Wageningen Academic P u b l i s h e r s
Beneficial Microbes, 2015; 6(3): 277-286
Bifidobacterium longum subsp. infantis BB-02 attenuates acute murine experimental http://www.wageningenacademic.com/doi/pdf/10.3920/BM2014.0070 - Sunday, October 08, 2017 11:50:45 PM - Göteborgs Universitet IP Address:130.241.16.16
model of inflammatory bowel disease S.D.A. Elian1#, E.L.S. Souza1#, A.T. Vieira1,2, M.M. Teixeira2, R.M.E. Arantes3, J.R. Nicoli1 and F.S. Martins1* 1Department
of Microbiology, Institute of Biological Sciences, Federal University of Minas Gerais, Avenida Presidente Antônio Carlos 6627, Pampulha Campus UFMG, 31270-901 Belo Horizonte, MG, Brazil; 2Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais, Avenida Presidente Antônio Carlos 6627, Pampulha Campus UFMG, 31270-901 Belo Horizonte, MG, Brazil; 3Department of General Pathology, Institute of Biological Sciences, Federal University of Minas Gerais, Avenida Presidente Antônio Carlos 6627, Pampulha Campus UFMG, 31270-901 Belo Horizonte, MG, Brazil; [email protected]; #authors contributed equally to this work Received: 26 May 2014 / Accepted: 25 August 2014 © 2014 Wageningen Academic Publishers
RESEARCH ARTICLE Abstract Inflammatory bowel diseases (IBD) are chronic inflammatory conditions, characterised by remissions and relapses episodes, whose main manifestations are ulcerative colitis and Crohn’s disease. Ulcerative colitis (UC), one of the main forms of IBD, has as standard treatment the use of corticosteroids and anti-inflammatory drugs. The use of antibiotics has been also reported, but the possible adverse effects, such as disturbance of the indigenous microbiota or resistance induction, should be taken into consideration, and thus the use of probiotics emerges as a possible alternative option of treatment. In this study, the oral administration of Bifidobacterium longum subsp. infantis BB-02 was evaluated as a preventive strategy for acute experimental UC induced in female BALB/c mice by ingestion of 3.5% dextran sulphate sodium in drinking water during 7 days. During this time, the daily disease activity index was evaluated, and on the seventh day the animals were euthanised to collect intestines and liver for analysis. Treatment with the probiotic resulted in clinical improvement of the animals. The histological and morphometric analyses showed a reduction of lesions and oedema in the gut, but there was no increase in the production of mucin. The dosage of secretory immunoglobulin A was significantly higher in the colitis group and reduced in the group treated with the probiotic. There was also a reduction in the inflammation of the colon, as demonstrated by a decrease in neutrophils infiltration, and KC/CXCL-1 levels. The intestinal permeability, which is typically increased during the onset of IBD, was also reduced by treatment with probiotic. Based on these data, it can be concluded that the bacterium B. infantis BB-02 has a probiotic potential for the attenuation of UC, but further studies should be conducted to verify the mechanism of protective action of the bacterium. Keywords: Inflammatory bowel diseases, ulcerative colitis, Bifidobacterium infantis, inflammatory cytokines
1. Introduction Inflammatory bowel diseases (IBD) are a group of inflammatory conditions of the large and small bowel. The major types of IBD are Crohn’s disease (CD) and ulcerative colitis (UC) (Xavier and Podolsky, 2007). The aetiology of both IBD still remains largely unclear, but it is accepted that a combination of genetic susceptibility characteristics and an altered immune response driven by microbial factors in the enteric environment contributes to the initiation and
chronification of the disease (Wirtz et al., 2007). UC has as standard treatment the use of corticosteroids and antiinflammatory drugs. The use of antibiotics has also been reported, but some of their possible adverse effects, such as induction of antimicrobial resistance and of ecological disturbance in the indigenous microbiota, should be taken into consideration. For this reason, the use of probiotics has emerged as a real alternative treatment, especially in mild cases of the disease.
