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TOXLET 9078 1–11 Toxicology Letters xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet

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Toxicity of oral cadmium intake: Impact on gut immunity

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Marina Ninkova , Aleksandra Popov Aleksandrova , Jelena Demeneskua , Ivana Mirkova , Dina Mileusnica , Anja Petrovicb , Ilijana Grigorovb , Lidija Zolotarevskic, Maja Tolinackid, Dragan Kataranovskia,e, Ilija Brceskif , Milena Kataranovskia,g,*

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a Immunotoxicology group, Department of Ecology, Institute for Biological Research “Sinisa Stankovic”, University of Belgrade, 142 Bulevar Despota Stefana, 1000 Belgrade, Serbia b Department of Molecular Biology, Institute for Biological Research “Sinisa Stankovic”, University of Belgrade, 142 Bulevar Despota Stefana, 11000 Belgrade, Serbia c Institute of Pathology, Military Medical Academy, 17 Crnotravska, 11000 Belgrade, Serbia d Laboratory for Molecular Microbiology, Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, 444a Vojvode Stepe, 11010 Belgrade, Serbia e Institute of Zoology, Faculty of Biology, University of Belgrade, 16 Studentski Trg, 11000 Belgrade, Serbia f Faculty of Chemistry, University of Belgrade, 3 Studentski Trg, 11000 Belgrade, Serbia g Institute of Physiology and Biochemistry, Faculty of Biology, University of Belgrade, 16 Studentski Trg, 11000 Belgrade, Serbia

H I G H L I G H T S








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Examination of the effect of oral cadmium intake on gut immune responses in rat. Tissue damage and inflammation were evident in homogenates of duodenum. Cadmium intake primes adaptive immune activities of mesenteric lymph node cells. Innate immune activities of mesenteric lymph node cells were observed. Cadmium-induced gut immune activity depicts its significance as health risk factor.

A R T I C L E I N F O

A B S T R A C T

Article history: Received 9 April 2015 Received in revised form 2 June 2015 Accepted 2 June 2015 Available online xxx

Gastrointestinal tract is one of the main targets of cadmium (Cd), an important food and drinking water contaminant. In the present study, the effect of subchronic (30 days) oral (in water) intake of 5 ppm and 50 ppm of cadmium on immune responses in the gut was examined in rats. Cadmium consumption resulted in reduction of bacteria corresponding to Lactobacillus strain, tissue damage and intestinal inflammation [increases in high mobility group box 1 (HMGB1 molecules), superoxide dismutase (SOD) and catalase (CAT) activity and proinflammatory cytokine (TNF, IL-1b, IFN-g, IL-17) content]. Draining (mesenteric) lymph node (MLN) stress response was observed [elevation of MLN glutathione (GSH) and metallothionein (MT) mRNA levels] and stimulation of both adaptive [cellularity, proliferation, proinflammatory (IFN-g and IL-17) MLN cell cytokine responses] as well as innate immune activity (increases in numbers of NK and CD68+ cells, oxidative activities, IL-1b). In contrast to proinflammatory milieu in MLN, decreased or unchanged antiinflammatory IL-10 response was observed. Stimulation of immune activities of MLN cells have, most probably, resulted from sensing of cadmium-induced tissue injury, but also from bacterial antigens that breached compromised intestinal barrier. These effects of cadmium should be taken into account when assessing dietary cadmium as health risk factor. ã2015 Published by Elsevier Ireland Ltd.

Keywords: Oral cadmium intake Rats Intestinal (duodenum) inflammation Mesenteric lymph nodes immune priming

* Corresponding author at: Immunotoxicology group, Department of Ecology, Institute for Biological Research “Sinisa Stankovic”, University of Belgrade, 142 Bulevar Despota Stefana, 11000 Belgrade, Serbia. Fax: +381 11 2761433. E-mail address: [email protected] (M. Kataranovski).

1. Introduction

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Cadmium (Cd) is one of the most toxic metals widely Q3 distributed in the environment. Although it affects the number of organs and tissues (WHO, 1992), cadmium toxicity is examined primarily in liver and kidneys (Kayama et al., 1995a,b). Oxidative

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http://dx.doi.org/10.1016/j.toxlet.2015.06.002 0378-4274/ ã 2015 Published by Elsevier Ireland Ltd.

