2.3 Lipopolysaccharide
2.3.2 Characters and functions of LPS
LPS, as a component of the outer bacterial membrane, acts as a stimulator of the innate immunity of the host but in high doses it can induce septic shock (ALEXANDER and RIETSCHEL 2001). LPS stimulates the immune cells to produce specific mediators in response to its systemic effect like reduced oxygen species, bioactive lipids and proteins such as interleukins (SCHLETTER et al. 1995). The bacterial membrane plays an important role in nutrient transport and mediates the interaction between the bacteria and the host organism (RIETSCHEL et al. 1994). Also LPS acts as a permeability barrier against different external factors like antibiotics (ALEXANDER and RIETSCHEL 2001). LPS is a pyrogenic molecule which may cause increase in the host temperature approximately from 1 to 1.5 °C (TAYLOR et al. 1991; NORIMATSU et al. 1995; WRIGHT et al. 2000; GARCIA-HERRERA et al. 2004; ABAD et al. 2002; JOHNSON and BORELL 1994). LPS is able to form extremely stable aggregates in aqueous environment (GUTSMANN et al. 2007).
2.3.3 The chemical structure of LPS
The lipopolysaccharide consists of a lipophilic hydrophobic region called lipid A part through which LPS is connected to the bacterial cell membrane (KIRSCHNING and BAUER 2001;
ULEVITCH and TOBIAS 1995) and a hydrophilic region called polysaccharide side chain (HITCHCOCK et al. 1986; TOMLINSON and BLIKSLAGER 2004; HODGSON 2006; SCHLETTER et al. 1995; RIETSCHEL et al. 1994). The polysaccharide side chain consists of a core oligosaccharide and the terminal O-specific chain antigen (Figure 6).
Figure 6: The general chemical structure of LPS of Enterobacteria (ALEXANDER and
RIETSCHEL 2001). Abbreviations: GlcN, glucosamine; Kdo, 2-keto-3-deoxyoctonic acid; Hep, D-glycero-D-manno-heptose.
2.3.3.1 Lipid A part
Lipid A region is the lipid component of LPS molecule that is responsible for the biological activity of LPS (RAETZ et al. 2006). It consists of a hydrophilic carbohydrate region and a hydrophobic fatty acid region (KABANOV and PROKHORENKO 2010). The hydrophobic membrane-anchoring region makes LPS forms aggregates in aqueous environment without biological activity (HODGSON 2006). The most active form of lipid A consists of two phosphorylated N-acetylglucosamine (NAG) molecules at positions 1´ and 4´ (Figure 7) (HODGSON 2006; RICHARD 1999); the molecules are connected via a β 1-6 linkage. Lipid A molecule carries about 6 β-hydroxy fatty acids (FAs) as that found in the pathogenic bacteria like Escherichia coli and Salmonella species and attached on carbon 2 via amide linkage and on carbon 3 via ester linkage (RIETSCHEL et al. 1994; HODGSON 2006). All the fatty acids in Lipid A are saturated. Some FAs are attached directly to the NAG dimer and others are esterified to the 3-hydroxy fatty acids that are characteristically present. The structure of Lipid A is highly conserved among Gram-negative bacteria, and is responsible for peptide mediator induction (RIETSCHEL et al. 1994) or the endotoxic activity of Gram-negative bacteria (GALANOS et al. 1985) either the synthetic or the natural lipid A portion.
Figure 7: General chemical structure of lipid A. R1 to R4 represent β-OH fatty acids (HODGSON 2006)
2.3.3.2 The oligosaccharide core
It is a hetero-oligosaccharide portion and in turn subdivided into an outer core and an inner core (RIETSCHEL et al. 1994; MAYEUX 1997). The outer core contains hexoses or neutral sugars (D-glucose, D-galactose and N-acetyl-D-glucosamine). It is the attachment site for O-antigen (RAETZ and WHITFIELD 2002). While the inner core consists of heptoses (Hep) and 2-keto-3-deoxyoctonic acid (Kdo) that considered as a characteristic marker of the LPS molecule and is connected directly with the lipid A part (MAYEUX 1997; ALEXANDER and RIETSCHEL 2001; WYCKOFF et al. 1998).
2.3.3.3 O-antigen
It is a long polysaccharide chain consisting of repeating units of hexoses (up to 50 units) bound together by α1-4 or β1-6 linkages forming either homopolymers or heteropolymers (i.e.
from one to eight monosaccharides) (HODGSON 2006; HITCHCOCK et al. 1986; ALEXANDER
and RIETSCHEL 2001; RAETZ and WHITFIELD 2002). The structure of these repeating units varies among bacterial strains and thus allows structural variability (RIETSCHEL et al. 1994).