ISSN 1876-2833 print, ISSN 1876-2891 online, DOI 10.3920/BM2014.0070277
http://www.wageningenacademic.com/doi/pdf/10.3920/BM2014.0070 - Sunday, October 08, 2017 11:50:45 PM - Göteborgs Universitet IP Address:130.241.16.16
S.D.A. Elian et al.
Since the discovery of the beneficial effect of sulfasalazine in UC, the number of agents used for treatment of IBD has increased (Macpherson and Harris, 2004). One of the latest additions to these therapies is the use of probiotics. According to the definition recommended by the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO), probiotics are ‘live microorganisms which when administered in adequate amounts confer a health benefit to the host’ (FAO/WHO Report, 2002). Lactic acid bacteria and bifidobacteria are the most common and first types of microbes used as probiotics. Bifidobacteria were first isolated at the beginning of the 20th century by Henry Tissier, who showed that bifidobacteria are predominant in the gut microbiota of breast-fed babies, recommending administration of such bacteria to infants suffering from diarrhoea (Martins et al., 2009). Various Bifidobacterium strains have shown important probiotic properties in biological and clinical assays (Vieira et al., 2013), and some of them are already used in the food industry. The strain BB-02 as well as Bifidobacterium lactis BB-12 and Bifidobacterium longum 51A have been tested in well controlled clinical trials (randomised, double-blind, placebo-controlled, multicentre study), showing a reducing effect on incidence and severity of necrotising enterocolitis, antibiotic-associated diarrhoea frequency and constipation symptoms, respectively (Bin-Nun et al., 2005; Corrêa et al., 2005; Guerra et al., 2011; Jacobs et al., 2013; Ofek Shlomai et al., 2014). In the present study, oral administration of B. longum subsp. infantis BB-02 was evaluated as a possible preventive strategy for UC attenuation in an experimental murine model of acute colitis induced by dextran sulphate sodium (DSS).
2. Materials and methods Animals Female 6 weeks-old BALB/c mice were obtained from CEBIO (Center for Bioterism), Federal University of Minas Gerais, Belo Horizonte, Brazil. Water and commercial autoclavable diet were sterilised by steam and administered ad libitum, and animals were maintained in a ventilated animal caging system (Alesco Ltda., Campinas, SP, Brazil) with controlled lighting (12 h light, 12 h dark), humidity (60-80%) and temperature (22±1 °C) (Martins et al., 2013). All experimental procedures were carried out according to the standards set forth by the Brazilian Society of Laboratory Animal Science/Brazilian College for Animal Experimentation (SBCAL/COBEA, 2006). The study was approved by the Ethics Committee in Animal Experimentation of the Federal University of Minas Gerais (CEUA/UFMG, protocol no. 047/2011).
278
Bacterial strain, growth conditions and treatment B. infantis BB-02 (also known as strain CHCC2228, Patent No. WO2010023178 A1) (Bennedsen et al., 2011) was supplied by Christian Hansen A/S (Horsholm, Denmark). For the experiments, B. infantis BB-02 was grown on De Man, Rogosa and Sharpe (MRS) broth (Difco, Sparks, MD, USA) during 48 h in an anaerobic chamber (Forma Scientific Company, Marietta, OH, USA,) containing an atmosphere of 85% N 2, 10% H2 and 5% CO2, at 37 °C without agitation. For treatment, mice were fed by oral gavage once a day with 0.1 ml of a suspension containing 9.0 log10 colony forming units (cfu)/ml resuspended in phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4), 10 days before colitis induction, and treatment was continued during the induced disease. Control and DSS groups received 0.1 ml PBS by oral gavage.
Induction of dextran sulphate sodium colitis Colitis was induced by the addition of DSS (36,000 to 50,000 kDA; MP Biomedicals, Solon, OH, USA) 3.5% (w/v) in drinking water ad libitum for 7 days (Wirtz et al., 2007). DSS solution was changed on days 3 and 5 of disease induction.