Please cite this article in press as: Ninkov, M., et al., Toxicity of oral cadmium intake: Impact on gut immunity. Toxicol. Lett. (2015), http://dx. doi.org/10.1016/j.toxlet.2015.06.002

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stress and inflammation are the underlying mechanisms of toxicity to these tissues (Horiguchi et al., 2000; Kayama et al., 1995b; Rikans and Yamano, 2000). Food-borne cadmium as well as drinking water containing this metal are the main sources of exposure to this environmental toxicant (Asar et al., 2000; Lalor, 2008; Nordberg, 2009; Park et al., 2002; Satarug et al., 2010). This allocates gastrointestinal tract (GIT) as cadmium main target where it can exert toxicity. According to present data, the majority of ingested cadmium is retained in the GIT mucosa and only a small Q4 amount (up to 3%) is absorbed (Andersen et al., 1988; Goon and Klaassen, 1989). Early studies showed that orally administered or perfused cadmium resulted in histologically evident intestinal toxicity in mice expressed as changes in architecture of villi, hemorrhagic gastritis and intestinal epithelial cell necrosis (Andersen et al., 1988; Valberg, 1977). Inflammatory intestinal response, characterized by induction of chemotactic cytokine MIP2 and subsequent neutrophil infiltration seems responsible for intestinal toxicity in response to single oral administration of moderate to high doses (25–100 mg/kg) of cadmium to mice (Zhao et al., 2006). Cadmium-induced production of IL-8 by human epithelial cell line in vitro (Hyun et al., 2007) suggests contribution Q5 of proinflammatory cytokines to intestinal toxicity of this metal. Several studies showed negative effect on gut microbiota (decrease in total intestinal microflora as well as changes in the ratio of probiotic to pathogenic bacteria) in mice administered with 20–50 mg/kg of cadmium for three to eight weeks (Breton et al., 2013a; Fazeli et al., 2011; Liu et al., 2014). Cadmium-induced dysbiosis might have been related to intestinal inflammation as increases in TNF colonic content of mice following 21-day oral administration of 20 or 100 mg/kg of cadmium has been reported (Liu et al., 2014). However, a decrease in TNF (and IL-1b) expression was noted in duodenum of mice administered with 100 mg/kg of cadmium for 4–12 weeks (Breton et al., 2013b). This leaves the mechanisms of induction of intestinal inflammatory response largely unknown. Intestine-draining mesenteric lymph nodes (MLN) are central sites of immune responses induction in the gut (Mowat, 2003). The activities of MLN cells are central for immune tolerance induction in the intestine and for induction of local protective responses (Mowat, 2003). Tolerogenic activity in these lymph organs, which prevails in basal conditions, forms the border between this compartment and the rest of immune system, precluding systemic immune priming (MacPherson and Smith, 2006). Having this in mind, the effect of oral cadmium administration on intestine and draining (mesenteric) lymph node activity in rats has been examined. The local effects of cadmium intake were evaluated in the homogenates of duodenum, the intestinal region most reactive to cadmium (Zhao et al., 2006) and included measurements of census of Lactobacilli (probiotic bacteria involved in immune homeostasis in the gut), concentration of necrotic marker high mobility group box 1 (HMGB1) molecule, activity of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) and proinflammatory cytokine contents. Basic parameters of MLN cell activity [cellularity, proliferation, T-cell derived proinflammatory (IFN-g and IL-17) and antiinflammatory (IL-10) cytokine production and mRNA levels, and selected innate immune effector activities] were determined in rats that consumed cadmium orally (in water) at 5 ppm or 50 ppm for 30 days. The dose of 5 ppm is considered as equivalent to the dose of exposure of women in Japan suffering from Itai-Itai disease (Bhattacharyya et al., 1988), while 50 ppm is equivalent to cadmium dose noted in the subjects living in highly polluted areas or that are professionally exposed to this metal (Wang et al., 2003). According to environmental animal studies (Blanusa et al., 2002; DamekPoprawa and Sawicka-Kapusta, 2004; Lukacinova et al., 2011) these doses cover environmentally relevant cadmium

