Page 1Articles of 29 in PresS. Am J Physiol Gastrointest Liver Physiol (July 19, 2007). doi:10.1152/ajpgi.00132.2007
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The Anti-inflammatory Properties of Lactobacillus gasseri Expressing Manganese Superoxide Dismutase (MnSOD) Using the Interleukin 10-Deficient Mouse Model of Colitis
Ian M. Carroll1, Jason M. Andrus2, José M. Bruno-Bárcena3,4, Todd R. Klaenhammer5, Hosni M. Hassan4 and Deborah S. Threadgill1.
1
Department of Genetics, CB# 7264, University of North Carolina at Chapel Hill, Chapel Hill,
NC 27599. USA; 2Department of Biology, Augusta State University, Augusta, GA 30904. USA; 3
Biomanufacturing Training & Education Center (BTEC), CB# 7928, North Carolina State
University, Raleigh, NC 27606. USA; 4Department of Microbiology, North Carolina State University, CB# 7615, Raleigh, NC 27606, USA. 5Department of Food Science, North Carolina State University, CB# 7624, Raleigh, NC 27695, USA;
Running Head: L. gasseri-MnSOD treatment for colitis
Correspondence to: D. S. Threadgill (
[email protected]). Department of Genetics, CB#7264, University of North Carolina, Chapel Hill, NC 27599, USA. Phone: 919843-4680, Fax: 919-966-3292.
Copyright © 2007 by the American Physiological Society.
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2 ABSTRACT Emerging evidence has implicated reactive oxygen species (ROS) in the pathogenesis of inflammatory bowel disease (IBD). Although intestinal epithelial cells produce the ROSneutralizing enzyme superoxide dismutase (SOD) the protein and activity levels of copper/zinc (Cu/Zn) and manganese (Mn) SOD are perturbed in inflamed tissues of IBD patients. Thus, we investigated the ability of MnSOD from Streptococcus thermophilus to reduce colitis symptoms in Interleukin (Il) 10-deficient mice using Lactobacillus gasseri as a delivery vehicle. Cohorts of 13-15 Il10-deficient mice were left untreated or supplemented with native L. gasseri or L. gasseri expressing MnSOD for four weeks. Colonic tissue was collected and inflammation was histologically scored.
The presence of innate immune cells was investigated by
immunohistochemistry and the host antioxidant response was determined by quantitative PCR. It was demonstrated that L. gasseri was stably maintained in mice for at least three days. L. gasseri producing MnSOD significantly reduced inflammation in Il10-deficient mice compared to untreated controls (P < 0.05), while the anti-inflammatory effects of both native and MnSOD producing L. gasseri were more pronounced in males. The anti-inflammatory effects of L. gasseri were associated with a reduction in the infiltration of neutrophils and macrophages. Transcripts of antioxidant genes were equivalent in colonic tissues obtained from control and probiotic-treated Il10-deficient mice. This study demonstrates that L. gasseri producing MnSOD has significant anti-inflammatory activity that reduces the severity of colitis in the Il10-deficient mouse.
Keywords: Probiotic, Lactobacilli, Oxidative Stress, Inflammatory Bowel Disease.