Experimental design Four groups of mice (12 animals per group) were evaluated: CTL, not treated with B. infantis BB-02 and without colitis induction; Bifido, only treated with B. infantis BB-02; DSS, only submitted to colitis induction; and Bifido+DSS, treated with B. infantis BB-02 and submitted to colitis induction. Animal weight, faeces consistency, and presence of blood in faeces were assessed daily during the colitis period for determination of the disease activity index (DAI). Animals were sacrificed 7 days after colitis induction, and colon was removed and analysed for histological scoring, measurement of myeloperoxidase (MPO, as indicator of the extent of neutrophil infiltration) and eosinophil peroxidase (EPO, as indicator of the extent of eosinophil infiltration) activities and cytokines profile (Eotaxin/CCL-11 and KC/ CXCL-1). Contents from the small intestine were used for secretory immunoglobulin A (IgA) determination. Colon length was also analysed. Another set of the same groups (6 animals per group) was used to determine in vivo permeability using the fluorescein isothiocyanate (FITC)labelled dextran method.
Daily disease activity index Mice were monitored clinically every day after colitis induction. DAI assessment was carried out according to the scoring system by Albert et al. (2010) and Cooper et al. (1993), with some modifications. The index (0-10) was based on the cumulative scores from 3 different parameters: Beneficial Microbes 6(3)
http://www.wageningenacademic.com/doi/pdf/10.3920/BM2014.0070 - Sunday, October 08, 2017 11:50:45 PM - Göteborgs Universitet IP Address:130.241.16.16
faeces consistency (0 = normal, 1 = soft but still formed, 2 = very soft, 3 = liquid), presence of blood in faeces (0 = negative hemoccult, 1 = positive hemoccult, 2 = blood traces visible in faeces, 3 = rectal bleeding) and weight loss (0 = no weight loss, 1 = loss from 1 to 5% of body weight, 2 = 5 to 10%, 3 = 10 to 20%, 4 = >20%). Faecal blood was tested using Hemaccult cards (INLAB-Diagnostica, São Paulo, SP, Brazil).
Histologic and morphometric analysis Samples from the small intestine, colon and liver were fixed in 4% formaldehyde and processed for inclusion in paraffin. For analysis of intestinal lesions, the tissues was rolled up (filled rolls) and fixed in Bouin. Histological sections (3-5 µm) were stained with haematoxylin and eosin or by the periodic acid Schiff (PAS)/Alcian Blue (AB) method, coded and analysed by one pathologist who was unaware of the experimental conditions for each group. The representative histopathological aspects of intestines and liver were documented using a digital camera (Olympus BX51; Olympus, Tokyo, Japan) and the software Image-Pro Express 4.0 for Windows (Media Cybernetics, Bethesda, MD, USA). For the evaluation of mucins, the combination of PAS and AB was used to distinguish neutral from acid mucin. After deparaffinised, slides containing colon sections were washed with running water for 5 min. Then, the sections were dipped in the equilibrium solution (3% acetic acid) for 3 min, immersed in 1% AB (pH 2.5) and washed with equilibration solution and distilled water. After this step, the slides were dipped in aqueous solution of 0.5% periodic acid for 5 min, incubated in Schiff reactive for 10 min, counterstained with Harris haematoxylin and washed with running water for 15 min. After staining, sections were dehydrated and mounted on Entellan slides (Merck). For the morphometric examination (areas with injury, oedema and production of mucin), the measures in the photomicrographs were done by using the program ImageJ (v. 1.47f, Wayne Rasband/National Institutes of Health, Bethesda, MD, USA). The relationship among the total length observed in the micrograph and the partial length where injuries or oedema could be observed was considered.
