Estudio del efecto directo independiente de su efecto prebiótico de oligosacáridos no digeribles sobre células de la mucosa intestinal

Resumen

Introduction Non digestible oligosaccharides (NDOs) are products used as nutraceuticals or functional food due to their prebiotic effect. A prebiotic effect is defined as `the selective stimulation of growth and/or activity(ies) of one or a limited number of microbial genus(era)/species in the gut microbiota that confer(s) health benefits to the host¿ [1]. Most commercialized prebiotics are complex oligosaccharides that are not degraded by intestinal enzymes, reaching the colon, where they are digested by colonic microbiota [2]. Prebiotics have been claimed to exert healthy effects in obesity, allergy and gastrointestinal disorders (infections, irritable bowel syndrome, inflammatory bowel disease) [1]. FOS, inulin and GOS are simple oligosaccharides widely used for their prebiotic effects and their properties as soluble fermentable fibers. GOS, inulin and FOS are rather simple oligosaccharides when compared human milk oligosaccharides in terms of molecular structure and degree of ramification. It has been previously shown than goat¿s milk oligosaccharides (GMOS) are more complex and similar to human milk oligosaccharides that those of other mammalian species [3]. A study carried out in colitic rats showed that GMOS exert prebiotic effects [4]. A significant number of natural compounds have been found to inhibit bacterial growth, although their mechanism of action remains unclear in most cases [5, 6] FOS and inulin have been found to inhibit the growth of pathogenic bacteria such as Salmonella typhimurium [7], S. enteritidis [8] Listeria monocytogenes or the fungus Candida albicans [9] FOS and inulin could also inhibit the growth and other virulence factors in P. aeruginosa. P. aeruginosa is an opportunistic pathogen able to infect different animals and plants [10, 11], being a frequent cause of hospital-acquired infections including ventilator associated pneumonia [12] and catheter infections in immuno-compromised patients. In addition to their well documented prebiotic actions, NDO may be expected to be in contact with intestinal epithelial cells (IECs) in the colon and the small intestine. It is thus possible that these compounds exert direct, non-prebiotic actions on the intestinal epithelium. Insight into the immunomodulatory effects of non absorbable oligosaccharides on IECs could open new perspectives in the treatment of intestinal inflammatory related diseases such as inflammatory bowel disease or allergies, and in the design of infant formulas. Overall appearance and physiology are essentially normal in GF rats and mice although GF condition have been known to affect brain development and behaviour, metabolism, obesity, food digestion and overall resistance to stress and injury [13]. The lymphoid tissue of the gut of GF animals is dormant and hypoplastic but is capable of responding to antigenic stimulation [14]. The GF rodent has chronically loose stool, is hemoconcentrated and has increased water intake pointing out an abnormal water homeostasis. The cecum is enlarged and this could be related to altered cecal fluid transport. It has been suggested that absorption of water and Na is increase in cecum of GF compared to conventional rats. Any abnormality in cecal water and Na transport in germ free could be caused by abnormal cecal contents, which are characterised by an extremely low concentration of exchangeable anions, or by an abnormality intrinsic to the GF rat cecum itself [15]. The aims of this study were: ¿ Characterise the effect of FOS and inulin in different mechanisms of pathogenicity of P. aeruginosa ¿ Characterise the direct immunomodulatory effects of NDO in intestinal epithelial cells ¿ Characterise the direct effect (independent of their prebiotic actions) of FOS and GOS in vivo As a consequence of the results obtained in the GF mice experiment, we added a new aim to this thesis: ¿ Study of the role of microbiota in hydroelectrolytic transport in colon in basal condition and experimental induce colitis in mice. Results Effects of FOS and inulin in different mechanisms related with the virulence of pseudomonas aeruginosa The addition of FOS and inulin (20mg/ml) inhibited the growth and the biofilm formation of P. aeruginosa. FOS (5mg/ml) also inhibited swarming and twitching motility while inulin had a lower effect or none. Inulin reduced the IL-6 secretion in macrophages infected with P. aeruginosa while FOS also inhibited TNF¿ and IL-10 production via modulation of NF-¿B. The inhibition as a result of FOS and inulin addition was more pronounced in the mutant ptxS (the more virulent strain) comparing