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3 INTRODUCTION Crohn’s disease (CD) and ulcerative colitis (UC), collectively known as Inflammatory Bowel Diseases (IBD), are disorders with unknown causes and are characterized by recurrent intestinal inflammation. IBD is believed to result from one or a combination of factors including geneticbased host susceptibility, enteric microflora and a dysfunctional immune response (33). In addition to these factors, emerging evidence has suggested a role for oxidative stress in the initiation and progression of IBD since increased levels of oxidized proteins, lipids and nucleic acids are found in the inflamed mucosa of IBD patients (10, 16). It is well established that inflammation in the gut is associated with the recruitment of activated phagocytic leukocytes and the production of inflammatory mediators such as proteases, cytokines, arachidonic acid metabolites and reactive oxygen species (ROS) (34). Recent studies have demonstrated that ROS, such as the superoxide anion, are not merely by-products of inflammation but are central to its pathogenesis (16, 21, 30). The normal intestinal mucosa is equipped with a network of antioxidant enzymes that neutralize ROS in a two-step pathway. First, superoxide dismutases (SODs) convert the primary superoxide anion (O2-•) into the more stable metabolite, hydrogen peroxide (H2O2). Second, H2O2 is converted to water by catalase (CAT) or glutathione peroxidase (GPO). The activities of these enzymes are usually balanced to maintain a low and continual steady-state level of ROS. However, the levels of SOD in IBD patients are frequently depleted (14, 17), highlighting the potential for elevated levels of this enzyme to function as a therapeutic for IBD. Three isoforms of SOD are found in humans, copper/zinc (Cu/Zn)-SOD, manganese (Mn)-SOD and extracellular (EC)-SOD. The abundance and activity of all three isoforms of SOD in paired inflamed and non-inflamed tissues from CD and UC patients compared with
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4 samples from normal control mucosa were recently investigated (13, 14). The level of Cu/ZnSOD protein was mildly reduced in the lamina propria of inflamed tissue whereas MnSOD protein was increased, although this was not accompanied by an increase in activity. Since Cu/ZnSOD is a key anti-oxidant and is reduced in inflamed colonic tissues, the potential therapeutic effects of this enzyme as a treatment for IBD have been investigated. Interestingly both protective (15, 34, 35) and antagonistic (12) effects have been reported for Cu/ZnSOD. The variability of efficacy of Cu/ZnSOD in these studies may be due to the different experimental models used and the mode of delivery of the enzyme. Similar studies have not been reported for MnSOD. Another important characteristic of IBD is an aggressive reaction to enteric microflora, an aspect that is reiterated in all current mouse models of colitis. Although specific members of the gut flora can be more colitogenic than others (33), it is not known which members are most relevant in human patients. As the microflora plays an important role in IBD, numerous studies have focused on how to utilize commensal bacteria or ‘probiotics’ as a treatment strategy for IBD. Probiotics are live microorganisms which when administered in adequate amount confer a health benefit on the host (FAO 2001, www.fao.org). It is believed that increasing the numbers of specific organisms within the gut microbiota can have a beneficial effect either directly by regulating the host immune system (23, 28, 41, 44) or indirectly by altering the composition of the gut flora (7, 8). The beneficial effects of the Lactobacilli, members of the lactic acid bacteria (LAB), have received particular attention. These organisms are present at relatively low levels in the human colon, but recent studies have demonstrated their ability to modulate inflammation in the Il10-deficient mouse model of colitis using Lactobacillus reuteri (18), a combination of L. reuteri and L. paracasei (27), L. plantarum (25) and L. salivarius (19, 23) and to help prevent
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5 enteric infections using L. casei (29). The use of microorganisms as vehicles to deliver biologically active molecules for the treatment of gastrointestinal diseases has also been demonstrated (39). Consequently, the current study was designed to investigate the therapeutic effects of L. gasseri producing the MnSOD from S. thermophilus in the Il10-deficient mouse model of colitis.
MATERIALS AND METHODS Bacterial Strains. The probiotics used in these studies were L. gasseri NC1500 and L. gasseri NC1501 (3, 4). L. gasseri NC1500 harbors the empty plasmid pTRK563 while L. gasseri NC1501 harbors the plasmid pSodA. pSodA was constructed by cloning the superoxide dismutase gene, sodA, from S. thermophilus into pTRK563. It has been demonstrated that L. gasseri expressing MnSOD offers protection to this organism from the superoxide radical, hydrogen peroxide and the hydroxyl radical, in vitro (3, 4). Both strains of L. gasseri were grown aerobically in MRS broth containing erythromycin at a final concentration of 2 µg/ml at 37°C. The L. gasseri strain used in this study was chosen due to its human origin (3) and its effective ability to colonize the mouse GI tract.
Il10-deficient Mouse Model. The wild-type C57BL/6J mice and its congenic Interleukin 10deficient (Il10-/-) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were bred and maintained under specific pathogen free conditions (SPF) in ventilated racks with HEPA-filtered air at constant temperature and humidity on a 12-hour light/12-hour dark cycle in AAALAC-approved facilities at the University of North Carolina at Chapel Hill. Mouse cages were fitted with wire bottoms at the initiation of experiments to prevent coprophagy. Mice were
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6 fed Purina 5058 rodent chow and water ad libitum under SPF conditions. All studies were performed under IACUC approved protocols.
Detection of L. gasseri. Colonization/persistence of each of the L. gasseri strains (NC1500 and NC1501) in the gut of the wild-type mice was tested by orally administering 1 × 109 CFU of each strain in 20 µl of sterile PBS to C57BL/6J mice (n = 5 mice for each strain) every day for five days. Fecal pellets were collected from before, during and up to seven days after the probiotic administration. Each fecal pellet was homogenized in 1 ml of sterile PBS. The homogenates were serially diluted and plated onto MRS agar containing 2 µg/ml erythromycin. The daily average CFUs of either probiotic strain in mouse feces were determined.