Myeloperoxidase assay The extent of neutrophil accumulation in the colon tissue was measured by assaying MPO activity, as previously described (Vieira et al., 2005). Briefly, the tissue was removed and snap-frozen in liquid nitrogen. Upon thawing, the tissue was homogenised with 1.9 ml buffer, pH 4.7 (0.1 M NaCl, 0.02 M NaH2PO4•1H2O, 0.015 M Na2-EDTA) and centrifuged at 12,000×g for 10 min. The supernatant was discarded and the precipitate was subjected to hypotonic lysis. After further centrifugation, the precipitate was resuspended in a 0.05 M NaH2PO4 buffer (pH 5.4) Beneficial Microbes 6(3)
Bifidobacterium infantis effects in IBD model
containing 0.5% hexadecytrimethyl-amonium bromide (HTAB) (Sigma-Aldrich, St. Louis, MO, USA), 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 for MPO activity by measuring the change in optical density at 450 nm using tetramethylbenzidine (Sigma-Aldrich). Results were expressed as arbitrary units (AU) denoting activity of MPO related to casein-elicited murine peritoneal neutrophils processed in the same way.
Eosinophil peroxidase assay EPO assay was performed as previously described (Strath et al., 1985). Briefly, 100 mg tissue was weighed, homogenised in 1.9 ml PBS, and centrifuged at 12,000×g for 10 min. The supernatant was discarded, and the erythrocytes were lysed by using a hypotonic solution. The samples were then centrifuged, the supernatant discarded, and the pellet suspended in 1.9 ml 0.5% HTAB in PBS buffer. 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, Sigma-Aldrich) (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.
Cytokine analysis Colon (100 mg) was homogenised in 1 ml PBS containing anti-proteases (0.1 mM PMSF, 0.1 mM benzethonium chloride, 10 mM EDTA, and 20 Kallikrein Inhibitor Units of aprotinin A, all purchased from Sigma-Aldrich) and 0.05% Tween 20 (Sigma-Aldrich). The samples were then centrifuged for 10 min at 12,000×g, and the supernatant was used immediately for assays. The concentration of cytokines (KC/CXCL-1 and Eotaxin/CCL-11) was measured by enzyme-linked immunosorbent assay (ELISA) using commercially available antibodies and according to the procedures supplied by the manufacturer (R&D Systems, Minneapolis, MN, USA) (Martins et al., 2013).
In vivo permeability assay In vivo permeability assay to assess barrier function was performed using dextran-labelled with fluorescein isothiocyanate (FITC-dextran, FD4, MW 3,000-5,000; Sigma-Aldrich) method, as described by Yan et al. (2009). Food and water were withdrawn for 4 h and mice were gavaged with FITC-dextran (60 mg/100 g body weight). After 3 h, blood was collected by cardiac puncture, processed for serum collection, and fluorescence intensity of each sample measured (excitation, 492 nm; emission, 525 279
S.D.A. Elian et al.
nm; Spectromax M3; Molecular Devices Inc., Sunnyvale, CA, USA).
http://www.wageningenacademic.com/doi/pdf/10.3920/BM2014.0070 - Sunday, October 08, 2017 11:50:45 PM - Göteborgs Universitet IP Address:130.241.16.16
Secretory immunoglobulin A determination The small intestine was removed by cutting the gastroduodenal and ileocaecal junction. The contents were removed, weighed and diluted with PBS, pH 7.2, containing anti-proteases in a 500 mg content per 2.0 ml of PBS. Then, they were centrifuged (2,000×g, 30 min, 4 °C) and the supernatant collected and frozen at -80 °C for later determination of immunoglobulins. The determination of total secretory immunoglobulin A (sIgA) level was performed by ELISA as previously described by Martins et al. (2009).