concentrations. Data obtained in the present study showed that oral cadmium intake resulted in intestine tissue damage and inflammation. Induction of mesenteric lymph node cell proliferation and expression of proinflammatory cytokine and innate effector cell activities were observed in these animals as well, which is a novel finding. Immune priming of mesenteric lymph nodes, most probably, resulted from sensing of cadmium-induced intestine injury and bacterial antigens that reached this compartment and might contribute to protection of vulnerable intestine from bacterial overgrowth. Such an activity in normally tolerogenic lymphoid environment, however might be an introduction to perturbation of local immune homeostasis as well.

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2. Materials and methods

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2.1. Chemicals

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Cadmium chloride (CdCl2) and tris free base—tris (hydroxymethyl) amino methane were purchased from Serva, Feinbiochemica (Heidelberg, Germany). Lipopolysaccharide (LPS; type 0111: B4 from Escherichia coli), Concanavalin A (ConA), L-epinephrine, hexadecyltrimethylammonium bromide (HTAB), N-(1-naphtyl) ethylenediamine dihydrochloride, sulfanilamide (p-aminobenzenesulfonoamide), o-dianisidine dihydrochloride, myeloperoxidase (MPO) were purchased from Sigma (Sigma Chemical Co., St. Louis, MO, USA). N,N,N0 ,N0 -ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate was obtained from USB Corporation (Cleveland, OH, USA). Blotto (non-fat dry milk) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Saccharose was obtained from Lachner (Neratovice, Czech Republic). Sodium nitrite, 5,50 -dithiobis (2-nitrobenzoic acid) (DTNB) and reduced L-Glutathione (GSH) were purchased from Fluka Chemika (Buchs, Switzerland). For use in experiments, LPS and ConA were dissolved in RPMI-1640 medium (PAA Laboratories, Pasching, Austria), and used in final concentration of 100 ng/ml and 1 mg/ml, respectively. All solutions for cell culture experiments were prepared under sterile conditions and sterile filtered (Minisart, pore size 0.20 mm, Sartorius Stedim Biotech, Goettingen, Germany) before use. Hydrogen peroxide (H2O2) was purchased from Zorka Farma (Sabac, Serbia). Culture medium supplemented with 2 mM glutamine, 20 mg/ml gentamycine (Galenika a.d., Zemun, Serbia), 5% (v/v) heat inactivated fetal calf serum (PAA Laboratories, Pasching, Austria) was used in cell culture experiments. Annexin V apoptosis detection kit (Allophycocyanin conjugated Annexin V and Propidium iodide); fluoroisothiocyanate (FITC)-labeled mouse antibodies to rat CD4 and mouse anti-rat granulocyte marker (HIS48); phycoerythrin (PE)-labeled mouse antibodies to rat CD8, mouse anti-rat CD314 (NKG2D), and F(ab0 ) 2 goat anti-mouse IgG were purchased from eBioscience (eBioscience Inc., San Diego, CA, USA). Mouse anti-rat CD163 (ED2) and Alexa Fluor 488-labeled mouse anti-rat CD68 (ED1) were purchased from AbD Serotec (Serotec Ltd., Oxford, UK). Dihydrorhodamine 123 (DHR 123) was purchased from Life Technologies Corp. (Carlsbad, CA, USA).

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2.2. Animals

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All animal procedures were complied with the Directive 2010/ 63/EU on the protection of animals used for experimental and other scientific purposes and approved by the Ethical Committee of the Institute for Biological Research “Sinisa Stankovic”, University of Belgrade. Male Dark Agouti (DA) rats 10–12 weeks old (at the beginning of treatment), bred and conventionally housed in a controlled environment at the Institute for Biological Research “Sinisa Stankovic” (Belgrade, Serbia) were used in the present study. They were maintained at 12 h photoperiod, 21/24  C temperature control and 60% relative humidity. All rats had ad

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libitum access to standard rodent chow and distilled water throughout the study. Four to six animals were assigned to each treatment group per experiment, in at least two independent experiments.