So that it acts as a surface antigen that protects the bacteria from phagocytosis and serum
complement, and determines the serological specificity or the bacterial serotype (SCHLETTER
et al. 1995; ALEXANDER and RIETSCHEL 2001). O-antigen chain also differ in length upon which LPS was divided into three types. Smooth LPS in which the O-antigen specific chain shows a full length, semi-rough LPS with restricted antigen and rough LPS without O-antigen such as in H. influenzae and is called lipooligosaccharide or LOS (HODGSON 2006;
ALEXANDER and RIETSCHEL 2001). LOS contains inner core from which one or more mono- or oligosaccharide branches extended (RAETZ and WHITFIELD 2002).
2.3.4 Mode of action of LPS
LPS is considered as a molecular pattern related to the pathogen-associated molecular patterns (PAMPs) of the bacterial cell wall structure through which the bacteria can be recognized via specific host receptors called pattern-recognition receptors (PRRs) that detect the pathogenic bacterial structure and specifically bind to the PAMPs (ECKMANN 2004;
COLLIER-HYAMS and NEISH 2005). One group of the PRRs are the toll-like receptors (TLRs) which activate cellular signal transduction pathways after binding with LPS and trigger innate defence mechanisms.
The lyses of Gram-negative bacteria cause them to release LPS from the outer membrane of their cell wall. The monomer form of LPS binds to binding protein (LBP) forming LPS-LBP complex. LPS-LBP is a 60-Kd glycoprotein, synthesized in hepatocytes and released into the blood circulation (TOMLINSON and BLIKSLAGER 2004). LBP presents in normal human serum at a concentration of 5-10 µg/ml; this level increases to approximately 200 µg/ml in case of acute phase response (GUTSMANN et al. 2001). The low concentrations of LBP enhance LPS-induced activation of mononuclear cells while acute concentrations inhibit such effect (GUTSMANN et al. 2007). It facilitates the diffusion of LPS and catalyzes its binding to the receptor antigen CD14, either the membrane-bound form (mCD14) or the soluble form (sCD14) (YU et al. 1997; ULEVITCH andTOBIAS 1994; SZALMÁS 2000).
2.3.4.1 LPS pathway through mCD14-bearing cells
LPS-LBP complex is recognized by a receptor membrane-bound molecule, mCD14 or cluster differentiation antigen 14 and is bound to it through the lipid A portion. mCD14 is a 55-KD
glycoprotein (MAYEUX 1997; TOMLINSON and BLIKSLAGER 2004; GUTSMANN et al. 2007;
SZALMÁS 2000) linked to the membrane of myeloid cells (tissue macrophages and peripheral monocytes) by a glycosyl-phosphatidyl-inositol (GPI) anchor (YU et al. 1997). In case of mCD14-expressing cells, the GPI tail lacks a transmembrane region and can not transmit a signal into the cell (RICHARD 1999), so that mCD14 must conjugate to another cell membrane component in order to form a functional LPS receptor able to transmit signals (TOMLINSON
and BLIKSLAGER 2004; GUTSMANN et al. 2007). The toll like receptor (TLR) is a transmembrane structure (TOMLINSON and BLIKSLAGER 2004), expressed on the surface of the intestinal epithelial cells (CARIO et al., 2002) and approximately 9 to 10 different TLRs were identified in mammals (BRIGHTBILL and MODLIN 2000; HERSHBERG 2002), one of them is TLR4 which is the major receptor mediating the LPS signaling pathway (TOMLINSON and BLIKSLAGER 2004) in association with myeloid differentiation protein 2 (MD2) (BRIGHTBILL
and MODLIN 2000; DZIARSKI et al. 2001; WERLING and JUNGI 2003; SHIMAZU et al. 1999;
ABREU et al. 2001; 2003; ECKMANN 2004) that enabled TLR4 to respond to LPS molecule.