Statistical analysis All experiments were repeated at least three times and the graphs represent a typical experiment. Data were expressed as means ± standard error of the means and analysis performed using the statistical software GraphPad Prism 5.00 (GraphPad Software, San Diego, CA, USA). Differences between means were evaluated using analysis of variance (ANOVA test), followed by Newman-Keuls test. Results with P
Beneficial Microbes, 2015; 6(3): 277-286
Bifidobacterium longum subsp. infantis BB-02 attenuates acute murine experimental http://www.wageningenacademic.com/doi/pdf/10.3920/BM2014.0070 - Sunday, October 08, 2017 11:50:45 PM - Göteborgs Universitet IP Address:130.241.16.16
model of inflammatory bowel disease S.D.A. Elian1#, E.L.S. Souza1#, A.T. Vieira1,2, M.M. Teixeira2, R.M.E. Arantes3, J.R. Nicoli1 and F.S. Martins1* 1Department
of Microbiology, Institute of Biological Sciences, Federal University of Minas Gerais, Avenida Presidente Antônio Carlos 6627, Pampulha Campus UFMG, 31270-901 Belo Horizonte, MG, Brazil; 2Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais, Avenida Presidente Antônio Carlos 6627, Pampulha Campus UFMG, 31270-901 Belo Horizonte, MG, Brazil; 3Department of General Pathology, Institute of Biological Sciences, Federal University of Minas Gerais, Avenida Presidente Antônio Carlos 6627, Pampulha Campus UFMG, 31270-901 Belo Horizonte, MG, Brazil; [email protected]; #authors contributed equally to this work Received: 26 May 2014 / Accepted: 25 August 2014 © 2014 Wageningen Academic Publishers
RESEARCH ARTICLE Abstract Inflammatory bowel diseases (IBD) are chronic inflammatory conditions, characterised by remissions and relapses episodes, whose main manifestations are ulcerative colitis and Crohn’s disease. Ulcerative colitis (UC), one of the main forms of IBD, has as standard treatment the use of corticosteroids and anti-inflammatory drugs. The use of antibiotics has been also reported, but the possible adverse effects, such as disturbance of the indigenous microbiota or resistance induction, should be taken into consideration, and thus the use of probiotics emerges as a possible alternative option of treatment. In this study, the oral administration of Bifidobacterium longum subsp. infantis BB-02 was evaluated as a preventive strategy for acute experimental UC induced in female BALB/c mice by ingestion of 3.5% dextran sulphate sodium in drinking water during 7 days. During this time, the daily disease activity index was evaluated, and on the seventh day the animals were euthanised to collect intestines and liver for analysis. Treatment with the probiotic resulted in clinical improvement of the animals. The histological and morphometric analyses showed a reduction of lesions and oedema in the gut, but there was no increase in the production of mucin. The dosage of secretory immunoglobulin A was significantly higher in the colitis group and reduced in the group treated with the probiotic. There was also a reduction in the inflammation of the colon, as demonstrated by a decrease in neutrophils infiltration, and KC/CXCL-1 levels. The intestinal permeability, which is typically increased during the onset of IBD, was also reduced by treatment with probiotic. Based on these data, it can be concluded that the bacterium B. infantis BB-02 has a probiotic potential for the attenuation of UC, but further studies should be conducted to verify the mechanism of protective action of the bacterium. Keywords: Inflammatory bowel diseases, ulcerative colitis, Bifidobacterium infantis, inflammatory cytokines
1. Introduction Inflammatory bowel diseases (IBD) are a group of inflammatory conditions of the large and small bowel. The major types of IBD are Crohn’s disease (CD) and ulcerative colitis (UC) (Xavier and Podolsky, 2007). The aetiology of both IBD still remains largely unclear, but it is accepted that a combination of genetic susceptibility characteristics and an altered immune response driven by microbial factors in the enteric environment contributes to the initiation and
chronification of the disease (Wirtz et al., 2007). UC has as standard treatment the use of corticosteroids and antiinflammatory drugs. The use of antibiotics has also been reported, but some of their possible adverse effects, such as induction of antimicrobial resistance and of ecological disturbance in the indigenous microbiota, should be taken into consideration. For this reason, the use of probiotics has emerged as a real alternative treatment, especially in mild cases of the disease.