with ptxR and WT strains. Finally, FOS reduced the ToxA intracellular levels in IEC-18 cells cocultured with P. aeruginosa while inulin had no effect. Inmunomodulatory effect of NDOs in intestinal epithelial cells. All tested NDOs increase GRO¿ and MCP1 secretion in IEC18 cells, in the order of efficacy GMOS>inulin>GOS>FOS, with the only exception of FOS, that had no effect on MCP1. The effect was concentration dependent and was maximum at 5 mg/ml in all cases. The addition of NDOs also resulted in an upregulation of MIP2 but no general effect on ICAM, IL6, eotaxin, CXCL10 or RANTES. The inhibitor of NF-¿B produced a dramatic inhibition of GRO¿ and MCP1 secretion evoked by inulin, GOS or GMOS. In contrast, there was no general inhibition by the PI3K. The MAPK inhibitors tested also inhibited the cytokine secretory effect of prebiotics but to a lower extent than NF-¿B inhibition. MyD88 and TLR4 expression was silenced by shRNA and the inductor effect of NDO was inhibited in a 42-77%. We also tested NDOs effect in human cell lines. HT29 cells responded to NDOs secreting IL-8 to a lower extend that IEC while Caco-2 and Caco-2/TC7 cell lines failed to respond to NDOs. RT-PCR analysis of TLR4 expression showed a correlation between the production of IL-8, GRO¿ and NHE3 and the expression of TLR4 in every cell line. MCP1 secretion was lower in colon explants of Tlr4 KO mice than in controls after NDOs addition. Effect of NDO in vivo After weaning GF mice were administered a diet enriched in FOS (5%) and GOS (5%) for 4 weeks. Mice also received a sterile bacterial homogenate (SBH) and its combination with FOS or GOS to evaluate the effect of NDOs in the presence of bacterial antigens in lumen. Conventional animals were used as control. In GF conditions, NDO±SBH enlarged jejunum villi in concordance with an increase in mg protein per gr of tissue. FOS ±SBH also reduced total IgG levels in plasma in GF mice. Transcriptome analysis showed significant changes in the expression of genes that codifies barrier function proteins, microbiota modulatory genes and gluthatione and lipid metabolism genes in jejunum and colon of mice treated with NDOs. Some of these results were validated by RT-PCR in colon of GF and conventional animals showing an increase in the expression of genes that codify tight junction proteins (ocln, cldn4, cldn5) and proteins that modulate microbiota in GF conditions. NDOs induced Cld4 and Cxcl10. Post genomic validation also showed increased levels of expression of different genes related with hydroelectrolytic transport named edn1, Scnn1b, Scnn1g (ß, ¿ Exact), Aqp8, Slc9a3 (NHE)-3 and Atp1a1 (the ¿ subunit of Na/K ATPase) in colon of GF mice that were partially normalized in the SBH group Aldosterone levels in plasma were reduced in GF mice while corticosterone were nearly doubled. Study of the role of microbiota in the hydroelectrolytic transport in colon Conventional mice were administered an antibiotic cocktail to induce microbiota depletion. Pseudo sterile (PS) mice showed similar characteristics to GF mice such as cecum enlargement or loose stools. The expression of molecules related with hydroelectrolytic transport was also increased in colon: Edn1, Aqp8, Scnn1a, Scnn1g (¿ and ¿ subunits of ENaC), Atp1a, Atp1b1 (¿ and ß subunits of Na/K ATPase; and cecum: Slc26a3 (DRA), Scnn1a, Scnn1g (¿ and ¿ ENaC) and Slc9a3 (NHE3) in PS mice while a not significant reduction of colon absorption was detected. DSS colitis was induced in GF and conventional mice resulting in different manifestation. While conventional animals lost weight, showed a doses dependent increase in macroscopic damage index and inflammation biochemical markers such as MPO and AP, GF mice had lower symptoms of inflammation. However, animals seemed sicker, mortality rate was higher and showed signs of loss of blood. Inflammatory proteins expression was also induced by DSS in conventional animals in a higher extend than in the GF ones. The expression of molecules related with barrier function (ZO-1, and occludin) was inhibited by DSS in conventional mice while there were no effects in GF conditions. DSS tended to reduce the expression of molecules related with hydroelectrolytic transport in both conditions. PS mice developed lower signs of inflammation comparing with control animals when DSS colitis was induced (based on body weight loss, MDI, MPO activity, inflammatory molecules expression and histological score). However, MUC3, ZO-1 and occludin were induced by DSS in PS mice. Colonic water absorption was reduced in PS mice while DSS tended to increase this parameter in both conditions. The expression of molecules related with hydroelectrolytic transport was reduced in both conditions. Discussion