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2.3. Cadmium treatment

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Cadmium chloride (CdCl2) was prepared in distilled water at concentration of 5 ppm (5 mg/l) and 50 ppm (50 mg/l) of Cd (II) ion and was given to rats for 30 days. Control rats were given distilled water solely. Solutions of Cd and water were replaced twice a week with freshly prepared ones. All functional measurements were carried out 24 h following a 30-day period of oral intake, in tissues taken from animals anesthetized by i.p. injection of 40 mg/kg b.w. of thiopental sodium (Rotexmedica, Tritau, Germany).

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2.4. Cadmium determination

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Cadmium content in blood, lungs, intestine, mesenteric lymph nodes, spleen, kidneys and liver was determined by atomic absorption spectrometry graphite tube technique (AAS Varian 1275; graphite tube, GTA-95, Palo Alto, Ca, USA). Lyophilized tissue samples were homogenized and digested in a microwave digestion system (MBS-9, CEM Innovators, U.K.) in a mix of concentrated HCl and HNO3 (metal-free). Following filtration all dilutions were done using metal-free ultrapure water. Reference materials were used as control samples: SeronormTM Trace Elements Serum L-1 and ClinChek Plasma Control. Method limit of detection (LOD) was 0.1 mg/kg (0.00089 mmol/kg) and limit of quantification (LOQ) 0.3 mg/kg (0.00267 mmol/kg). The concentrations were expressed as mmol of Cd per kg of wet tissue or blood weight.

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2.5. Histology

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Samples of duodenum collected at necropsy were fixed in 4% formaldehyde (pH 6.9) and processed for embedding in paraffin wax for subsequent sectioning at 5 mm. Hematoxylin and eosin (H&E)-stained histology slides were subsequently analyzed by a certified histopathologist in a blinded manner using a Coolscope digital light microscope (Nikon Co., Tokyo, Japan).

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2.6. Preparation of intestinal homogenates

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Segments of duodenum (approximately 4 cm distal to the gastric pylorus) were removed, washed through the lumen with ice-cold non-pyrogenic physiological saline, cut through, weighed, snap frozen in liquid nitrogen and stored at 80  C until use. Tissue samples were homogenized by IKA T18 Basic Homogenizer (IKA Works Inc., Wilmington NC, USA) in ten volumes of sucrose buffer (10 mM Tris–HCl pH 7.6, 1 mM EDTA, 250 mM sucrose) on ice. The homogenates were sonicated and then ultra-centrifuged at 100,000  g for 1 h and 45 min, at 4  C. Supernatants were used for cytokine measurements and determination of antioxidant enzyme activity.

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2.7. Denaturing gradient gel electrophoresis (DGGE) analysis

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Genomic DNA was extracted from intestine samples by the QIAamp DNA stool mini kit (Qiagen, Hilden, Germany). PCR with isolated genomic DNA as a template was performed according to Heilig et al. (2002) using Lactobacillus-specific primer set Lab0159f (primer sequence GGA AAC AG (A/G) TGC TAA TAC CG) and Uni-0515r (primer sequence ATC GTA TTA CCG CGG CTG CTG GCA) from Metabion International, Martinsried, Germany. The PCR reaction was performed in thermal cycler Gene AmpR PCR system 2700 (Applied Biosystems, Foster City, CA). Amplification stage was

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done using KAPA Taq DNA polymerase (KAPA Biosystems, Cape Town, South Africa). Bacterial DNA was then set on denaturing gradient gel prepared according to Lukic et al. (2013) and glass plates for DGGE apparatus DGGE-2001 (C.B.S. Scientific, San Diego, CA) were used.

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2.8. Determination of superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6) activity

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Activity of SOD was determined by the epinephrine method (Misra and Fridovich, 1972). One unit of activity was defined as the amount of enzyme necessary to decrease the rate of epinephrine auto-oxidation by 50% at pH 10.2. CAT activity was determined by the rate of H2O2 decomposition measured spectrophotometrically at 240 nm as described (Beutler, 1982). One unit of CAT activity was defined as the amount of enzyme that decomposed 1 mmol H2O2 per minute at 25  C and pH 7.0. The measurements of SOD and CAT activities were accomplished using Shimadzu UV-160 spectrophotometer (Kyoto, Japan). Protein concentration was determined by Lowry assay (Lowry et al., 1951) using bovine serum albumin (Fraction V obtained from Sigma, Sigma Chemical Co., St. Louis, MO, USA) as a reference.

