recapitulate and enlarge the description of sepsisinduced lymphocyte dysfunctions in patients.

Most importantly, in these ex vivo experiments, cell incubation with rhIL-7 significantly restored every sepsis-induced lymphocyte alterations. Indeed, rhIL-7 significantly increased lymphocyte response to stimulation as measured by cell proliferation, IFN-g production, STAT5 phosphorylation or BCL2 expression in septic patients. Importantly, this restoring effect of rhIL-7 was observed in survivors as well as nonsurvivors (data not shown). To our knowledge, these results are the first to show that septic patients' cells remain responsive to rhIL-7 treatment and that rhIL-7 is efficacious ex vivo in restoring sepsis-induced lymphocyte dysfunctions. Although performed ex vivo, they suggest that rhIL-7 could represent an innovative therapy in sepsis.

These data are very complimentary to results obtained in murine models of sepsis [START_REF] Unsinger | Interleukin-7 ameliorates immune dysfunction and improves survival in a 2-hit model of fungal sepsis[END_REF][START_REF] Kasten | Interleukin-7 (IL-7) treatment accelerates neutrophil recruitment through gdTcell IL-17 production in a murine model of sepsis[END_REF][START_REF] Unsinger | IL-7 promotes T cell viability, trafficking, and functionality and improves survival in sepsis[END_REF]. Indeed, in mice, in vivo treatment with rhIL-7 was associated with significant improvement in T cell viability, trafficking and functionality after bacterial as well as fungal sepsis [START_REF] Unsinger | Interleukin-7 ameliorates immune dysfunction and improves survival in a 2-hit model of fungal sepsis[END_REF][START_REF] Unsinger | IL-7 promotes T cell viability, trafficking, and functionality and improves survival in sepsis[END_REF]. Importantly, in these experiments, survival was significantly improved as well. Interestingly and as observed in the current study, rhIL-7 treatment was associated with restoration of IFN-g production and increased BCL2 expression. More generally, these animal studies showed that, although targeting adaptive immune cells, rhIL-7 therapy could impact every aspect of the immune response, including innate immune cells such as neutrophils [START_REF] Kasten | Interleukin-7 (IL-7) treatment accelerates neutrophil recruitment through gdTcell IL-17 production in a murine model of sepsis[END_REF]. Although not tested in our study, this suggests that rhIL-7 therapy could efficiently restore both innate and adaptive immune dysfunctions after sepsis. Moreover, results observed in ongoing clinical trials in other pathologies sharing similar immune dysfunctions further support the potential beneficial effect of rhIL-7 in sepsis. In particular, beside the improvement of T cell viability and functionality, this molecule has been shown to improve TCR repertoire diversifica- tion, whereas septic patients present with a marked decrease in TCR repertoire diversity [START_REF] Venet | Decreased T cell repertoire diversity in sepsis: a preliminary study[END_REF]. Similarly, the effect of rhIL-7 involves the downregulation of expression of immunoregulatory receptors such as PD-1, whereas PD-1 expression has been shown to be increased on circulating cells from septic patients [START_REF] Huang | PD-1 expression by macrophages plays a pathologic role in altering microbial clearance and the innate inflammatory response to sepsis[END_REF]. Importantly, PD-1 knockout mice have been shown to be more resistant to sepsis than wild-type animals [START_REF] Huang | PD-1 expression by macrophages plays a pathologic role in altering microbial clearance and the innate inflammatory response to sepsis[END_REF]. Finally, rhIL-7 therapy also leads to a relative decrease in the frequency of Treg while an increased percentage of circulating Treg has been repeatedly shown in septic patients [START_REF] Monneret | Marked elevation of human circulating CD4 + CD25 + regulatory T cells in sepsis-induced immunoparalysis[END_REF].