NDOs, are compounds that are not digested by intestinal enzymes reaching the colon intact where they act as prebiotics. Prebiotics are known for their ability to selectively influence bacterial proliferation in the gut conferring health benefits to the hosts. Antimicrobial properties have been described for a number of oligosaccharides [16]. We were able to show that FOS (1) inhibits P. aeruginosa growth, biofilm formation and motility; (2) limits the P. aeruginosa evoked NF-¿B dependent cytokine secretion in macrophages; and (3) decreases exotoxin A levels in P. aeruginosa infected IEC18 cells. These effects have also been observed for inulin, but in general the magnitude of the changes induced by FOS was superior to that of inulin. This indicates that the length of the oligosaccharide chains is an essential determinant for the magnitude of the biological activities observed. Our data suggest that FOS may be a useful component of a drug cocktail to combat P. aeruginosa infection. Besides their prebiotic effect, NDO may have additional actions that are independent of their interaction with the colonic microbiota. Our data demonstrate that the NDOs studied, namely FOS, GOS, GMOS and inulin, induce the secretion of proinflammatory cytokines by intestinal epithelial cells. Using IEC18 as an in vitro model, MCP1, GRO¿ and MIP2 were upregulated by NDOs, while ICAM, IL-6, eotaxin, CXCL10 and RANTES were generally unaffected. Pharmacological and molecular evidence indicates that the effects on MCP1/GRO¿ were dependent chiefly on TLR4/MyD88/NF-¿B and secondarily on MAPK. We hypothesized that NDOs could act as TLR4 ligands based on the fact that the canonical agonist LPS is a saccharide itself. The biological consequences of TLR4 ligation by NDOs (or other agonists) are debatable. In principle, TLR4 activation produces proinflammatory signal. Since the intestinal epithelium is constantly exposed to bacteria and bacterial components on its apical membrane, this would naturally lead to inflammation. However this does not happen and instead an homeostatic equilibrium is established between the microbiota and the host via a number of mechanisms not well characterized yet [17]. IECs respond to both pathogenic and commensal bacteria. IEC or nonhematopoietic-specific deletion of TLR4, MyD88 or NEMO results in defective defense against bacterial infection and/or inflammation [18-20]. TLR4 has been reported to be expressed at low levels in basal conditions in vivo but it is upregulated in Crohn¿s disease. Thus TLR4 may play a determinant role in intestinal inflammation and defense. All NDOs apparently can act as agonists, although the efficacy at the receptor tends to be higher as the structure becomes more complex. To evaluate the direct effects, independent of the prebiotic actions of NDO in vitro , we conducted an experiment in germ free conditions. After weaning germ free mice were administered a diet enriched in FOS and GOS (5%). We also included a combination of the NDO with a sterile bacterial homogenate (GOS+SBH and FOS+SBH) and the consequent control group SBH (0,016%) to study the effects of NDO in presence of antigens. Finally, we conducted a control experiment with conventional mice. We studied the role of NDOs on intestinal enzymes activities since about 12% of components of human milk are soluble fiber that could affect intestinal maturation [21, 22]. Although, our results confirm some actions of NDOs in epithelial intestinal maturation they were not consistent in all intestinal segments. We might consider that oligosaccharides from human milk are structurally more complex than FOS and GOS. NDO±SBH enlarged jejunum villi in concordance with an increase in mg protein per gr of tissue suggesting a trophic action of these compounds. Our results pointed out reduced levels of IgG in GF mice administered FOS±SBH. Previous studies have shown how FOS and GOS can modify Ig levels in serum [23, 24] thus we show that FOS can module immune system independently of its prebiotic effect. Next we complete this analysis with the transcriptome study in colon and jejunum by means microarrays. The transcriptome analysis confirmed the direct effects of NDOs, SBH and its combinations since a substantial amount of genes were modified by the treatments in jejunum and colon. Ingenuity analysis showed that some functions were modified by the three treatments such as molecular transport, cell growth and proliferation, cell movement or lipid metabolism. NDOs modified genes related to lipid transport, glutathione metabolism, barrier function and defense against pathogens. However, NDOs and SBH exerted inversed actions. While the SBH inhibited the lipoprotein (Apo), defensins, tight junctions and