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2.9. Western immunoblot analysis

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The duodenum tissue was homogenized at 4  C in 1 ml ice-cold sucrose homogenization buffer containing a mixture of protease inhibitors (Protease inhibitors mix G, Serva Electrophoresis GmbH, Heidelberg, Germany). The homogenates were centrifuged at 9700  g for 20 min at 4  C and the resulting supernatants were aliquoted, snap-frozen in liquid nitrogen and stored at 80  C. For analysis, whole homogenates (40 mg) were diluted in 2  Laemmli buffer (1:1 v/v) and heated at 95  C for 5 min. For immunodetection, protein samples were separated by 12% SDS-PAGE and transferred onto PVDF membranes (Hybond-P, Amersham Pharmacia Biotech, Buckinghamshire, England). The membranes were blocked for 1 h at room temperature with 5% non-fat dry milk in blotto base buffer (0.1% Tween 20, 20 mM Tris–HCl pH 7.6, 137 mM NaCl) and then incubated for an additional 2 h at room temperature in the same buffer containing rabbit polyclonal anti-HMGB1 (Abcam, ab18256). After washing three times with blotto base buffer containing 1% non-fat dry milk, horseradish peroxidaseconjugated goat anti-rabbit IgG H&L secondary antibody (Abcam, ab6721) was applied for 1 h at room temperature. Membranes were washed extensively in blotto base buffer and antibody binding was detected on x-ray film by enhanced chemiluminescence using the WSCL detection system (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The bands were visualized and quantified with Total Lab (Phoretix) electrophoresis software. Protein concentration was determined by Lowry assay (Lowry et al., 1951).

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2.10. Mesenteric lymph node cell preparation and culture

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Mesenteric lymph nodes were weighed on precision balance (0.0001 g). Cell suspensions were prepared by mechanical teasing of tissue over nylon mesh (70 mm nylon, BD Bioscience, Bedford, USA) and cells were counted by the improved Neubauer hemocytometer. The number of viable cells, determined by using a trypan blue exclusion assay, always exceeded 95%. For proliferation measurements, MLN cells (0.3  106/well) were cultured in medium (spontaneous proliferation) for 48 h. 0.5 mCi [3H]-thymidine (GE Healthcare, UK) per well was added after 48 h of culture and incubated for additional 16 h. Incorporation of [3H]-thymidine was measured by liquid scintillation counting (1219 RackBeta, LKB Wallac, Turku, Finland) and proliferation was expressed as counts per minute (c.p.m.).

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670 nm in a 96-well ELISA plate reader. The amount of present nitrite was obtained by extrapolation from a standard curve constructed in parallel with known concentration of sodium nitrite solutions.

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For cytokine production, MLN cells (1 106/well) were cultured in 96-well plates for 48 h in medium solely (spontaneous production) and in the presence of 1 mg/ml of ConA (stimulated production).

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2.14. ELISA

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Cytokine (TNF, IL-1b, IFN-g and IL-17) concentration in intestine tissue homogenates and IL-1b, IFN-g, IL-10 and IL17 in media conditioned by MLN cells were evaluated using enzyme-linked immunosorbent assays (ELISA) for rat TNF, rat IFNg and for mouse IL-17 cross-reactive with rat IL-17 (eBioscience Inc., San Diego, CA, USA), rat IL-1b and rat IL-10 (R&D systems, Minneapolis, USA) according to the manufacturer's instructions. Cytokine titers were calculated by a reference to standard curve constructed using known amounts of respective set-provided recombinant cytokines.