ISSN 1876-2833 print, ISSN 1876-2891 online, DOI 10.3920/BM2014.0070277
http://www.wageningenacademic.com/doi/pdf/10.3920/BM2014.0070 - Sunday, October 08, 2017 11:50:45 PM - Göteborgs Universitet IP Address:130.241.16.16
S.D.A. Elian et al.
Since the discovery of the beneficial effect of sulfasalazine in UC, the number of agents used for treatment of IBD has increased (Macpherson and Harris, 2004). One of the latest additions to these therapies is the use of probiotics. According to the definition recommended by the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO), probiotics are ‘live microorganisms which when administered in adequate amounts confer a health benefit to the host’ (FAO/WHO Report, 2002). Lactic acid bacteria and bifidobacteria are the most common and first types of microbes used as probiotics. Bifidobacteria were first isolated at the beginning of the 20th century by Henry Tissier, who showed that bifidobacteria are predominant in the gut microbiota of breast-fed babies, recommending administration of such bacteria to infants suffering from diarrhoea (Martins et al., 2009). Various Bifidobacterium strains have shown important probiotic properties in biological and clinical assays (Vieira et al., 2013), and some of them are already used in the food industry. The strain BB-02 as well as Bifidobacterium lactis BB-12 and Bifidobacterium longum 51A have been tested in well controlled clinical trials (randomised, double-blind, placebo-controlled, multicentre study), showing a reducing effect on incidence and severity of necrotising enterocolitis, antibiotic-associated diarrhoea frequency and constipation symptoms, respectively (Bin-Nun et al., 2005; Corrêa et al., 2005; Guerra et al., 2011; Jacobs et al., 2013; Ofek Shlomai et al., 2014). In the present study, oral administration of B. longum subsp. infantis BB-02 was evaluated as a possible preventive strategy for UC attenuation in an experimental murine model of acute colitis induced by dextran sulphate sodium (DSS).
2. Materials and methods Animals Female 6 weeks-old BALB/c mice were obtained from CEBIO (Center for Bioterism), Federal University of Minas Gerais, Belo Horizonte, Brazil. Water and commercial autoclavable diet were sterilised by steam and administered ad libitum, and animals were maintained in a ventilated animal caging system (Alesco Ltda., Campinas, SP, Brazil) with controlled lighting (12 h light, 12 h dark), humidity (60-80%) and temperature (22±1 °C) (Martins et al., 2013). All experimental procedures were carried out according to the standards set forth by the Brazilian Society of Laboratory Animal Science/Brazilian College for Animal Experimentation (SBCAL/COBEA, 2006). The study was approved by the Ethics Committee in Animal Experimentation of the Federal University of Minas Gerais (CEUA/UFMG, protocol no. 047/2011).
278
Bacterial strain, growth conditions and treatment B. infantis BB-02 (also known as strain CHCC2228, Patent No. WO2010023178 A1) (Bennedsen et al., 2011) was supplied by Christian Hansen A/S (Horsholm, Denmark). For the experiments, B. infantis BB-02 was grown on De Man, Rogosa and Sharpe (MRS) broth (Difco, Sparks, MD, USA) during 48 h in an anaerobic chamber (Forma Scientific Company, Marietta, OH, USA,) containing an atmosphere of 85% N 2, 10% H2 and 5% CO2, at 37 °C without agitation. For treatment, mice were fed by oral gavage once a day with 0.1 ml of a suspension containing 9.0 log10 colony forming units (cfu)/ml resuspended in phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4), 10 days before colitis induction, and treatment was continued during the induced disease. Control and DSS groups received 0.1 ml PBS by oral gavage.
Induction of dextran sulphate sodium colitis Colitis was induced by the addition of DSS (36,000 to 50,000 kDA; MP Biomedicals, Solon, OH, USA) 3.5% (w/v) in drinking water ad libitum for 7 days (Wirtz et al., 2007). DSS solution was changed on days 3 and 5 of disease induction.
