LPS and cytokines

Intraperitoneal injection of 5 µg LPS/kg BW in pigs resulted in increasing levels of plasma cytokines, including TNF-α and IL-6 at 2 h after injection with elevated levels of plasma urea nitrogen (PUN) at 8 and 12 h after LPS injection (WEBEL et al. 1997). In another study it was shown that intravenous injection of different doses of LPS (up to 2 mg/kg BW) in pigs induced apoptosis in the lymphocytes and this was accompanied by elevation in serum TNF-α levels, mostly at 1 h post-injection in a dose dependant manner, and this increase remained for 6 h post-injection in pigs treated with 2 mg LPS/kg BW (NORIMATSU et al. 1995). Similarly, plasma TNF- α level was increased at 2 and 4 h after intraperitoneal injection of 100 µg/kg BW of growing pigs (WRIGHT et al. 2000). In vitro studies revealed that 100 ng LPS/ml induced TNF-α, IL-6, IFN-γ and IFN-β mRNA at 2 and 7h (CHUNG et al. 2003). In vitro studies in human revealed that LPS resulted in increasing levels of TNF- α, IL-1β and IL-6 in septic patients (CASEY et al. 1993). The infection of T84 cells with invasive gram-negative bacteria (Salmonella dublin) resulted in increased levels of IL-8 mRNA (JUNG et al. 1995).

LPS was found to be able to stimulate p38 in murine macrophage cell line, reached peak activity after 20 min and maintained its effect up to 4h (BROOK et al. 2000). Primed mice with LPS resulted in production of serum TNF-α and IFN-γ in a dose-dependent manner (KIENER

et al. 1988).

2.4. The interaction effect between DON and LPS

A synergistic effect between DON and LPS was found in induction of apoptosis in lymphoid tissues of mice (ZHOU et al. 2000). In vitro DON induced higher proinflammatory cytokine expression in LPS primed cells (PESTKA and ZHOU 2006) than in case of DON or LPS alone.

Mice primed intraperitoneally with 1 mg LPS/kg BW and then treated 8h later with oral DON at concentrations from 0.5 to 25 mg/kg BW showed increasing levels of the pro-inflammatory

cytokines IL-1β, IL-6 and TNF-α after 2 hours (ISLAM and PESTKA 2006) with reduction in the dose of DON required to induce plasma IL-1β, IL-6 levels from 2 mg/kg BW to 0.5 mg DON/kg BW, and that required for induction of TNF-α from 12.5 to 2 mg DON/kg BW, suggesting that LPS reduced the minimum dose required of DON to induce cytokine response and reflecting that LPS increased the sensitivity to DON and prolonged the cytokine response.

Co-exposure to intraperitoneal LPS at concentrations of 1 and 5 mg/kg BW with oral DON at 5 or 25 mg/kg BW in mice elevated gene expression of TNF-α significantly than administration of LPS or DON alone (ZHOU et al., 1999).

In murine macrophage cell line, DON at different concentrations was able to induce TNF-α mRNA at 2 h and much less at 7 h in a dose-dependent manner (CHUNG et al., 2003), while under application of DON with LPS, TNF-α mRNA levels remained elevated at 2- and 7-h time points.

DON was found to increase the bacterial translocation as a result of its effect on the integrity of enterocytes (KOLF-CLAUW et al. 2009; PINTON et al. 2009). A synergistic effect was found between DON and LPS in induction of pro-inflammatory cytokines TNF-α and IL-1β in porcine alveolar macrophages (DÖLL et al. 2009a). Comparing to LPS alone, robust responses of IL-1β, IL-6 and TNF-α were obtained in murine macrophages that primed with LPS for 4-16h then treated with 250 ng DON/ml for 2h (PESTKA and ZHOU 2006). DON was able to reduce the viability of the cells (Kupffer cells) significantly at concentration of 4000 nM in the presence of 1 µ g LPS/ml at 48h incubation (DÖLL et al. 2009b).

