Wageningen Academic P u b l i s h e r s Beneficial Microbes, 2015; 6(6): 807-815 http://www.wageningenacademic.com/doi/pdf/10.3920/BM2015.0018 - Monday, November 13, 2017 5:06:58 AM - Göteborgs Universitet IP Address:130.241.16.16 Effect of Saccharomyces cerevisiae strain UFMG A-905 in experimental model of inflammatory bowel disease F.C.P. Tiago1 , B.A.A. Porto1, N.S. Ribeiro1, L.M.C. Moreira1, R.M.E. Arantes2, A.T. Vieira3, M.M. Teixeira3, S.V. Generoso4, V.N. Nascimento4, F.S. Martins1 and J.R. Nicoli1* 1Department of Microbiology, Institute of Biological Sciences, Federal University of Minas Gerais, Avenida Presidente Antônio Carlos 6627, C.P. 486, Pampulha – Campus UFMG, 31270-901, Belo Horizonte, MG, Brazil; 2Department of General Pathology, Institute of Biological Sciences, Federal University of Minas Gerais, Avenida Presidente Antônio Carlos 6627, C.P. 486, Pampulha – Campus UFMG, 31270-901, Belo Horizonte, MG, Brazil; 3Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais, Avenida Presidente Antônio Carlos 6627, C.P. 486, Pampulha – Campus UFMG, 31270-901, Belo Horizonte, MG, Brazil; 4School of Pharmacy, Federal University of Minas Gerais, Avenida Presidente Antônio Carlos 6627, C.P. 486, Pampulha – Campus UFMG, 31270-901, Belo Horizonte, MG, Brazil; jnicoli@icb.ufmg.br Received: 12 February 2015 / Accepted: 17 June 2015 © 2015 Wageningen Academic Publishers RESEARCH ARTICLE Abstract In the present study, the protective potential of Saccharomyces cerevisiae strain UFMG A-905 was evaluated in a murine model of acute ulcerative colitis (UC). Six groups of Balb/c mice were used: not treated with yeast and not challenged with dextran sulphate sodium (DSS) (control); treated with S. cerevisiae UFMG A-905 (905); treated with the non-probiotic S. cerevisiae W303 (W303); challenged with DSS (DSS); treated with S. cerevisiae UFMG A-905 and challenged with DSS (905 + DSS); and treated with S. cerevisiae W303 and challenged with DSS (W303 + DSS). Seven days after induction of UC, mice were euthanised to remove colon for enzymatic, immunological, and histopathological analysis. In vivo intestinal permeability was also evaluated. An improvement of clinical manifestations of experimental UC was observed only in mice of the 905 + DSS group when compared to animals from DSS and W303 + DSS groups. This observation was confirmed by histological and morphometrical data and determination of myeloperoxidase and eosinophil peroxidase activities, intestinal permeability and some proinflammatory cytokines. S. cerevisiae UFMG A-905 showed to be a potential alternative treatment for UC when used in an experimental animal model of the disease. Keywords: Saccharomyces cerevisiae, ulcerative colitis, dextran sulphate sodium, inflammatory bowel disease, probiotic 1. Introduction In recent decades, much attention has been given to the normal intestinal microbiota modulation by live microbial adjuvants called probiotics. Probiotics are defined as live microorganisms which when administered in adequate amounts confer health benefits to the host (FAO/WHO, 2002). It is known that a major concern of the World Health Organization is the search for new therapies that do not act as strong selective pressure favouring the selection of pathogens increasingly aggressive and resistant. There is now a wide variety of bacteria used as probiotics such as bifidobacteria and lactobacilli. The only commercially available yeast for worldwide probiotic use in humans is Saccharomyces cerevisiae var. boulardii, which protects the intestinal ecosystem against various infectious and inflammatory disorders (McFarland, 2010; Pothoulakis, 2009), whereas some other S. cerevisiae strains are most used in animal production. Studies carried out by Martins et al. (2005, 2007, 2011) and Generoso et al. (2010) showed that S. cerevisiae strain UFMG A-905, isolated from ‘cachaça’ production (local sugar cane spirit), was able to survive and colonise ISSN 1876-2833 print, ISSN 1876-2891 online, DOI 10.3920/BM2015.0018807 http://www.wageningenacademic.com/doi/pdf/10.3920/BM2015.0018 - Monday, November 13, 2017 5:06:58 AM - Göteborgs Universitet IP Address:130.241.16.16 F.C.P. Tiago et al. the gastrointestinal tract of conventional and germ-free mice, respectively, and to protect these animals against experimental infection by