lysozyme expression, NDOs induced the expression of these genes. The role of NDOs in barrier functions is controversial [25, 26]. Our results showed an upregulation in the expression of genes that codify tight junctions proteins (Cldn4 and Cld5) in colon in conventional and GF mice administered FOS. NDOs also increased the expression of Cxcl10 and Reg3g in colon in conventional and GF mice. Cxcl10 is a chemokine while Reg3g is an antibacterial lectine thus the upregulation of these genes could be related to a better barrier function. We also observed that SBH modified the expression of several molecules related to transport in colon. These results were quite interesting since it has been previously established a defect in the hydroelectrolytic transport in GF mice. Besides, the SBH partially reverted this effect. Next we conducted some experiments in mice whose microbiota depletion (PS mice) was induced by administering an antibiotic cocktail. PS mice shared some characteristics with GF mice such us caecum enlargement or the presence of mild feces [27] . The expression of molecules related with hydroelectrolytic transport in PS mice was up regulated in colon and caecum. This increase in carriers expression would produce a higher water absorption in colon and caecum. Colon loop experiment confirmed a reduced absorption in PS mice. These mice did not show diarrhea or modified ionic and protein serum levels showing that the defect in absorptive capacity might be functionally compensated. A lower water absorption in vivo and higher electrogenic Na absorption in caecum of GF rats was observed in previous studies. Simonetta et al published higher levels of ATPase Na/K in ileum and caecum in GF rats that was related to a higher efficiency in absorption [28] The water and sodium loss induce the renin-angiotensin-aldosterone system that increases the arterial pressure by means not only actions in kidney but also in distal colon increasing the electrogenic Na absorption through ENaC. We hypothesize that the defect in water absorption in GF conditions could be compensated by a secondary hiperaldosterism. However, our results showed lower levels of aldosterone in GF mice. On the other hand, costicosterone levels were increased in GF mice in concordance with previous studies [29, 30]. Some studies have suggested than GF mice are resistant to glucocorticoid explaining the elevated levels of this hormone in GF mice. Moreover, Glucocorticoids induce the absorption of water and electrolytes in bowel per se. Besides high levels of corticosterone can induce aldosterone effects. Next, we decided to study the alterations in the hydroelectrolytic transport in GF mice in an experimental model of colitis. However, since inflammation often depends on microbiota we needed to evaluate the degree of inflammation in GF mice. Our results showed that DSS response in GF mice was attenuated although general condition of the mice was damaged due to a loss of blood as a consequence of a defect in barrier function. PS mice also showed a light response to DSS although their general condition was not damaged. We related this phenomenon with the presence of Treg cells in mucosa of the PS mice due to the previous presence of microbiota. The DSS administration had an inhibitory effect in the carriers expression in conventional animals. This data is supported by other studies in IBD patients and in experimental models of colitis [31-33] although not all of them agree [32, 34]. We observed similar results in PS and GF mice suggesting that the inhibition could be caused by the DSS damage exerted in epithelial barrier instead of by the inflammation or the bacterial presence. It is also possible that a low level of inflammation is enough to repress the expression of carriers The effect was proportionally more important in conventional mice since PS control mice already had a lower percentage of dry matter in feces. However, these changes were not correlated to lower water absorption. Finally we can conclude that: ¿ FOS reduces P. aeruginosa pathogenicity through distinct mechanisms ¿ NDOs induce the secretion of proinflammatory cytokines by intestinal epithelial cells in vitro via TLR4/MyD88/NF-¿B activation. ¿ FOS and GOS exert direct actions independent of their prebiotic effect in vivo and interact with microbiota antigens. ¿ The lack of microbiota results in an induction of the expression of hydroelectrolytic carriers as a possible compensatory mechanism to water homeostasis alteration observed in germ free mice. ¿ DSS inhibits the expression of hydroelectrolytic carriers in colon independently of the presence of microbiota or degree of inflammation. This inhibition did not correlate with lower water absorption.