Probiotic Therapy. C57BL/6J-Il10-/- mice begin developing inflammation around four weeks of age when housed in the UNC animal facilities. Although the C57BL/6J-Il10-/- mice are regarded as colitis resistant (1), the presence of antagonistic gut flora like Helicobacter hepaticus (H. hepaticus) can dramatically accelerate colitis. Random sampling of feces from of our Il10 -/- mice demonstrated the presence of H. hepaticus and Enterococcus faecalis (potentially colitogenic) in this mouse colony. Consequently, L. gasseri administration was carried out on three-week-old mice before the onset of inflammation and continued for four weeks. Fresh cultures of L. gasseri NC1500 and NC1501 were grown in MRS broth containing 2 µg/ml erythromycin. The bacterial cells were pelleted and washed twice with PBS. The concentration of each L. gasseri strain was adjusted to 1 × 109 CFU/ml based on OD600 readings which had previously been correlated with CFU numbers (data not shown). Next, 1 ml of each suspension was pelleted by centrifugation
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7 and then resuspended in 20 µl of PBS. Mice were administered sterile PBS (control group; 8 male and 7 female), L. gasseri NC1500 (5 male and 8 female) or L. gasseri (MnSOD) NC1501 (7 male and 7 female). Each mouse was supplemented with 20 µl PBS (control) or 1 × 109 CFU (of either L. gasseri strain) orally in 20 µl PBS every day for five days and then every third day until the completion of the study. After four weeks of treatment the colon from each CO2 euthanized mouse was removed, flushed, weighed and splayed open longitudinally before processing for histological analysis. The composition of the gut microflora can vary between mice from different litters (5), much the same way it varies between unrelated humans (45). Therefore, a control (PBS treated) mouse from each litter was used to control for inter-litter heterogeneity.
Histopathology. Cecal and colonic tissues from wild-type and Il10-/- mice were fixed in 10 % neutral buffered formalin and embedded in paraffin before cutting 7 µm sections and staining with haematoxylin and eosin (H&E) for light microscopic examination. Evidence of colitis was based upon the presence of lymphocyte infiltration, goblet cell depletion, crypt architectural distortion and ulceration. The degree of inflammation in each tissue section was graded on a scale from 0 to 4 based on the following criteria: 0, no evidence of inflammation; 1, low level of lymphocyte infiltration, minimal epithelial hyperplasia; 2, generalized infiltration of lymphocyte cells in the lamina propria, obvious epithelial hyperplasia, mild goblet cell depletion; 3, pronounced lamina propria cell infiltration, marked epithelial hyperplasia, marked decrease in goblet cells, architectural distortion; 4, all the criteria from grade 3 plus the presence of crypt abscesses and mucosal ulceration. Inflammatory scores were recorded for a generalized state of the entire colon as well as for the proximal and distal colon. All samples were masked until
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8 grading of inflammation was complete.
Immunohistochemistry. To quantify inflammatory cells, myeloperoxidase (MPO) and cyclooxygenase-2 (COX2) were detected by immunohistochemistry (IHC) in paraffin embedded tissues as previously described (11). Briefly, slides with sectioned colonic tissues were deparaffinized in xylene and graded alcohols. After a five minute microwave exposure in 10 mM citrate buffer, the slides were immersed in buffer containing 3 % H2O2 to inhibit endogenous peroxidase activity. Blocking was performed for one hour with normal goat serum and then the slides were incubated with a primary antibody overnight at 4°C. Rabbit anti-MPO (DakoCytomation, Denmark) was used to detect neutrophils (11) and rabbit anti-COX2 (Cayman Laboratories, MI) was used to detect activated macrophages (36). After brief washes the slides were incubated with biotinylated goat anti-rabbit secondary antibody for one hour then with avidin-horseradish-peroxidase (Vectastain™, CA). After additional washes, the slides were developed with 3,3 -diaminobenzidine (DAB; Biomedia, CA). Slides were then counter-stained with hematoxylin, dehydrated and mounted with a coverslip. Normal epithelial tissue and test tissues lacking a primary antibody incubation step were used as controls for both MPO and COX2 staining.