3. Material and Methods

3.1 Animals

A total of 22 pigs (Deutsches Edelschwein) were used in the present study. The animals were 2-3 months old. For the first series 6 pigs were used and in the second series 16 pigs were used. The animals were obtained from The Institute of Animal Nutrition, Friedrich Loeffler Institute, and were housed in the stables of the Department of Physiology, University of Veterinary Medicine (Hannover) for about one week. Feed and water were offered for ad libitum intake (For the composition of the diet see Table 5). The animals in the second series were used to estimate the effect of DON on weight gain. The mean body weight for control group (8 pigs) was 15.6 ± 1.2 kg and for DON-fed group (8 pigs) was 16.4 ± 2.9 kg at the start of the experimental period.

3.2 Experimental design

In order to assess the effects of DON on the electrophysiological parameters in the small intestine of the pigs in vitro, duodenum, mid jejunum and ileum were examined with two different intestinal tissue segments per pig in the first series. In the second series segments of mid jejunum were examined. The animals were divided into two groups, control group (8 pigs) and DON-fed group (8 pigs). The mean body weights for the animals in both groups were 15.6 ± 1.2 and 16.4 ± 2.9 kg, respectively, at the start of the experimental period. The animals were further subdivided into four groups, the first group including control animals without previous treatment, the second as the LPS group in which animals were injected intraperitoneally with LPS 3 hours before slaughtering, the third group was DON-fed group in which pigs fed on DON-containing diet for 2 weeks at concentration of 2.901 mg DON/kg feed and the last group was DON/LPS including pigs fed on DON-contaminated diet and injected intraperitonealy with LPS. Experimental design in details is shown in table 6.

Table 5: Compositions of the experimental diet

pantothenic acid, 7.5 mg; choline chloride, 125 mg; biotin, 50 µg; folic acid, 0.5; vitamin C, 50 mg.

Table 6: Experimental design of the whole study

Series DON (mg/kg diet)

LPS (µg/kg BW)

In vitro DON

(ng/ml) Tissue

First series

1^st Experiment 0 0 0, 5, 10, 20 Duodenum, mid

jejunum, ileum

2^nd Experiment 0 0 38, 77, 154, 308 Duodenum, mid

jejunum, ileum Second series

Control 0 0 0, 4000, 8000 Mid jejunum

LPS 0 5 (ip) 0, 4000, 8000 Mid jejunum

DON 2.901 0 0, 4000, 8000 Mid jejunum

DON/LPS 2.901 5 (ip) 0, 4000, 8000 Mid jejunum

3.3 Electrophysiological measurements

3.3.1 Chemicals and solutions for the electrophysiological experiments

DON was purchased from (Sigma-Aldrich D-0156, München, Germany) diluted in isotonic saline. LPS was used from E. coli serotype 0111:B4 (Sigma-Aldrich L-2630, München, Germany). The buffer solutions (Modified Krebs-Henseleit solution) that bathed the mucosal and serosal surfaces of the epithelial tissues are shown in table 6. All solutions were prepared with Indomethacin (10^-5M) to reduce prostaglandin synthesis and were adjusted to an osmolality of 300 mosmol/kg with mannitol to maintain the same osmotic pressure in both sides of the chambers. The PH of the buffer was adjusted to 7.45 using HCl. The buffers were continuously gassed with carbogen (95% O2, 5% CO2) at 38.4°C all over the experiment.

Glucose was added to the serosal buffer to provide an energy substrate and achieve active transport conditions.

Table 7: Compositions of Krebs-Henseleit buffer solution for mucosal and serosal sides

Mucosal buffer (mmol/L)

Serosal buffer (mmol/L)

NaCl 113.6 113.6

KCl 5.4 5.4

HCl 0.25 0.4

MgCl2.6 H2O 1.2 1.2

CaCl₂.2 H₂O 1.2 1.2

NaHCO₃ 21.0 21.0

Na2HPO4.2 H2O 1.2 1.2 NaH2PO4.H2O 0.3 0.3

mannitol 31.96 23

glucose - 10

3.3.2 Tissue sampling and preparation

The animals were slaughtered by stunning, bleeding for about 2 min during which blood samples were collected from each pig in two Lithium-Heparin tubes. The gastrointestinal tract was removed within 5 min after bleeding. In the first experimental series intestinal segments of about 80 cm length were immediately taken from the duodenum, mid jejunum and ileum.