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2.15. Reverse transcription—real time polymerase chain reaction (RTPCR)

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2.12. Reduced glutathione (GSH) measurements

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GSH content was estimated in MLN cell lysates (1 106 freshly isolated cells, lysed in 10 mM HCl), following protein precipitation by addition of 5% sulphosalicylic acid. The supernatant was used to quantify GSH level using DTNB in Tris–HCl (pH 8.9) and reduced glutathione as standard (Anderson, 1986) spectrophotometrically (Shimadzu Corporation, Lakewood, USA) at 412 nm. The levels of GSH were expressed as mmol of GSH/106 cells.

Total RNA was isolated from MLN cells using mi-Total RNA Isolation Kit (Metabion, Martinsried, Germany) according to the manufacturer’s instructions. Reverse transcription of isolated RNA was conducted using random hexamer primers and MMLV (Moloney Murine Leukemia Virus) reverse transcriptase following the manufacturer’s instructions (Fermentas, Vilnius, Lithuania). Amplification of prepared cDNA was done in a total volume of 20 ml using Power SYBR1 Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems). Thermocycler conditions were: an initial step at 50  C for 5 min, followed by a step at 95  C for 10 min and subsequent 2-step PCR program at 95  C for 15 s and 60  C for 60 s for 40 cycles. PCR primers (forward/reverse) used in the study were: IFN-g: 50 -AACAGTAAAGCAAAAAAGGATGCA-30 /50 -TGTGCTGG ATCTGTGGGTTGT-30 ; IL-17: 50 -CTACCTCAACCGTTCCACTTCAC-30 / 50 -CCTCCCAGATCACAGAAGGATATC-30 ; IL-1b: 50 -CACCTCTCAAGCAGAGCA-30 /50 -GGGTTCCATGGTGAAGTCAAC-30 ; IL-10: 50 -GAAGACCCTCTGGATACAGCTGC-30 /5 0 -TGCTCCACTGCCTTGCTTTT-3 0 ; Metallothionein (MT) -1: 50 -GAACTGCAAATGCACCTCCTGC 30 /50 CAAGACTCTGAGTTGGTCCG-30 ; Metallothionein (MT) -2: 50 -TGCA AGAAAAGCTGCTGTTCC-30 /50 -TTACACCATTGTGAGGACGCC-30 ; bactin (housekeeping reporter gene): 50 -CCCTGGCTCCTAGCACCAT-30 /50 -GAGCCACCAATCCACACAGA-30 PCR products were detected in real-time and results were analyzed with 7500 System Software (Applied Biosystems) and calculated as 2dCt where dCt was the difference between Ct values of specific gene and the endogenous control (b-actin). Data for mRNA from cells of cadmium treated animals were expressed as relative value to mRNA from control animals.

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Aliquots of 1 106 of MLN cells were incubated on ice for 30 min with FITC- conjugated antibodies to rat CD4 and HIS48, and PE labeled antibodies for rat CD8 and mouse anti-rat NKG2D. After washing, the cells were fixed with 1% paraformaldehyde (Serva, Heidelberg, Germany). Expression of CD163 molecules was measured on MLN cells (1 106) incubated with mouse anti-rat CD163 for 30 min, following staining with FITC-conjugated F(ab0 )2 goat anti-mouse IgG for further 30 min. For detection of CD68+ cells, MLN cells (1 106) were fixed in 1% paraformaldehyde in PBS, permeabilized for 15 min with PBS containing 0.2% Tween-20 (Sigma Chemical Co., St. Louis, MO, USA) and then incubated for 30 min with mouse anti-rat CD68-Alexa Fluor 488. Following staining, the cells were washed twice with PBS, fixed with 1% paraformaldehyde and kept in the dark at 4  C until analysis. For detection of apoptosis, freshly isolated cells (2  106) were incubated with Annexin V and Propidium iodide according to manufacturer’s instructions. Fluorescence intensity was assayed in the CyFlow Space system (Partec). A minimum of 10,000 events/ sample was acquired each time and analyzed using FlowMax software (Partec, Munich, Germany).