Salmonella enterica subsp. enterica serovar Typhimurium and Clostridium difficile, as well as against the consequences of an experimental intestinal obstruction. The results obtained by Martins et al. (2010, 2011) and Generoso et al. (2010) suggest that many of the beneficial effects may be due to an antiinflammatory capacity through the ability of the yeast to bind the pathogenic bacteria onto its cell surface (Tiago et al., 2012), but also by the production of soluble extracellular compound with anti-inflammatory properties. Similar capacity has been previously described in Saccharomyces boulardii by Pothoulakis (2009). Inflammatory bowel disease (IBD) forms a group of inflammatory conditions of the small and large intestine, and the major types of IBD are Crohn’s disease (CD) and ulcerative colitis (UC) (Xavier and Podolsky, 2007). Although the exact cause of IBD is unknown, an abnormal inflammatory response and a disrupted mechanism of host tolerance to the non-pathogenic resident microbiota in a genetically susceptible host have been proposed (Fiocchi, 2003; Ogura et al., 2001; Sartor, 2006). The fundamental role of intestinal microbiota in IBD is demonstrated by the fact that experimental animals remain healthy when raised in germ-free conditions and only develop the disease after colonisation with a commensal pathogen-free microbiota (Kawada et al., 2007). Mesalazine is mainly used for the treatment of UC but suffers from the drawback of having poor bioavailability. Faecal microbiota transplantation is an alternative option with promising results in therapy for Clostridium difficile infection, but questions remain regarding its efficacy and safety in IBD patients. On the other hand, the indication of probiotics in cases of IBD is based on a series of studies in humans and animals which also suggest that the intestinal microbiota plays a central role in the pathogenesis of CD and UC (Damaskos and Kolios, 2008; Do et al., 2010; Hedin et al., 2007; Sang et al., 2010). Probiotics, such Escherichia coli Nissle and VSL#3 (Bibiloni et al., 2005) have been suggested as substitute to mesalazine for maintenance of remission in patients with UC (Kruis et al., 2004). On the other hand, bacterial genera frequently used as probiotics, such as Bifidobacterium and Lactobacillus were increased in active IBD patients and, for this reason, should be used more cautiously during the active phase of IBD. Additionally, Sartor (2006) reports that bacterial probiotics induced an inflammation increase in the duodenum and colon of animals with colitis, suggesting that yeasts might be more adequate candidates to be administrated in these situations as probiotics (Guslandi et al., 2000, 2003; Steinhart et al., 1996). In relation to the possible mechanisms of action, probiotics were likely to play a key role in protecting UC by reducing the inflammatory factor TNF-α expression through inhibiting the TLR4 expression in the colon tissue (Segain et al., 2000). 808 In the present study, the protective potential of S. cerevisiae strain UFMG-905 was evaluated in a murine model of acute UC induced by dextran sulphate sodium (DSS), based on clinical signals, as well as on enzymatic, immunological, and histopathological analysis. 2. Materials and methods Yeasts S. cerevisiae strain UFMG A-905 belongs to the collection of Dr. Carlos Augusto Rosa (Departamento de Microbiologia, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil). S. cerevisiae W303 was provided by Dr. Ieso Miranda Castro and Dr. Rogelio Lopes Brandão (Universidade Federal de Ouro Preto, Ouro Preto, Brazil), and was used as a negative control for probiotic properties. The yeasts were grown in YPD (yeast extract 1%, peptone 2% and dextrose 2%) broth during 24 h at 37 °C on a shaker adjusted to 150 rpm. Afterward, the culture was concentrated tenfold by centrifugation to obtain 9.0 log10 cfu/ml. Mice Ten weeks old conventional Balb/c mice of both sexes were used. Water and commercial autoclavable diet (Nuvital, Curitiba, Brazil) were sterilised by steam and offered ad libitum to animals which were maintained in a ventilated animal caging system (Alesco Ltda., Campinas, Brazil) with controlled lighting (12 h light, 12 h dark) and temperature (22.0±0.5 °C). All experimental procedures were carried out according to the standards set forth by the