Duodenum was about 20 cm after pylorus, mid jejunum was 3 meters after pylorus and ileum was one meter proximal to ileo-caecal valve. In the second series segments from the mid jejunum were taken (the fourth meter after pylorus). The segments were immediately rinsed with ice-cold saline (0.9% NaCl) and kept in a modified glucose-containing Krebs-Henseleit buffer solution at 4°C, being continuously gassed with carbogen (95% O2, 5% CO2) until mounting in Ussing chambers. After longitudinal incision along the mesenteric border, the intestinal segments were washed free of any remaining intestinal contents and the muscle and serosal layers were stripped off by scraping the serosal surface with the edge of a glass slide before mounting the mucosal layer into Ussing chambers keeping the tissues wet by applying ice-cold buffer.

The blood samples were centrifuged for 10 minutes at 3600 rpm and 4°C and plasma was separated in four Eppendorf tubes (2 ml each) for each pig and stored at -20°C until analysis.

3.3.3 Ussing Chamber Technique

The aim of this study was to estimate the effects of DON on the electrophysiological parameters across the porcine small intestines in vitro. The electrogenic transport of glucose and alanine were assessed and used as indicators for such transport processes. These nutrients are co-transported with sodium ions across the mucosal surface of the epithelial cells via specific transporters. The tissue conductance (Gt) represents the reciprocal of the electrical tissue resistance and considered as a useful measure of the integrity of the intestinal barrier including the transcellular and paracellular pathways. The physiological approach that was used to investigate the previous parameters was Ussing chamber technique. It is a valuable method used for measuring nutrient transport across epithelial tissues (CLARKE 2009). The chambers are made of two acrylic glass fragments and were connected to glass circulation reservoirs through silicon tubes (Figure 10). The glass reservoirs were filled with 12.5 ml of modified Krebs-Henseleit buffer solution at both sides of the tissues and were water jacketed to enable warming of the intestinal preparation at 38.4°C and gassed permanently with carbogen (95% O₂, 5%CO₂) to maintain continuous circulation and the pH at 7.45. The electrophysiological experiments were carried out by computer controlled voltage/current clamps (scientific instrument, Dipl.-Ing. K. Mußler, Aachen, www.Kmsci.de). The clamps were connected to the chambers through 3M KCl-containing agar bridges and Ag/AgCl electrodes (Mettler Toledo Prozessanalytik GmbH, Germany) through which the short circuit currents were recorded. Fluid resistance and potentials were measured before mounting the tissue segments and corrected for during the experiments. To apply the standard experiment, the electrical and chemical gradients must be abolished. This was achieved by operating the chambers with the buffers in the absence of the intestinal preparations. During this period the bubbles were removed and the buffer resistance was compensated. The adaptation time for the buffers was about 10 min after which the buffers reached the desired temperature 38.4°C.

Clamping the voltage to 0 mV eliminate the electrical gradient.

For determination of electrophysiological parameters, the tissue preparations were mounted in Ussing chambers with the mucosal surface upward and with an exposed surface area of 1.13

cm² with silicon rings and nets to prevent tissue damage and bulging. The tissues were allowed to adapt and recover under open circuit conditions for 10-20 minutes and were then clamped to 0 mV in order to eliminate the electrical gradients. Identical buffer solutions were used on both sides in order to abolish the chemical gradient. Thus the tissues were incubated in the absence of the transepithelial electrochemical gradient.