2.16. Data display and statistical analysis

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Results were expressed as median values and range (min-max). Statistical analysis was performed using GraphPad Prism (version 6; GraphPad Software, San Diego, USA). Statistical significance was defined by Kruskal–Wallis test with a Dunn’s multiple comparison post hoc test. A P value 10 mg/ kg in rats (Goon and Klaassen, 1989) which can explain higher MLN cadmium load at 50 ppm (compared to 5 ppm). Increase in cellular GSH and in mRNA for both forms of metallothionein in MLN cells reflects stress response to cadmium. Elevated concentrations of GSH noted in the present study are in line with data that showed that chronic exposure of rats to cadmium resulted in increased levels of GSH in liver and kidneys (Mendieta-Wejebe et al., 2013; Shaikh et al., 1999) and might possibly be related to protective effects shown for GSH (Rana and Verma, 1996) or its precursor Nacetyl cysteine (Kaplan et al., 2008; Wang et al., 2009). Increases in metallothionein isoforms might be related to tissue retention of cadmium (Klaassen et al., 2009) and to the role of this small protein as efficient intracellular scavenger involved in intracellular detoxification via binding to cadmium (Nordberg and Nordberg, 2000). Elevated levels of GSH and MT might bear relevance not solely as markers of cadmium-induced stress but as regulators of immune cell responses as postulated in lung inflammation and inflammatory bowel diseases (Rahman and MacNee, 2000; Waeytens et al., 2009). Increases in MLN mass observed in the present study are in contrast to the early data that showed lack of the effect of cadmium on MLN (Yamada et al., 1981). Acute regimen of administration (single intraperitoneal dose) employed in that study might be responsible for this difference. MLN cell proliferation might have accounted for increased lymph node cellularity, though cell accumulation might be responsible as well. Lack of changes in the ratio of CD4+ to CD8+ cells implies involvement of both populations in rise in MLN cellularity. Differential effects on proinflamatory (IFN-g and IL-17) vs. antiinflammatory (IL-10) cytokine production and expression depict up-regulation of inflammatory/immune activity in this lymphoid compartment. As a comparison, nearly the opposite effect was

Table 4 Basic aspects of innate immune activities of mesenteric lymph node cells. Parameter

Cadmium dose (ppm) 0

5

50

DHR positive cells (%) MPO (U/106 cells)

0.5 (0.4–0.5) 2.9 (1.5–4.0)

0.8* (0.6–1.1) 3.1 (2.1–4.4)

0.9* (0.8–1.6) 3.8* (3.3–6.0)

NO production (mM) Spontaneous LPS-stimulated

2.5 (2.2–2.9) 4.0 (2.7–5.5)

2.2 (2.0–2.3) 3.9 (3.1–4.5)

2.6 (2.3–2.9) 5.5* (4.3–6.1)

IL-1b production (pg/ml) Spontaneous IL-1b mRNA expression (relative values)

327.0 (147.0–457.0) 1.0 (0.7–1.4)

552.0* (527.0–577.0) 1.8*** (1.6–3.0)

677.0* (487.0–867.0) 2.3* (0.7–4.5)

Results are presented as median and range (min–max) from three independent experiments with five to six animals per group per experiment. Significantly different at: P < 0.05, and ***P < 0.001 vs. control animals (Cd dose 0 ppm).