Brazilian College for Animal Experimentation (COBEA, 2006). The study was approved by the Ethics Committee in Animal Experimentation of the Federal University of Minas Gerais (CETEA/UFMG). Experimental design Sixty conventional animals were divided into the following six groups (10 mice in each group): (1) not treated with yeast and not challenged with DSS (control); (2) treated with S. cerevisiae UFMG-905 (905); (3) treated with S. cerevisiae W303 (W303); (4) challenged with DSS (DSS); (5) treated preventively with S. cerevisiae UFMG-905 and challenged with DSS (905 + DSS); and (6) treated preventively with S. cerevisiae W303 and challenged with DSS (W303 + DSS). These animals were used for clinical, enzymatic, immunologic, and histopathological analysis. For permeability analysis, a second set of the following groups was used (6 mice per group): control, 905, DSS and 905 + DSS. Beneficial Microbes 6(6)  http://www.wageningenacademic.com/doi/pdf/10.3920/BM2015.0018 - Monday, November 13, 2017 5:06:58 AM - Göteborgs Universitet IP Address:130.241.16.16 Treatments For preventive probiotic treatment, conventional mice received by oral gavage a daily dose of 0.1 ml containing 9.0 log10 cfu/ml 10 days before challenge, and treatment was continued during all the remaining experimental infection. For induction of colitis, mice treated or not with the yeasts received DSS 3-4% (w/v) (36,000 to 50,000 kDA; MP Biomedicals, Santa Ana, CA, USA) diluted in water (filtered and autoclaved) during seven days ad libitum, and control animals only water before sacrifice. Seven days post-challenge with DSS, animals of each group were killed by cervical displacement. At sacrifice, the colon was removed and analysed for histological scoring, measurement of myeloperoxidase (MPO, as indicator of the extent of neutrophil infiltration), eosinophil peroxidase (EPO, as indicator of the extent of eosinophil infiltration), as well as for determinations of some chemokines and cytokines. Samples from colon were also used for histological and morphometric analysis. Disease activity index The daily disease activity index (DAI) assessment was carried out according to the classic scoring system described by Cooper et al. (1993) with some modifications. DAI was based on the cumulative scores (0-10) from three different parameters: faeces consistency (0 = normal, 1 = soft but still formed, 2 = very soft, 3 = diarrhoea), presence of blood in faeces (0 = negative hemoccult, 1 = positive hemoccult, 2 = blood traces visible in faeces, 3 = rectal bleeding) and weight loss (0 = negative weight loss, 1 = loss from 1 to 5% of body weight, 2 = 5-10%, 3 = 10-15%, 4 = 15-20%) Cytokines, chemokines, myeloperoxidase and eosinophil peroxidase determinations The concentrations of interferon-gamma (IFN-γ), tumour necrosis factor alpha (TNF-α), chemokine (C-X-C motif ) ligand 1 (CXCL1/KC) and eotaxin-1 (CCL11) in colon of mice were measured by ELISA, using commercially available antibodies and according to the procedures supplied by the manufacturer (R&D Systems, Minneapolis, MN, USA). The colon tissue (100 mg) was homogenised in PBS (0.4 M NaCl and 10 mM NaPO4) containing anti-proteases (0.1 mM phenylmethylsulphonyl fluoride, 0.1 mM benzethonium chloride, 10 mM EDTA and 20 kallikrein inhibitor units of aprotinin A) and 0.05% Tween-20 (100 mg of tissue/ ml of solution). The samples were then centrifuged for 10 min at 3,000×g and the supernatant immediately used for ELISA assays. A segment of colon was removed upon dissection, weighed and immediately flash-frozen. Samples were kept at -70 °C Beneficial Microbes 6(6) Effect of yeast probiotic on experimental UC until determination of MPO concentrations using an ELISA kit (Cell Sciences, Canton, USA), as described by Arrieta et al. (2009). EPO assay was performed as previously described (Strath et al., 1985). Briefly, 100 mg of colon tissue was weighed, homogenised in 1.9 ml of phosphate buffered saline (PBS), and centrifuged at 12,000×g for 10 min. The supernatant was discarded, and the erythrocytes were lysed. The samples were then centrifuged, the supernatant discarded, and the pellet suspended in 1.9 ml of 0.5% hexadecyltrimethyl ammonium bromide in PBS saline. The samples were frozen three times in liquid nitrogen and centrifuged at 4 °C at 12,000×g for 10 min. The supernatant