Quantitative PCR. Total colonic RNA was extracted from Il10-/- mice administered either PBS (control), L. gasseri NC1500 or L. gasseri (MnSOD) NC1501 (n = 3, for each group) with Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Briefly, each colon was homogenized in 1mL Trizol solution using a Retsch MM300 bead beating apparatus (Retsch, Inc., Newton, PA). 220 µl chloroform was added to the homogenate, mixed and then centrifuged
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9 at 12 000 × g for 15 min at 4°C. RNA was precipitated from the aqueous layer by adding an equal volume of 2-propanol and incubating the mixture at room temperature for 10 min. The mixture was then centrifuged at 12,000 g for 10 min at 4°C. The RNA pellet was washed with 1mL 70% ethanol and resuspended in 100 µL of nuclease free water. The concentration of each RNA preparation was determined by measuring optical density at 260 nm. The High Capacity cDNA Archive Kit (Applied BioSystems, Foster City, CA) was used to reverse transcribe 500 ng of total colonic RNA into first-strand complementary DNA (cDNA) with 250 U of reverse-transcriptase, 1 × random primers and 1mM dNTPs in a 100 µl reaction mixture at 37°C for 120 min. Quantitative polymerase chain reaction (PCR) was carried out with a real-time MX 3000P™ thermocycler (Stratagene, La Jolla, CA) using a fluorescently labeled (FAM) oligonucleotide probe (Applied BioSystems, Taqman® Gene Expression Assays). The genes investigated were Sod2 (Mm00449726_m1), Sod3 (Mm00448831_m1), Cat (Mm00437992_m1) and Gpx1 (Mm00656767_g1). The PCR reaction mixture contained 10 ng cDNA, 1 × primer/probe mix and 0.5 U of AmpliTaq Gold® PCR master mix in a final volume of 15 µl. An initial denaturing step of 10min at 95°C was followed by 40 cycles of amplification at 95°C for 15 s and 60°C for 1 min. PCR products were quantified using cDNA standards of known concentration for each gene investigated. Each gene analyzed was normalized with Gapdh yielding a relative transcript level. Gapdh has been previously used to normalize oxidative stress related genes in different organs in the mouse (2, 20, 42).
Statistical Analysis. Experimental groups were analyzed for significance of differences between the means of treatment groups and control groups by ANOVA with Tukey’s multiple comparison test or an unpaired T-test using GraphPad (v4.0a; Prism,, San Diego, CA).
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RESULTS Gastrointestinal Transit. The presence of L. gasseri NC1500 and L. gasseri NC1501 could be detected within the feces of wild type C57BL/6J mice for up to four days after cessation of treatment following five consecutive days of feeding 1 × 109 CFU (Fig. 1). Indeed, the 16S rDNA gene was sequenced from random colonies obtained on the erythromycin containing MRS plates to confirm the selectivity of this method for the administered L. gasseri strains. A fluctuation was observed in the CFU levels of each organism during the first four days of probiotic administration. However, CFU levels of the two L. gasseri cultures stabilized on day five (last day of probiotic feeding) through day nine, showing little variation between the viable counts of either strain in feces. It was concluded that after five days of supplementation both L. gasseri NC1500 and L. gasseri NC1501 (MnSOD) persisted within the mouse GI tract for the same length of time and at approximately the same viable cell numbers. Additionally, no obvious differences were observed in bacterial fecal densities or persistence in the GI tract between male and female mice in this study. A feeding regimen was constructed based on the knowledge that bacterial numbers significantly decline three days after the cessation of bacterial supplementation (Fig. 1). Thus, each test mouse received 1 × 109 CFU of the respective L. gasseri strain every third day after an initial five day administration ensuring that either bacterium would be present in the mouse gut for the duration of the experiments (Fig. 2). The presence of either L. gasseri strain was detected in the feces of randomly sampled test mice throughout the four weeks of bacterial supplementation (data not shown).
L. gasseri and MnSOD biotherapy. Following four weeks of treatment, the dissected colons
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11 from L. gasseri-treated and control mice displayed distinct phenotypic differences. The colons from PBS-treated control mice were distinctly thicker than the colons dissected from littermates fed either L. gasseri strain (Fig. 3A). This observation was quantified when the wet weight of the majority of colons in each group was measured revealing that the wet weights of the colons from L. gasseri fed mice were lower than those from corresponding control mice. Although the colons from mice fed with either strain displayed a marked decrease in weight these reductions were not statistically significant (colonic weights: control = 0.5546 ± 0.0518 g, n = 5 males and 7 females; L. gasseri NC1500 = 0.4540 ± 0.0195 g, n = 4 males and 7 females; L. gasseri NC1501 (MnSOD) = 0.4504 ± 0.03172 g, n = 7 males and 5 females).