Experimental design Four groups of mice (12 animals per group) were evaluated: CTL, not treated with B. infantis BB-02 and without colitis induction; Bifido, only treated with B. infantis BB-02; DSS, only submitted to colitis induction; and Bifido+DSS, treated with B. infantis BB-02 and submitted to colitis induction. Animal weight, faeces consistency, and presence of blood in faeces were assessed daily during the colitis period for determination of the disease activity index (DAI). Animals were sacrificed 7 days after colitis induction, and colon was removed and analysed for histological scoring, measurement of myeloperoxidase (MPO, as indicator of the extent of neutrophil infiltration) and eosinophil peroxidase (EPO, as indicator of the extent of eosinophil infiltration) activities and cytokines profile (Eotaxin/CCL-11 and KC/ CXCL-1). Contents from the small intestine were used for secretory immunoglobulin A (IgA) determination. Colon length was also analysed. Another set of the same groups (6 animals per group) was used to determine in vivo permeability using the fluorescein isothiocyanate (FITC)labelled dextran method.
Daily disease activity index Mice were monitored clinically every day after colitis induction. DAI assessment was carried out according to the scoring system by Albert et al. (2010) and Cooper et al. (1993), with some modifications. The index (0-10) was based on the cumulative scores from 3 different parameters: Beneficial Microbes 6(3)
http://www.wageningenacademic.com/doi/pdf/10.3920/BM2014.0070 - Sunday, October 08, 2017 11:50:45 PM - Göteborgs Universitet IP Address:130.241.16.16
faeces consistency (0 = normal, 1 = soft but still formed, 2 = very soft, 3 = liquid), presence of blood in faeces (0 = negative hemoccult, 1 = positive hemoccult, 2 = blood traces visible in faeces, 3 = rectal bleeding) and weight loss (0 = no weight loss, 1 = loss from 1 to 5% of body weight, 2 = 5 to 10%, 3 = 10 to 20%, 4 = >20%). Faecal blood was tested using Hemaccult cards (INLAB-Diagnostica, São Paulo, SP, Brazil).
Histologic and morphometric analysis Samples from the small intestine, colon and liver were fixed in 4% formaldehyde and processed for inclusion in paraffin. For analysis of intestinal lesions, the tissues was rolled up (filled rolls) and fixed in Bouin. Histological sections (3-5 µm) were stained with haematoxylin and eosin or by the periodic acid Schiff (PAS)/Alcian Blue (AB) method, coded and analysed by one pathologist who was unaware of the experimental conditions for each group. The representative histopathological aspects of intestines and liver were documented using a digital camera (Olympus BX51; Olympus, Tokyo, Japan) and the software Image-Pro Express 4.0 for Windows (Media Cybernetics, Bethesda, MD, USA). For the evaluation of mucins, the combination of PAS and AB was used to distinguish neutral from acid mucin. After deparaffinised, slides containing colon sections were washed with running water for 5 min. Then, the sections were dipped in the equilibrium solution (3% acetic acid) for 3 min, immersed in 1% AB (pH 2.5) and washed with equilibration solution and distilled water. After this step, the slides were dipped in aqueous solution of 0.5% periodic acid for 5 min, incubated in Schiff reactive for 10 min, counterstained with Harris haematoxylin and washed with running water for 15 min. After staining, sections were dehydrated and mounted on Entellan slides (Merck). For the morphometric examination (areas with injury, oedema and production of mucin), the measures in the photomicrographs were done by using the program ImageJ (v. 1.47f, Wayne Rasband/National Institutes of Health, Bethesda, MD, USA). The relationship among the total length observed in the micrograph and the partial length where injuries or oedema could be observed was considered.