was used in the enzymatic assay. Briefly, o-phenylenediamine (OPD) (10 mg) was dissolved in 5.5 ml distilled water, and then 1.5 ml of OPD solution added to 8.5 ml of Tris buffer (pH 8.0), followed by the addition of 7.5 µl of H2O2. Using a 96-well plate, 100 µl of substrate solution was added to 50 µl of each sample. After 30 min, the reaction was stopped with 50 µl of 1 M H2SO4 and the absorbance read at 492 nm. Histologic and morphometric analysis Samples of colon were taken for histological analysis, fixed in a 4% buffered formalin solution, dehydrated, cleared, embedded in paraffin, cut into sections of 4-5 µm thick, stained by hematoxylin and eosin (H&E), and coded and analysed by optical microscopy by a single pathologist who was unaware of the experimental conditions for each group. For morphometric analysis, the images were obtained by a JVC TK-1270/RGB microcamera (JVC, Wayne, NJ, USA) and the KS 300 Software built into a Kontron Elektronick/ Carl Zeiss image analyser (Oberkohen, Germany). Lesion extents in the different groups were determined as the percentage of colonic lesion length (μm) in relation to the entire length of the colon. Similarly, oedema area were determined as the percentage of areas affected by oedema (μm2) in relation to the total area of the colon. Permeability assay To evaluate in vivo intestinal permeability, all mice received 0.1 ml of diethylenetriamine pentaacetic acid (DTPA) solution labelled with 18.5 MBq of 99mTc by gavage. After 90 min, the animals were anesthetised and 500 µl of blood was collected and placed in appropriate tubes for radioactivity determination (Viana et al., 2009). Data were expressed as % of administrated dose, using the following equation: % of administrated dose found in blood = cpm found in blood × 100 cpm of administrated dose Where cpm is counts per minute. 809 F.C.P. Tiago et al. a high percentage of lesions was also observed in the W303 group, but without oedema. The results were expressed as the average of at least two independent experiments. Results are shown as mean ± standard error of the mean. Normalised data were compared by using analysis of variance (ANOVA) followed by a Student Newmann Keuls post hoc analysis. Results were considered significant at P<0.05. Figure 2 shows one of the representative histopathological aspects of colon of control, DSS, 905 + DSS, W303 and W303 + DSS groups. Epithelial tissues were clearly preserved in the colon of mice from control, 905 and 905 + DSS groups when compared to animals from DSS and W303 + DSS groups which presented numerous lesions and degeneration of microvilli. The high percentage of lesions without oedema, shown in Figures 1B and 1C for the W303 group, was confirmed in the histopathological aspects of the colon mucosa of these mice. 3. Results Figure 1A (and Supplementary Figure S1 for details) shows that probiotic treatment reduced the daily DAI by day seven only in the 905 + DSS group (P<0.05). When colitis activity was assessed as a percentage of colon with lesions (Figure 1B) and oedema area (Figure 1C), a difference was again observed between mice from the 905 + DSS group and animals from the DSS and W303 + DSS groups. Curiously, Daily disease activity index score 10 8 c 6 b 4 2 a 0 1 2 3 4 15 b a 5 b a a Control DSS 5 6 7 C b b 10 0 Days B 15 Control DSS 905 W303 905 + DSS W303 + DSS d 905 905 + DSS W303 W303 + DSS Oedema area % perimeter of the colon 20 Colonic MPO and EPO activities were measured as indicators of the extent of neutrophil and eosinophil infiltration into the mucosa, respectively. After a 7-day period of induced colitis, MPO and EPO activity was A 0 Lesion % perimeter of the colon http://www.wageningenacademic.com/doi/pdf/10.3920/BM2015.0018 - Monday, November 13, 2017 5:06:58 AM - Göteborgs Universitet IP Address:130.241.16.16 Statistical analysis 10 c 5 a a a 0 Control DSS 905 905 + DSS W303 W303 + DSS Figure 1. (A) Disease activity index, (B) percentage of lesions and (C) oedema area in the colon of mice not treated with yeast and not challenged with DSS (control); treated with Saccharomyces cerevisiae UFMG A-905 (905); treated with S. cerevisiae W303 (W303); challenged with DSS (DSS); treated preventively with 905 and challenged with DSS (905 + DSS); and treated with W303 and challenged with DSS (W303 + DSS). a, b, c and d statistically significant difference (P<0.05). 