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observed in spleen cells of animals used in the present study (a decrease in cell proliferation, lack of changes in IFN-g production and decrease in IL-17 production) in line with our recently published data (Demenesku et al., 2014) that underscored the significance of MLN proinflammatory cytokine commitment. The significance of IFN-g and IL-17 was corroborated further by the present study initial observations that showed elevated levels of mRNA for subunits of IL-12 and IL-23, cytokines needed for differentiation of IFN-g-producing and IL-17-producing cells, respectively. Namely, increases of relative values (expressed as change vs. control ratio) of p. 35, subunit of IL-12 (median 3.44, range 2.64–4.23, and median 6.39, range 1.26–9.19, at 5 ppm and 50 ppm, respectively, compared to median 0.97, range 0.54–1.47 in controls, P < 0.05), of p. 19 subunit of IL-23 (median 2.64, range 0.79–4.77, and median 2.74, range 0.76–8.33, at lower and higher cadmium doses, respectively, compared to median 0.85, range 0.19–2.31 in controls, P < 0.05) and in their common subunit (p. 40) at higher cadmium dose (median 4.29, range 2.15–10.80 vs. median 0.89, range 0.55–1.98 in controls, P < 0.05) were observed. Microenvironment of MLN in basal state is normally tolerogenic and establishes firewall between this lymphoid compartment and the rest of immune system (MacPherson and Smith, 2006). Increases of production of IFN-g and IL-17 (and IL-1b) along with lack of changes/decrease of IL-10, imply the shift from immune tolerogenic towards proinflammatory milieu. In analogy with the data showing that induction of effector (IFN-g- and IL-17producing) T cells in Crohn’s disease (CD) occurs in MLN (Sakuraba et al., 2009), this lymphoid tissue might be envisaged as site for generation of effector cells that subsequently infiltrated gut mucosa in rats that consumed cadmium. Damage to intestinal epithelium and necrosis might have accounted for stimulation of MLN proinflammatory cytokine responses. Cell necrosis is so-called immunogenic cell death, characterized by release of dying cell products which function as damage/danger associated molecular patterns (DAMPs) that prime inflammatory and T cell immune responses against dying cells (Green et al., 2009; Rock and Kono, 2008). HMGB1 is one of the best studied markers of immunogenic cell death (Green et al., 2009) and molecules that gained MLN by afferent lymph might have primed cytokine responses. Increased activity of innate immunity cells depicts further proinflammatory character of MLN environment. Detection of these activities in MLN indicates that both adaptive and innate immune responses are induced, which is in line with data that showed that both types of responses complemented each other to maintain gut immune homeostasis (Slack et al., 2009). Accumulation of CD68+ cells might have resulted from the need of these cells for development of inflammatory responses. Increased numbers of cells with this marker were noted in MLN of patients with malignant lymphoma (Bjerke et al., 1993) and in perivascular inflamed mucosa tissue specimens from patients with inflammatory bowel disease (Rutgveit et al., 1994). In addition, investigations in mice showed Th1 inducing potential of these cells (Mills et al., 2000). Increases in MLN cell oxidative activity (DHR oxidation, NO production) might rely on CD68+ cells, as these are major carriers of mentioned activities (Mills et al., 2000). Slight, but significant, elevation of NK cell numbers might have aided establishment of protective immune responses in MLN (MartinFontecha et al., 2004) as these cells are part of innate immune responses to antigens from necrotic cells (Green et al., 2009) and microbial antigens (Bancroft, 1993). Lack of changes in granulocytes is in contrast to data that showed accumulation of these cells in intestine following oral cadmium administration (Zhao et al., 2006). Granulocytes are cells of acute inflammatory responses (Sherwood and Toliver-Kinsky, 2004) and enrichment of intestine in these cells might reflect the

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response to acute regime (single dose) of cadmium administration in that study. Although no quantitative changes in granulocytes were observed in the present study, their activity might be enhanced and elevation in MPO activity in MLN cells from rats that consumed higher cadmium dose supports such considerations. Increases of reactive oxygen species producing (DHR+) cells and of cells that have increased intracellular MPO activity as well as increased enhanced responsiveness of NO production to LPS stimulation, most probably resulted from stimulation by products of necrotic tissue acquired from injured intestine and stimulation with antigens derived from microbial flora that gained access through injured gut epithelial barrier and trafficked to MLN where they can prime immune (cytokine) responses (Mowat, 2003).

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5. Conclusions

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Oral intake of cadmium by rats resulted in intestinal damage and inflammation. Immune priming of major gut-associated lymph nodes, mesenteric lymph nodes, was observed as well, which is a novel finding. Stimulation of immune activities of MLN cells have, most probably, resulted from sensing of potentially dangerous cadmium-induced intestinal damage, but from microbial antigens as well. Primed MLN cells might help protection of vulnerable intestine from bacterial overgrowth, but can contribute to perturbation of local immune homeostasis as well. These effects of cadmium should be taken into account when assessing dietary cadmium as health risk factor.

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Conflict of interest

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The authors declare that they have no conflict of interest.

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Acknowledgement

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This study was supported by the Ministry of Education, Science Q7 and Technological Development of the Republic of Serbia, Grant #173039.

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References

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