Figure 10: A schematic diagram of Ussing chamber

After an equilibration period of about 30 minutes, the basal values for potential differences, short circuit currents (Isc) and electrical tissue conductances (Gt) were recorded automatically. DON was added to the mucosal side of the Ussing chambers at the previously mentioned concentrations (Table 6) to reach a final volume of 13 ml in the glass reservoirs.

An equal volume of modified glucose-containing Krebs-Henseleit buffer was added to the serosal side. In order to assess the effects of DON and LPS on alanine and glucose transport, alanine (10 mM) was added to the mucosal side of the tissues in each Ussing chamber. Ten min later glucose (10 mM) was added to the luminal side. The experiment was ended 20

minutes after addition of glucose. The Isc and Gt values for alanine and glucose were recorded automatically and the responses of Isc and Gt after addition of alanine and glucose were calculated as differences between the constant values before nutrient addition and the maximal response (∆ Isc and ∆ Gt).

3.3.4 Calculations and Statistics

The differences between the basal values before addition of the nutrient and the maximal response were calculated using Microsoft Office Excel 2003 and represent ∆ Isc and ∆ Gt.

The previous data are analysed using 2- and 3-factorial analysis of variance (ANOVA) and presented as means ± standard deviation (SD) whereas n represents the number of pigs and p-values < 0.05 were considered to be significant.

3.4 DON transport study

The aim of this part of the experiment was to evaluate the mucosal uptake and serosal release of DON across porcine jejunal epithelia in vitro.

3.4.1 Sampling

In the second series buffer samples (1.5 ml each) were collected from both sides of the tissues in Eppendorf tubes. Time of sampling was at 7.5, 15, 30 and 90 min after addition of DON.

Only samples at 30 min and at 90 min were analysed to obtain about one hour incubation period without sampling in between. The samples were analysed by LC-ESI-MS/MS and the limit of detection was 0.2 ng/ml. The compositions of the buffer are in table 7.

3.4.2 Determination of DON in the buffer samples of Ussing-chambers with LC-MS-MS with purification over ChemElut cartridges

- Material:

- LC-ESI-MS/MS (API 4000 Qtrap, Applied Biosystems, Darmstadt, Germany, coupled with a 1200 series HPLC system, Aligent Technologies, Böblingen, Germany)

- ChemElut cartridge 1ml (Varian, Nr. 12198002, 100 Stk. 204 €) - Ethyl acetate (Roth Nr.73361, HPLC-grade)

- Water (LC/MS – Quality)

- Acetonitrile (LC-MS-Quality) - Ionized Water

- Reaction vials (1.5ml) - 10 ml conical flask - Rotatory evaporator

- PVDF-Membran filter (0.45 µm, 4 mm, Amchro)

- All flasks were rinsed with methanol (HPLC-Quality) and were left to dry overnight.

- Method:

- An aliquot of 500µl of the sample were diluted with 500 µl ionized water and mixed.

- The total volume of the diluted sample was transferred to a ChemElut cartridge and was left for 5 min to be absorbed and distributed over a solid support in the cartridge.

- The sample was extracted with 10 ml of Ethyl acetate. The collected extract was evaporated at 40°C and 300 mbr just to dryness.

- The residue was re-dissolved in 200 µl acetonitrile/water mixture (13/87, v/v) in an ultrasonic bath for 5 min.

- The sample was filtered through PVDF membrane filter (0.45 µm, 4 mm, Amchro) and was kept frozen until LC-MS-MS measurements.