810 Beneficial Microbes 6(6) Effect of yeast probiotic on experimental UC Control DSS 905 DSS + 905 W303 W 303 + DSS Figure 2. Histopathological aspects of the colon from mice not treated with yeast and not challenged with DSS (control); treated with Saccharomyces cerevisiae UFMG A-905 (905); treated with S. cerevisiae W303 (W303); challenged with DSS (DSS); treated preventively with 905 and challenged with DSS (905 + DSS); and treated with W303 and challenged with DSS (W303 + DSS). Hematoxilin and eosin, 10:100. higher and similar in all groups challenged with DSS when compared to the control, except in the 905 + DSS group (Figures 3A and 3B). However, only the difference in MPO activities between the DSS and 905 + DSS group was statistically significant (P<0.0001) (Figure 3A). To complete the evaluation of the recruitment of neutrophils and eosinophils to the inflammatory site, levels of CCL11 and CXCL1/KC were determined in the colon (Figures A 0.6 b EPO colon D.O. (492 nm) b 200 a 100 4A and 4B respectively). A significant increase in CXCL1/ KC concentration was observed in the DSS group when compared to the control and 905 + DSS groups (Figure 4B). Figure 4A shows a significant increase in CCL11 for all the groups treated with DSS as well as for the W303 group. Figure 4D shows an increase in IFN-γ levels in the colon of DSS and W303 + 303 animals, which was reduced to basal level in the 905 + DSS group. a a a B 0.4 0.2 DS S 30 3+ DS S W S DS 90 5+ 30 3 W 90 5 ntr S DS S 3+ DS 5+ 90 W 30 S DS 3 W 30 5 90 ntr ol Co ol 0.0 0 Co 300 MPO colon D.O. (450 nm) http://www.wageningenacademic.com/doi/pdf/10.3920/BM2015.0018 - Monday, November 13, 2017 5:06:58 AM - Göteborgs Universitet IP Address:130.241.16.16  Figure 3. (A) Myeloperoxidase (MPO) and (B) eosinophil peroxidase (EPO) activity levels in the colon of mice not treated with yeast and not challenged with DSS (control); treated with Saccharomyces cerevisiae UFMG A-905 (905); treated with S. cerevisiae W303 (W303); challenged with DSS (DSS); treated preventively with 905 and challenged with DSS (905 + DSS); and treated with W303 and challenged with DSS (W303 + DSS). a and b statistically significant difference (P<0.05). Beneficial Microbes 6(6) 811 CXCL1/KC pg/100 mg of colon 1.5 1.5 1.0 0.5 b b 1.0 0.5 a a a 0.4 0.2 DS S 30 3+ DS S 90 5+ DS S 30 3 W D W 0.8 90 5 Co ntr ol DS S W 30 3+ DS S 90 5+ DS S 30 3 W 90 5 C INF-γ pg/100 mg of colon 0.6 0.4 0.2 S 30 3+ DS S DS W 90 5+ S DS 3 30 W 5 90 ntr S W 30 3+ DS S 90 5+ DS S 30 W DS 3 5 90 ol ntr Co ol 0.0 0.0 Co TNF-α pg/100 mg of colon B 0.0 0.0 Co ntr ol Eotaxin-1 (CCL 11) pg/100 mg of colon A 2.0 0.6 Figure 4. (A) CCL11, (B) CXCL1/KC, (C) TNF-α and (D) INF-γ levels in colon of mice not treated with yeast and not challenged with DSS (control); treated with Saccharomyces cerevisiae UFMG A-905 (905); treated with S. cerevisiae W303 (W303); challenged with DSS (DSS); treated preventively with 905 and challenged with DSS (905 + DSS); and treated with W303 and challenged with DSS (W303 + DSS). a and b statistically significant difference (P<0.05). 15 Permeability (counts per minute/g of blood) Analysis of in vivo intestinal permeability was performed by evaluating radioactivity in blood after oral administration of 99mTc-DTPA. Figure 5 shows an increase in intestinal permeability in DSS group when compared to the control group, and a reduction to basal value in the 905 + DSS group (P<0.05). b 10 a 5 a a S 90 5+ DS 5 90 S DS tro l 0 Co n http://www.wageningenacademic.com/doi/pdf/10.3920/BM2015.0018 - Monday, November 13, 2017 5:06:58 AM - Göteborgs Universitet IP Address:130.241.16.16 F.C.P. Tiago et al. Figure 5. In vivo intestinal permeability of mice not treated with yeast and not challenged with DSS (control); treated with Saccharomyces cerevisiae UFMG A-905 (905); challenged with DSS (DSS); treated preventively 905 and challenged with DSS (905 + DSS). a and b statistically significant difference (P<0.05). 