Histological Evaluation. Similar to total colonic weights, the average histological scores for Il10/-
mice supplemented with either strain of L. gasseri showed a decrease in the degree of
inflammation compared to control mice; however only L. gasseri NC1501 (MnSOD) displayed a statistically significant difference (Fig. 3B; P < 0.05). Compared to wild-type mice (Fig. 3C), Il10-/- from the control group displayed strong inflammatory damage evident in the form of crypt architectural distortion, pronounced cell infiltration in the lamina propria, goblet cell destruction, hyperplasia and the presence of crypt abscesses (Fig. 3D). Although some tissues from mice fed L. gasseri NC1500 showed a reduction in inflammation, the majority of tissues from this group exhibited characteristics of severe inflammatory damage (Fig. 3E). In contrast, the tissues from mice fed L. gasseri expressing MnSOD, NC1501, displayed a substantial reduction in inflammation characteristics (Fig. 3F). Colons from L. gasseri 1501 (MnSOD)-treated mice exhibited infiltrating cells within the lamina propria, but crypt architecture was normal with intact goblet cells with a greatly reduced occurrence of hyperplasia.
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12 The level of inflammation in colonic tissues was also recorded for the proximal and distal ends of each colon revealing that the degree of inflammation was more pronounced at the distal end (Fig. 3G). Although not statistically significant, there was a slight reduction in inflammatory scores for the proximal colon for both probiotics. L. gasseri NC1501, however, produced a significant reduction in the inflammatory score for the distal colon (P < 0.05).
Sex Effects. To determine whether males and females respond equivalently, the inflammatory scores for whole colons was evaluated with respect to sex. The female Il10-/- control group displayed an inflammatory score that was markedly lower than the male controls (Fig. 4A versus B). Interestingly, L. gasseri NC1500 treatment was associated with an increase in the inflammatory score in female colons, but a significant reduction of inflammation in males (P < 0.05). The antagonistic and protective effects of L. gasseri NC1500 in female and male mice, respectively, were also observed in both the proximal and distal regions of the colon (data not shown). The SodA producing L. gasseri (NC1501) caused a reduction of inflammation in both male and female mice, but only males reached statistical significance (Fig. 4B; P < 0.01). A comparison of the male and female inflammatory scores from each treatment group revealed that the inflammation was in general more severe in male Il10-deficient mice (Fig. 4C).
Innate Immune Response. Colonic tissue sections from the majority of mice in each experimental group (control, n = 7: L. gasseri, n = 13; L. gasseri MnSOD, n = 13) were analyzed by immunohistochemistry (IHC) to evaluate the innate immune response. Since neutrophil infiltration in the lamina propria is a characteristic of the inflammation score, staining for myeloperoxidase (MPO), a marker for neutrophils (11), was used to support the H&E-based
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13 score. Compared with wild-type mice (Fig. 5A), colonic tissues from control mice contained a large number of MPO positive cells within the lamina propria (Fig. 5B). The number of neutrophils present in colonic tissues was correlated with the severity of inflammation and the histological score (Fig. 5B-D). Colonic tissue sections from the same mice used for MPO staining were also stained for cyclooxygenase-2 (COX2), a marker for macrophages (36) by IHC. Many infiltrating cells in the lamina propria stained positive for COX2 in inflamed colonic tissue (Fig. 6). These COX2 positive cells occurred in patches throughout the entire colon and consistent with MPO staining the number of these positive cell patches was reduced in less inflamed tissues (Fig. 6A-D) supporting the H&E inflammation scores. The COX2 positive cells were not as abundant as MPO positive cells and occurred mainly where cellular infiltrate in the lamina propria was dense or near breaches in the epithelial layer (Fig. 6B). Overall the presence of MPO and COX2 positive cells in the lamina propria of tissue sections tightly mirrored the histological grade applied to each section. For example, when each stained section was grouped by histological grade the number of MPO and COX2 positive cells progressively increased in tandem with the inflammatory histological scores applied. Although the number of neutrophils and macrophages were significantly decreased in the L. gasseri and L. gasseri MnSOD groups there was no significant difference observed between the two probiotic treated groups with respect to these inflammatory cells (Patches of MPO positive cells in tissue sections: control, 15 ± 4.925; L. gasseri, 5.5338 ± 1.403, P < 0.05; L. gasseri MnSOD, 5.231 ± 1.49, P < 0.05. Patches of COX2 positive cells in tissue sections: control, 10.75 ± 1.66; L. gasseri, 3.231 ± 1.105, P < 0.001; L. gasseri MnSOD, 2.846 ± 1.78, P < 0.001). The sex effects seen with histological grading were not fully reiterated by MPO and COX2 scoring, with the
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14 exception that the reduction in MPO and COX2 staining reached statistical significance in males but not females. Antioxidant Gene Expression. In response to inflammatory stimuli, several antioxidant genes involved in the neutralization of the superoxide radical (Sod2, MnSOD and Sod3, (EC) Cu/ZnSOD) and hydrogen peroxide (Cat, catalase and Gpx1, glutathione peroxidase) are expressed. Quantitative PCR analysis showed that the expression of the antioxidant genes associated with superoxide radicals (Sod2 and Sod3) increased in colons of untreated Il10deficient mice (Fig.