In total, our results strongly support the hypothesis that rhIL-7 could represent an innovative therapy in the treatment of sepsis as we show that IL-7 pathway is not significantly altered and is still functional in septic shock patients and that rhIL-7 treatment significantly restores sepsis-induced lymphocyte functions to a normal response. This supports the rational for the initiation of a clinical trial testing rhIL-7 in septic shock patients, although the adequate design of such study still deserves to be precisely determined.

FIGURE 1 .

 1 FIGURE 1. Plasmatic IL-7 concentration in septic shock patients. Plasmatic IL-7 concentration was measured in septic patients (n = 24) at day 1-2 (D1-2) and day 3-4 (D3-4) after the onset of shock in comparison with healthy volunteers (HV, n = 26) (A), in survivors (S, n = 15) versus nonsurvivors (NS, n = 9) (B), and in patients who will develop a secondary infection (NI, n = 4) versus patients remaining free of any nosocomial episode (NoNI, n = 20) (C). Nonparametric Mann-Whitney U test was used to compare results between groups.

FIGURE 2 .

 2 FIGURE 2. Lymphocyte expression of IL-7Ra-chain in septic shock patients. Cell surface expression of IL-7R a-chain (CD127) was measured as mean of fluorescence intensity on CD4 + and CD8 + T cells from septic patients (n = 20) at day 1-2 (D1-2) and day 3-4 (D3-4) after the onset of shock versus healthy volunteers (HV, n = 30) (A, B), from survivors (S, n = 12) versus nonsurvivors (NS, n = 8) (C, D), and from patients who will develop a secondary infection (NIn = 4) versus versus patients remaining free of any nosocomial episode (NoNI, n = 16) (E, F). Nonparametric Mann-Whitney U test was used to compare results between groups.

FIGURE 3 .

 3 FIGURE 3. Plasmatic concentration of soluble IL-7R a-chain in septic shock patients. Plasmatic concentration of the soluble fraction of IL-7R a-chain (sCD127) was measured in septic patients (n = 33) at day 1-2 (D1-2) and day 3-4 (D3-4) after the onset of shock versus healthy volunteers (HV, n = 16) (A), in survivors (S, n = 20) versus nonsurvivors (NS, n = 13) (B), and in patients who will develop a secondary infection (NI, n = 5) versus patients remaining free of any nosocomial episode (NoNI, n = 28) (C). Nonparametric Mann-Whitney U test was used to compare results between groups. Nonparametric Wilcoxon paired test was used to evaluate evolution over time within a group.

FIGURE 4 .

 4 FIGURE 4. Lymphocyte proliferation is restored by rhIL-7 in septic shock patients. Mononuclear cells were isolated from peripheral blood of septic shock patients (gray columns, n = 10) and healthy volunteers (open columns, n = 10). Cells were cultured for 5 d in the presence or not of anti-CD2/CD3/ CD28 Ab-coated beads (TCR Stim, ratio of beads/cells = 4:1) and rhIL-7 (100 ng/ml). Cell proliferation was evaluated by [ 3 H]thymidine incorporation. (A) Results are presented as mean 6 SEM of proliferative response evaluated for each cell culture condition and each experiment as the ratio versus nonstimulated cells response. (B) Results are presented as individual values of rhIL-7-induced proliferation evaluated for each experiment as the ratio of the proliferative response induced by TCR stimulation alone or in the presence of rhIL-7. Nonparametric Mann-Whitney U test was used to compare results between groups. Nonparametric Wilcoxon paired test was used to evaluate evolution over time within a group.

FIGURE 5 .