3.4.3 Analysis of buffer samples using LC/MSMS technique - LC-MS/MS-conditions: QTrap 4000 ABSciex

- Mode: ESI negative

- Acquisition Method: DON+de-DON_190508_delay.dam - HPLC-conditions: Agilent Technologie

- Column: BETASIL Phenyl-Hexyl, 3 µm, 100 x 2,1 mm (Thermo) - Guard column: Phenyl, 4x2 mm (Phenomenex, Nr. AJO-4350), with

guard column holder of Phenomenex - Eluent A: 0.13 mM NH₄ Acetat, pH 7,4

- Eluent B: Acetonitrile

- Gradient: 0 – 2.0 min: 4% B; 2.0 – 5.0 min: 4-95% B; 5.0 – 7.0 min: 95% B; 7.0 – 7.5 min: 95-4% B; 7.5 – 12.0 min: 4%

B

- Flow rate: 400 µl/min - Injection volume: 10 µl - Column oven temperature: 20°C

- tR DON/De-epoxy DON : ca. 4.4/4.6 min 3.4.4 Determination of plasma DON concentrations

DON was analyzed in porcine plasma by HPLC-DAD after clean-up with immunoaffinity columns (IAC) according to (VALENTA et al. 2003) with slight modifications.

- Material and reagents:

- Sodium acetate buffer (PH 5.5) - β-glucuronidase (Sigma, G 0876)

- ChemElut cartridge (Varian, Middelburg, Netherlands) - IAC (DONtest HPLC^®, VICAM, Watertown, MA, USA) - Method:

- One ml of sodium acetate buffer (PH 5.5) was added to the plasma sample (1.5 ml) - The previous mixture was incubated for 16 h with β-glucuronidase

- Then the sample was extracted with ethyl acetate on a ChemElut cartridge and cleaned-up by IAC.

- The samples were measured by HPLC-DAD.

- The mean recovery of DON was 91% ± 8% (n=12; 10-30 ng/ml). The results were not corrected for recovery.

3.4.5 Determination of plasma TNF-α concentration

TNF-α was determined using ELISA kit (R&D Systems, Minneapolis, USA) based on porcine specific matched pairs of antibodies in combination with recombinant standards.

All reagents and samples were prepared at room temperature according to manufacture instructions.

1- Preparation of the reagents

a- Control was dissolved in 1 ml of ionized H2O

b- Wash buffer (25 ml) was mixed with 600 ml of ionized water.

c- Substrate solution was prepared 15 min before use in dark place; color reagents A and B were mixed in equal parts.

2- Standard series

a- Standard was dissolved in 2 ml Calibrator diluent RD6-33 and was left for 5 min.

b- Six Polypropylene tubes were used.

c- 200 µl of Calibrator diluent were added in each tube to make a standard series d- A Blank tube was filled with Calibrator diluent.

3- 50 µl of Assay Diluent RD1-63 were added to the plate.

4- 50 µl Standards (in duplicate), control (in duplicate), Blank (in duplicate) and samples (single determination) + Blank

5- Shaking for 1 min.

6- The plate was sealed with foil and was incubated for 2h at room temperature.

7- The plate was washed 5 times with 400 µl wash buffer.

8- 100 µl Conjugate was added.

9- The plate was sealed with foil and was incubated for 2h at room temperature 10- The plate was washed 5 times with 400 µl wash buffer.

11- 100 µl Substrate Solution were added (15 min before start).

12- The plate was incubated for 30 min in dark place.

13- 100 µl stop solution were added.

14- The plate was measured (within 30 min) at 450 nm, 540 nm, 570 nm.

3.4.5 Calculations and statistics

The measured DON concentrations (ng/ml) were converted to amounts (ng) by multiplying the initial and final DON concentrations with the respective buffer volumes. The differences between the beginning and the end was calculated and presented as ∆ amount of DON [µg/h]

at both, the mucosal and the serosal side, thus providing data on mucosal uptake and serosal release. Those data were analysed using 2-factorial ANOVA analysis (DON in feed and ip LPS) including their interactions. One-factorial ANOVA analysis was applied to evaluate the effect of the group on both mucosal and serosal amounts of DON. All results were presented as means ± standard deviation (SD) whereas n represents the number of pigs and p-values <

0.05 were considered to be significant.