7A-B) while the genes involved with detoxifying hydrogen peroxide
decreased in expression (Fig. 7C-D). The transcript levels of the antioxidant genes in the colons from mice administered either L. gasseri NC1500 and L. gasseri NC1501 (MnSOD) were similar to those of the control mice.
DISCUSSION The implication of microbial antigens in the pathogenesis of IBD has led to the concept of using natural intestinal organisms to alter the composition of the gut flora or to deliver therapeutic molecules to target areas in the intestinal tract. The use of probiotics has been extensively investigated in animal models and clinical trials (7, 18, 19, 23, 27-29, 32, 41), including L. gasseri, which has been tested for therapeutic effects in a rat model of colitis (24). In addition, the use of genetically modified bacteria as delivery vehicles for biologically active molecules for immunization (37, 38) and to treat IBD (37, 43) has been demonstrated. In this study, we show that unmodified L. gasseri had a non-statistically significant effect on colitis in the Il10-deficient mouse model overall (i.e. with both males and females included), but did significantly reduce the inflammatory damage in male Il10- deficient mice. In contrast, modified L. gasseri showed
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15 significant effects on colitis reduction both overall, and in male mice thus supporting the preferred use of the modified strain. It has been suggested that an effective probiotic strain must transiently persist and reach high numbers at the target area of the gut (25). Previous studies have demonstrated that when administered orally L. salivarius spp. salivarius and L. plantarum can persist in mouse feces for up to two and eleven days, respectively, after cessation of probiotic administration (22, 25). We found that both L. gasseri strains can persist for up to four days in the mouse GI tract. Since, the engineered strain of L. gasseri does not display a colonization advantage in wild type C57BL/6J mice, the therapeutic benefits arising from the use of this organism may not be due to a prolonged retention time or higher viable numbers within the gut. However, the oxidative environment in Il10-deficient mice differs from wild type C57BL/6J mice, thus the retention time of L. gasseri producing MnSOD in the inflamed GI tract may also differ. Based on random bacterial sampling throughout the experimental period, there was no significant retention time difference (data not shown) for either strain in the inflamed GI tract, suggesting this is not a major factor in the treatment outcomes. The L. gasseri host strain used in this study is the neotype strain of this species and is of human origin (3). Indeed, the transit of an L. gasseri strain in the human intestinal tract has been demonstrated (26). It is possible that the retention time in the intestinal tract of the mouse of the L. gasseri strains used in this study may vary between other strains and animal hosts. Regardless, the transient presence of either strain of L. gasseri in the mouse gastrointestinal tract implies that this organism cannot overcome competitive exclusion by the autochthonous flora. Thus, it is unlikely these particular strains of L. gasseri reduce inflammation by permanently altering the composition of the gut flora, a mechanism that has been previously proposed for probiotics (6). It
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16 is more likely that L. gasseri introduced into the gut in relatively large numbers temporarily dominates a particular niche in the host gut flora altering its composition. Thus, as lactic acid bacteria are relatively unreactive organisms (40) the host is exposed to a less antigenic microbiota. This mechanism might also reduce exposure of the host immune system to highly antagonistic organisms such as Enterococcus faecalis (9) and Helicobacter hepaticus (31). The use of endogenous SOD as a therapeutic molecule for IBD has been investigated previously (12, 15, 34, 35). Attempts to replenish Cu/ZnSOD levels either by subcutaneous delivery or through transgenesis have demonstrated some protective effect (15, 35). Unlike the present study, these investigations did not demonstrate that SOD can reduce inflammatory damage at the histological level. An explanation for the partial success of Cu/ZnSOD therapy may be that in inflamed tissues of IBD patients the activity and protein levels of MnSOD are more significantly altered than Cu/ZnSOD (13). Thus, replenishing MnSOD levels in inflamed tissues would appear a better strategy. We describe herein a novel approach using bacterial delivery of a bacterial MnSOD to curtail inflammation in the Il10-deficient mouse model of colitis. Interestingly, we found that both the parental and MnSOD producing strain of L. gasseri exhibit a sex bias in the Il10-deficient mouse model of colitis. Consistent with our results, female mice are less responsive to bacterially-induced inflammation in a mouse model for liver cancer (31). Unexpectedly, when the composition of gut flora in female mice was altered by introducing the wild-type form of L. gasseri in large numbers, the resultant inflammation increased. This suggests that although the female Il10-deficient mice used in this study do not in general develop severe colitis, they are capable of developing an aggressive form of colitis when the right stimulus is applied.