Myeloperoxidase assay The extent of neutrophil accumulation in the colon tissue was measured by assaying MPO activity, as previously described (Vieira et al., 2005). Briefly, the tissue was removed and snap-frozen in liquid nitrogen. Upon thawing, the tissue was homogenised with 1.9 ml buffer, pH 4.7 (0.1 M NaCl, 0.02 M NaH2PO4•1H2O, 0.015 M Na2-EDTA) and centrifuged at 12,000×g for 10 min. The supernatant was discarded and the precipitate was subjected to hypotonic lysis. After further centrifugation, the precipitate was resuspended in a 0.05 M NaH2PO4 buffer (pH 5.4) Beneficial Microbes 6(3)
Bifidobacterium infantis effects in IBD model
containing 0.5% hexadecytrimethyl-amonium bromide (HTAB) (Sigma-Aldrich, St. Louis, MO, USA), 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 for MPO activity by measuring the change in optical density at 450 nm using tetramethylbenzidine (Sigma-Aldrich). Results were expressed as arbitrary units (AU) denoting activity of MPO related to casein-elicited murine peritoneal neutrophils processed in the same way.
Eosinophil peroxidase assay EPO assay was performed as previously described (Strath et al., 1985). Briefly, 100 mg tissue was weighed, homogenised in 1.9 ml PBS, and centrifuged at 12,000×g for 10 min. The supernatant was discarded, and the erythrocytes were lysed by using a hypotonic solution. The samples were then centrifuged, the supernatant discarded, and the pellet suspended in 1.9 ml 0.5% HTAB in PBS buffer. 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, Sigma-Aldrich) (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.
Cytokine analysis Colon (100 mg) was homogenised in 1 ml PBS containing anti-proteases (0.1 mM PMSF, 0.1 mM benzethonium chloride, 10 mM EDTA, and 20 Kallikrein Inhibitor Units of aprotinin A, all purchased from Sigma-Aldrich) and 0.05% Tween 20 (Sigma-Aldrich). The samples were then centrifuged for 10 min at 12,000×g, and the supernatant was used immediately for assays. The concentration of cytokines (KC/CXCL-1 and Eotaxin/CCL-11) was measured by enzyme-linked immunosorbent assay (ELISA) using commercially available antibodies and according to the procedures supplied by the manufacturer (R&D Systems, Minneapolis, MN, USA) (Martins et al., 2013).
In vivo permeability assay In vivo permeability assay to assess barrier function was performed using dextran-labelled with fluorescein isothiocyanate (FITC-dextran, FD4, MW 3,000-5,000; Sigma-Aldrich) method, as described by Yan et al. (2009). Food and water were withdrawn for 4 h and mice were gavaged with FITC-dextran (60 mg/100 g body weight). After 3 h, blood was collected by cardiac puncture, processed for serum collection, and fluorescence intensity of each sample measured (excitation, 492 nm; emission, 525 279
S.D.A. Elian et al.
nm; Spectromax M3; Molecular Devices Inc., Sunnyvale, CA, USA).
http://www.wageningenacademic.com/doi/pdf/10.3920/BM2014.0070 - Sunday, October 08, 2017 11:50:45 PM - Göteborgs Universitet IP Address:130.241.16.16
Secretory immunoglobulin A determination The small intestine was removed by cutting the gastroduodenal and ileocaecal junction. The contents were removed, weighed and diluted with PBS, pH 7.2, containing anti-proteases in a 500 mg content per 2.0 ml of PBS. Then, they were centrifuged (2,000×g, 30 min, 4 °C) and the supernatant collected and frozen at -80 °C for later determination of immunoglobulins. The determination of total secretory immunoglobulin A (sIgA) level was performed by ELISA as previously described by Martins et al. (2009).
Statistical analysis All experiments were repeated at least three times and the graphs represent a typical experiment. Data were expressed as means ± standard error of the means and analysis performed using the statistical software GraphPad Prism 5.00 (GraphPad Software, San Diego, CA, USA). Differences between means were evaluated using analysis of variance (ANOVA test), followed by Newman-Keuls test. Results with P