4. Chapter 1

Effects of deoxynivalenol and lipopolysaccharide on electrophysiological parameters in growing pigs

4. Chapter 1

Effects of deoxynivalenol and lipopolysaccharide on electrophysiological parameters in growing pigs

Amal Halawaª, Sven Dänicke^b, Susanne Kersten^b, Gerhard Brevesª

a Physiological Institute, University of Veterinary Medicine, Hannover, Germany

b Institute of Animal Nutrition, Friedrich Loeffler Institute (FLI), Federal Research Institute for Animal Health, Braunschweig, Germany

Submitted to Mycotoxin Research, at 06.05.2012, undergoing review

Corresponding author: Sven Dänicke, Institute of Animal Nutrition, Friedrich-Loeffler-Institute (FLI), Federal Research Friedrich-Loeffler-Institute for Animal Health, Bundesallee 50, D-38116 Braunschweig, Fax: + 49 531-596 3199, E-mail: sven.daenicke@fli.bund.de

4.1 Abstract

Deoxynivalenol (DON) is a major B-trichothecene that gains importance from its natural occurrence in cereals worldwide. It has many effects on rapidly dividing cells relying on intensive protein synthesis for growth and proliferation. Lipopolysaccharide (LPS) is an endotoxin released from most Gram-negative bacteria which plays a major role in induction of inflammation and sepsis under certain conditions. From our experiments we aimed at studying the effects of different concentrations of DON on electrogenic transport of nutrients and on tissue conductances using the Ussing chamber technique. The effect of DON contaminated feed on the respective parameters as well as the interactions between DON and LPS were assessed using porcine jejunal tissues. In vitro DON inhibited the absorption of alanine and glucose across pig jejunum at concentrations of 4000 and 8000 ng/ml, suggesting that DON had an inhibitory effect on the electrogenic transport of nutrients across porcine small intestines. Electrogenic transport of alanine and glucose across porcine small intestine varied regionally among intestinal segments with higher response in ileal tissues. A synergistic effect was observed between DON in feed and injected LPS on tissue conductances in response to glucose with higher short circuit currents across porcine jejunal mucosa were observed in nutrient stimulated conditions.

Keywords: deoxynivalenol; lipopolysacchaide; Ussing chamber; electrogenic transport.

4.2 Introduction

Deoxynivalenol (DON) is a secondary metabolite produced by different Fusarium species, such as Fusarium graminearum and Fusarium culmorum (BAKAN et al. 2002; BOTTALICO

1998; VISCONTI and BOTTALICO 1983), which grow on cereals such as corn, wheat, barley and maize (ABBAS et al. 1985; EFSA 2004; FDA 2010; MEGALLA et al. 1986; RICHARD 2007).

DON is known to affect both, the gastrointestinal tract and immune system (ROTTER et al.

1996) in which cells are mainly rapidly dividing such as lymphocytes, fibroblasts and epithelial cells (DÖLL et al. 2009b; ERIKSEN 2003; ROCHA et al. 2005) and depending in their proliferation on protein synthesis. DON is one of the most potent trichothecenes that has emetic activity (COPPOCK and JACOBSEN 2009). Swine are the most susceptible species to DON (ROTTER et al. 1996). Acute toxicity of DON, especially in pigs, is characterised by abdominal distress, vomiting and diarrhea (EFSA 2004;FORSYTH et al. 1977; WILLIAMS et al.

1988; YOUNG et al. 1983). Chronic toxicity of DON is characterised by anorexia, reduced weight gain, feed refusal and modulated immune function (ROTTER et al. 1996). DON is rapidly absorbed from the gastrointestinal tract, through paracellular or transcellular routes (AWAD et al. 2007a; SERGENT et al. 2006; VIDEMANN et al. 2007). The major absorption sites are the proximal parts of the small intestine of pigs after oral exposure (DÄNICKE et al.

2004a). DON can be detected in pig plasma within 30 min after both, oral and intragastric

2004a). DON can be detected in pig plasma within 30 min after both, oral and intragastric

Im Dokument Toxicological and immunological effects of DON and LPS at the intestinal barrier (Seite 41-0)