Activation of this complex results in triggering of a signal transduction through a serial of events including myeloid differentiation factor 88 (MyD 88), interleukin-1 (IL-1) receptor-associated kinase (IRAK) and TNF-α receptor-receptor-associated factor 6 (TRAF6).This is followed by activation of mitogen activated protein kinase (MAPK) pathway (WERLING and JUNGI
2003; ALEXANDER and RIETSCHEL 2001; CARIO et al. 2000) in which NF-кB-inducing kinase (NIK) is activated. The activated NIK activates IκB-α and β kinases resulting in proteolytic degradation of IκB and activation of the nuclear factor kappa B (NF-кB). NF-кB normally and in a steady state located in the cytoplasm in association with the inhibitory IκB protein in an inactive form (SCHEIDEREIT 1998). The activated transcription factor (NF-кB) then translocates to the nucleus of the cell (BRIGHTHILL and MODLIN 2000; CHANG and KARIN
2001; DAVIS 2000; ABREU et al. 2001; CARIO et al. 2000; ELEWAUT et al. 1999) and switches on gene expression for cytokines (i.e. activates the transcription of pro-inflammatory genes) and consequent release of proinflammatory mediators (HODGSON 2006) such as tumor necrosis factor-α (TNF-α) and interleukins (GUTSMANN et al. 2007) (Figure 8).
2.3.4.2 LPS pathway through sCD14
Another form in which GPI anchor is absent is sCD14. sCD14 is released in serum by mature phagocytes either by secretion or by proteolytic cleavage (ALEXANDER and RIETSCHEL 2001;
MAYEUX 1997) at concentrations of 3-4 µg/ml in normal human serum (BAŽIL et al. 1986;
SCHÜTT 1999). sCD14 affords recognition of LPS by non-CD14-bearing cells such as endothelial cells, dendritic cells, epithelial cells and smooth muscle cells ((SCHLETTER et al.
1995; ALEXANDER and RIETSCHEL 2001; MAYEUX 1997; FREY et al. 1992), but at the same time expressing toll-like receptor 4 (TLR4) and facilitate the systemic action of low serum LPS concentrations. While at higher concentrations, LPS follows the CD14-independent pathway (GUTSMANN et al. 2007).
TNF-α and IL-1β that are released in response to LPS cellular activation from CD14-bearing cells can in turn activate NF-κB even in CD14-non-bearing cells via cytokine receptors on the target cells such as IL-1R1 and TNFR1 or endothelial cytokine receptors and initiate inflammation and activate both the complement pathways and the coagulation pathway and this is considered as an indirect way for cellular response to LPS (VAN DE WALLE et al. 2008;
TOBIAS et al. 1997; STANCOVSKI and BALTIMORE 1997) (Figure 9).
Figure 8: A schematic diagram for the mechanism of action of LPS 2.3.4.3 LPS pathway and intestinal epithelial cells
Intestinal epithelial cells are the first line of defence against microbial invasion (TOMLINSON
and BLIKSLAGER 2004); at the same time the intestinal lumen contains many bacteria that the normal intestinal epithelial cells do not respond to. This could be explained by the intestinal epithelial cells express low levels of TLR4 and MD2 (ABREU et al. 2001; 2003) make the intestinal cells unresponsive to LPS. This can explain why LPS from commensally bacteria do not cause a frank inflammation to the exposed mucosa (ECKMANN 2004). So that increasing the expression of both TLR4 and MD2 is needed to make the intestinal epithelia responsive to LPS stimulation. It was found that interferon gamma (IFN-γ) increased the expression of MD2 and TLR4 in intestinal cell lines, also tumor necrosis factor alpha (TNF-α) increased the expression of TLR4 (ABREU et al. 2002; 2003).
The epithelial cells are devoid of membrane-bound form of CD14 (mCD14) antigen receptors (BÄCKHED et al. 2002) which is important for binding to LPS molecule and conveying it to TLR4-MD2 complex. So that there are two mechanisms suggested explaining the responsiveness of the intestinal cells to the LPS molecules. Firstly, it could be bound to LPS-LBP complex through the soluble form of CD14 (sCD14) forming sCD14-LPS-LPS-LBP complex then binding to the surface receptor resulting in cellular activation (ULEVITCH and TOBIAS
1995). Secondly, the LPS molecule could interact directly with TLR4 which are found on the apical surface of the intestinal epithelial cells (IMAEDA et al. 2002).
2.3.4.4 Neutralization of LPS
Neutralization of LPS results in inhibition in mononuclear cell-activation and thus reduces cytokine-release (GUTSMANN et al. 2001; NETEA et al. 1998). It is carried out via different pathways, including binding with LBP or sCD14 or by binding to the endogenous lipoproteins. LBP catalyzes the transfer of LPS directly to high-density lipoprotein (HDL) which is known to bind and inactivate LPS (WURFEL et al. 1994; FLEGEL et al. 1993).