812 4. Discussion One of the conventional treatments of patients with IBD is the use of antibiotics to manipulate the enteric microbiota and reduce damage to the epithelium. However, the use of antibiotics has limited value in cases of IBD, except in special situations, such as fistulae, abscesses, bacterial overgrowth and pouchitis. Moreover, prolonged use of antibiotics can cause serious side effects and dramatically alter the microbiota with undesirable consequences, such as antibiotic-associated diarrhoea, pseudomembranous colitis and induction of antimicrobial resistance. Hence, the use of probiotics would be a more appropriate alternative Beneficial Microbes 6(6) http://www.wageningenacademic.com/doi/pdf/10.3920/BM2015.0018 - Monday, November 13, 2017 5:06:58 AM - Göteborgs Universitet IP Address:130.241.16.16  option and the evaluation of candidate microorganisms for this objective can be done in an experimental animal model simulating IBD, such as mice challenged with DSS for UC. The clinical manifestations of UC induced by DSS include weight loss, diarrhoea, faecal blood (Perse and Cerar, 2012), acute inflammation of the colon (Vieira et. Al, 2009), and eventually death (Perse and Cerar, 2012). Some of these clinical signals were scored and used to build the daily DAI allowing a determination of the disease evolution. The yeast UFMG A-905 presents interesting characteristics to be applied for a probiotic use, promoting improvement in the clinical manifestations of experimental UC in a murine model. However, this protective effect was obtained in a preventive form and this fact reduces the possible use of viable yeast cells to prophylactic situation, such as maintenance of remission after a conventional treatment. Treatment with the yeast S. cerevisiae W303 did not show any improvement in clinical signs of the disease, confirming the importance of such negative control, rarely used in probiotic studies. Clinical results must be reinforced by histopathological and morphometric data, such as lesion and oedema extension as determined in the present work. Our histopathology results confirmed the data obtained in DAI determination, since in animals submitted to a DSS-induced colitis a protection was observed in mice previously treated with the yeast UFMG A-905 when compared to the DSS group. In these histopathological images, normal aspect of epithelial tissues of the colon was observed in control group, whereas animals from DSS group showed tissues with numerous injuries and degenerations of microvilli. Treatment with 905 yeast before DSS challenge led to a significant improvement in intestinal lesions, and this was confirmed by morphometric analysis of lesion size and oedema area in the colon which shows significant reduction between DSS group and 905 + DSS group. The absence of protection observed in the DAI evolution of W303 + DSS group was confirmed by the extense lesions in the colon epithelium of mice treated with this yeast. Treatment with 905 alone did not cause damage to the tissue of animals, but some morphometric data showed lesions in the W303 group (lesion perimeter in the colon). It is well known that histopathological changes caused by DSS-induced colitis, such as those described above, are accompanied by an infiltration of neutrophils in the lamina propria and crypt abscesses, providing an intense inflammation in intestinal mucosa and submucosa (Kobayashi, 2008; Perse and Cerar, 2012). The results of the present study showed a higher MPO activity in DSS animals when compared to the control group. Also a significant decrease was observed in this MPO activity in the 905 + DSS group in comparison to the DSS group, suggesting that the lower neutrophil recruitment caused less damage to the tissue. Clinical and experimental evidence suggest that Beneficial Microbes 6(6) Effect of yeast probiotic on experimental UC eosinophils play an important role in the interrelationship between the intestinal microbiota and IBD. Eosinophils express several pattern recognition receptors (e.g. Toll-like receptors), allowing them to recognise bacterial antigens and to produce pro-inflammatory cytokines and EPO, which have antibacterial properties (Hogan et al., 2012). The migration of eosinophils to the site of action is conducted by mechanisms involving chemoattractants, such as CCL11, which acts by recruiting eosinophils to target sites (Vieira et al, 2009). In this study, higher levels of EPO and CCL11 in animals with DSS-induced colitis were observed. Clinical and experimental studies indicate that eosinophils contribute to the pathogenesis of IBD, however it is not known how the eosinophilic host