 5 FIGURE 5. CD4 + and CD8 + lymphocyte proliferations are restored by rhIL-7 in septic shock patients. Mononuclear cells were isolated from peripheral blood of septic shock patients (gray columns, n = 7) and healthy volunteers (open columns, n = 4). Cells were stained with CFSE and cultured for 5 d in the presence or not of anti-CD2/CD3/CD28 Ab-coated beads (TCR Stim, ratio of beads/cells = 4:1) and rhIL-7 (100 ng/ml). Cell proliferation was evaluated by flow cytometry (decreased CFSE fluorescence intensity). Results are presented as mean 6 SEM of percentages of proliferating cells (i.e., with a decreased CFSE fluorescence intensity in comparison with nonstimulated cells). (A) CD4 + T cell proliferation. (B) CD8 + T cell proliferation. (C) One representative example of a CFSE staining after culture in one septic shock patient and one healthy volunteer is presented. Nonparametric Wilcoxon paired test was used to evaluate evolution over time within a group. (D) Numbers of CD4 + T cell divisions in septic shock patients and healthy controls after stimulation with anti-CD2/CD3/CD28 Ab-coated beads alone (stimulated cells) or with rhIL-7 (stimulated cells + rhIL-7).

FIGURE 7 .

 7 FIGURE 7. STAT5 phosphorylation and BCL2 induction in T lymphocytes are restored by rhIL-7 in septic shock patients. Mononuclear cells were isolated from peripheral blood of septic shock patients (gray columns, n = 10) and healthy volunteers (open columns, n = 10). (A) Cells were cultured overnight in the presence or not of anti-CD2/CD3/CD28 Ab-coated beads (TCR Stim, ratio of beads/cells = 4:1) and 15 min in the presence or not of rhIL-7 (100 ng/ml). STAT5 phosphorylation in T cells was then measured by flow cytometry (pSTAT5, intracellular staining). For each experiment and culture condition, means of fluorescence intensity of pSTAT5 were normalized by value measured in the control well. (B) One representative example of a pSTAT5 staining after culture in one septic shock patient and one healthy volunteer is presented. (C) Cells were cultured 48 h in the presence or not of anti-CD2/ CD3/CD28 Ab-coated beads (TCR Stim, ratio of beads/cells = 4:1) and rhIL-7 (100ng/ml). BCL2 expression in T cells was then measured by flow cytometry (intracellular staining). For each experiment and culture condition, means of fluorescence intensity of BCL2 were normalized by value measured in the control well. Results are presented as mean 6 SEM. Nonparametric Wilcoxon paired test was used to evaluate evolution over time within a group.

Table I .

 I Demographic, clinical, and immunological data for septic shock patients

	Clinical data
	Age at admission (y)		73 [62-80]
	Gender	Male	44 (63)
	SAPS II at diagnosis of shock		58 [47-67]
	SOFA score		11 [8-13]
	Comorbidities	0	23 (33)
	Initial infection	$1 Bacilli Gram 2	47 (67) 19 (27)
		Cocci Gram +	19 (27)
		Fungi	5 (7)
		Others	11 (16)
	Type of infection	Community-acquired	43 (61)
		Hospital-acquired	27 (39)
	Site of infection	Pulmonary	24 (34)
		Abdominal	17 (24)
		Others	29 (41)
	Mortality	Nonsurvivors	24 (34)
	Secondary infections	Occurrence	14 (20)
	Immunological data
	CD3 + CD4 + T lymphocytes	D3-4	355 [190-530]
	Treg (%)	D3-4	10 [8-14]
	mHLA-DR (%)	D3-4	41 [19-70]

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Acknowledgments

We thank Anne Portier and Dr. Caroline Guignant from the Immunology Laboratory of Hopital E. Herriot-Lyon for help in performing preanalytical handling of samples and He ´le `ne Vallin, Nathalie Panel, and Marion Provent (Clinical Research Center, Lyon-Sud) for work on patients' inclusion and clinical data acquisition. We also thank Angela Crawley and Jonathan Angel for sharing protocol details regarding sCD127 measurement.

This work was supported by funds from the Hospices Civils de Lyon, by the French Ministry of Health (Projet Hospitalier de Recherche Clinique 2008; to G.M. and A.L.), and by the European Society for Intensive Care Medicine (to F.V., Levi-Montalcini Biomedical Sciences Award 2011). These funding sources had no role in study design, in the collection, analysis and interpretation of data, in the writing of the report, and in the decision to submit the paper for publication.

Disclosures

The authors have no financial conflicts of interest.