Indirectly, LBP convey LPS to sCD14 which in turn transfers LPS to HDL (SCHÜTT 1999; DE
HAAS et al. 2000). While at low concentrations of LBP, it was suggested that serum amyloid P component (SAP) bound to HDL preventing its binding to LPS and played a role in balancing the amount of LPS (DE HAAS et al. 2000). Another protein, Bactericidal-permeability increasing protein (BPI) is found in neutrophil granules (ULEVITCH and TOBIAS 1995) and can bind to the cell wall of Gram-negative bacteria via the N-terminal region causing membrane disruption and subsequent death with releasing of its LPS. The released LPS then is bounded by the BPI protein and be neutralized (CANNY et al. 2002).
Figure 9: A schematic diagram for activation of NF-κB via some cytokines
2.3.5 LPS and the Intestinal Barrier
LPS can be absorbed through the intestinal epithelium (TOMLINSON and BLIKSLAGER 2004) to the general circulation through either the transcellular or the paracellular pathway. The fluorescence-labelled LPS was found to be taken up by the intestinal mucosa mostly by passive transcellular pathway (DREWE et al. 2001). On the other hand the paracellular permeability of Caco-2 human intestinal cell line to LPS was increased under hypoxic conditions.
When LPS was applied to the basolateral surface of the intestinal epithelium this resulted in impairment of the function of the tight junction barrier (KIMURA et al. 1997) with significant inhibition of the transepithelial electrical resistance (TEER) in IEC-6 cells of rats treated with 1.0 and 10 mg LPS/ml. This inhibition in TEER was not observed in case of exposing the apical surface to LPS. In contrast, a reduction in TEER was observed in epithelial Caco-2
cells monolayer when exposed to apical LPS for 24 h under low glucose conditions (YU et al.
2005) with increased apical-to-basolateral translocation of dextran in the epithelial monolayer that indicated increase in the paracellular permeability. In the same study, the sugar uptake activity was assessed using C14-labelled α-methyl glucopyranoside (α-MG) which is transported only by SGLT-1 evidenced by the inhibition of the sugar uptake after addition of phlorizin either in high or low glucose medium. Only under high glucose conditions the cells that exposed to LPS showed a two-fold increase in the α-MG uptake.
LPS could inhibit the intestinal uptake of 5 mM D-fructose across rabbit jejunum (GARCIA -HERRERA et al. 2003) when the tissues were pre-incubated with 3 µg/ml LPS for 12 min without morphological changes in the exposed intestinal tissues. At the same study, LPS did not modify the uptake of D-fructose across the brush border membrane vesicles either in control or LPS-treated group, suggesting that LPS had no direct effect. On the other hand, intravenous injection of 2 and 20 µg LPS/kg BW for 90 min inhibited absorption of 5 mM D-fructose in rabbits (GARCIA-HERRERA et al. 2008). Besides its inhibitory effect on sugar absorption, LPS decreased the intestinal active transport of L-leucine in rabbits (ABAD et al.
2001) but did not alter the simple diffusion of the amino acid. It could be attributed to that LPS decreased the activity of Na+/K+-ATPase at the basolateral membrane (ABAD et al. 2001;
AMADOR et al. 2008). The inhibitory effect of LPS on L-leucine uptake was increased by the pre-incubation of tissues with LPS, suggesting that the endotoxin needed a time to exert its
AMADOR et al. 2007; GARCIA-HERRERA et al 2008).Tumor necrosis factor alpha (TNF-α) that was released in response to the systemic action of LPS, was found to decrease the intestinal absorption of 0.5 mM L-leucine (approximately 40%) in rabbits after iv injection of 0.2 and 2 µg TNF-α /kg BW (ABAD et al. 2002). It was also found that TNF-α inhibited absorption of D-fructose (GARCIA-HERRERA et al. 2004) after intravenous administration of 2 and 4 µg TNF-α/kg BW into rabbits, which was attributed to the reduction in numbers of GLUT-5 in the brush border membranes of the treated animals (GARCIA-HERRERA et al 2004; 2008).
Similarly, TNF-α was found to inhibit the intestinal absorption of D-galactose (AMADOR et al.
2007) after intravenous injection of 2 µg TNF-α/kg BW through decreasing the number of SGLT1 molecules at the plasma membrane of intestinal cells.
2.3.6 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)
Series DON (mg/kg diet)