response reacts directly against infection and how this is related to tissue lesions or repair (Hogan et al., 2013). As expected from the analysis of MPO activity, the results obtained with the determination of CXCL1/KC levels in the colon showed higher values in the group treated with DSS compared to controls, and the previous treatment with the 905 yeast reduced this concentration, leading to a lower recruitment of neutrophils, and preserving the tissue. Neutrophil recruitment is generally accompanied by changes in the cytokine concentrations and in colon characteristics, modifying the permeability of the intestinal barrier and increased susceptibility to colitis (Danese, 2011; Turner, 2009). In this way, Matricon et al. (2010) and Perse and Cerar (2012) observed a significant increase in the expression of TNF-α and IFN-γ in the colon, one day after treatment with DSS. Probiotics may affect the mucosal immune system by reducing the production of intestinal inflammatory cytokines, such as TNF-α and IFN-γ, by modulating the nuclear factor κB pathway and by influencing the cross-talk between natural killer cells and dendritic cells. In the present study, such results were noted in mice challenged with DSS, which presented an increase in inflammatory cytokines, whereas in animals treated with the yeast 905 the concentration in IFN-γ was reduced, suggesting a probable link with a better preservation of the epithelial barrier integrity. The first line of defence against injuries from the intestinal lumen is the epithelial barrier. Due to its extreme toxicity, DSS, when used to induce experimental colitis, damages the integrity of the intestinal epithelial barrier and increases the permeability, even for large molecules. Here, the determination of intestinal permeability in vivo was performed using blood radioactivity measure after oral administration of 99mTc-DTPA. As expected, intestinal permeability was increased in the DSS group when compared to the control group, and physiological values were obtained back when mice were previously treated with 905 yeast before the challenge with DSS. The improvement of intestinal permeability by probiotics has been documented in several studies, and this effect was 813 http://www.wageningenacademic.com/doi/pdf/10.3920/BM2015.0018 - Monday, November 13, 2017 5:06:58 AM - Göteborgs Universitet IP Address:130.241.16.16 F.C.P. Tiago et al. apparently linked with an increased expression of ZO-1 in epithelial cells (Zyrek et al., 2007). Vilela et al. (2008) evaluated the role of S. boulardii ingestion on intestinal permeability in patients with Crohn’s disease, and results showed that patients treated with yeast obtained beneficial results, showing a reversion of the increased intestinal permeability associated with the pathology. In the present study, a similar effect was obtained in a murine model when the treatment with the 905 yeast was performed before the challenge with DSS. 5. Conclusions The data obtained with S. cerevisiae UFMG A-905 were promising for a potential alternative treatment of IBD, as demonstrated when used preventively in the present animal experimental model of UC, which showed an improvement of clinical manifestations (DAI), confirmed by histological and morphometrical data, as well as by results from determinations of MPO and EPO activities, intestinal permeability and some pro-inflammatory cytokines. Studies on the exact mechanisms responsible for these beneficial effects are needed for a better use of the yeast and this is currently carried out in our laboratory. Supplementary material Supplementary material can be found online at http:// dx.doi.org/10.3920/BM2015.0018. Figure S1. Score evolution of three parameters used for disease activity index calculation: presence of blood in faeces, faeces consistency and weight loss. Acknowledgments The authors are grateful to Clélia N. Silva for valuable technical help. This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundacão de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG). FCPT was the recipient of a post-doctoral fellowship from CNPq. References Arrieta, M.C., Madsen, K., Doyle, J. and Meddings, J., 2009. Reducing small intestinal permeability attenuates colitis in the IL10 genedeficient mouse. Gut 58: 41-48. 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