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17 The additional beneficial effects exerted by the MnSOD producing strain of L. gasseri relative to the native strain can potentially occur via one of two mechanisms. The MnSOD produced in L. gasseri may act directly to relieve oxidative stress in the gut or indirectly by enhancing the natural beneficial properties of L. gasseri. The direct mechanism implies that SOD produced by the engineered L. gasseri reduces the levels of superoxide radicals in the gut imposed by specific members of the gut flora (9, 31) and the immune system. Consistent with this mechanism, we found that colonic tissues from mice administered MnSOD producing L. gasseri show a noticeable infiltrate of immune cells, but retention of normal goblet cell numbers and crypt architecture. The increased transcriptional levels of host antioxidant genes in inflamed colons from Il10-deficient mice implies that the superoxide radicals are the primary source of oxidative stress in this model. This supports the importance of a molecule that can disarm oxygen free radicals as therapy for colitis. Since the response in control or probiotic treated groups is equivalent regardless of inflammatory status, there may be a threshold level of endogenous antioxidant activity supporting the observed benefit of additional MnSOD. An alternative mechanism by which MnSOD may function is indirectly by enhancing the probiotic activity of L. gasseri. We have previously demonstrated that the MnSOD producing strain of L. gasseri has increased survival rates in vitro in the presence of both superoxide radicals and hydrogen peroxide (3, 4). As oxidative stress is abundant in the mammalian gut, particularly in inflamed tissues, the elevated level of MnSOD may overcome the oxidative insult from the immune system allowing for the engineered form of L. gasseri to establish a more intimate contact with the colonic epithelium. As the native strain of L. gasseri is more sensitive than its MnSOD-producing strain to superoxide radicals and hydrogen peroxide, the native L. gasseri may be unable to overcome the host defenses in order to exert greater beneficial effects
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18 (3). In summary the present study outlines the use of L. gasseri treatment of inflammation in the colon of Il10-deficient mice. The use of MnSOD to enhance the therapeutic properties of L. gasseri for colitis was also demonstrated.
ACKNOWLEDGEMENTS This work was supported by a subcontract from National Institutes of Health grant U01 CA084239 to DST; by the North Carolina Agricultural Research Service to HMH. The authors thank Chevonne Eversley, Erica Rinella, Ming Yu, Angelika Jährig, Kristin Ellis and Letycia Nunez-Argote for technical assistance and David Threadgill for Il10-deficient founder mice and for comments and suggestions on the manuscript.
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20 16. Kruidenier L and Verspaget HW. Review article: oxidative stress as a pathogenic factor in inflammatory bowel disease--radicals or ridiculous? Aliment Pharmacol Ther 16: 19972015, 2002. 17. Lih-Brody L, Powell SR, Collier KP, Reddy GM, Cerchia R, Kahn E, Weissman GS, Katz S, Floyd RA, McKinley MJ, Fisher SE, and Mullin GE. Increased oxidative stress and decreased antioxidant defenses in mucosa of inflammatory bowel disease. Dig Dis Sci 41: 2078-2086, 1996. 18. Madsen KL, Doyle JS, Jewell LD, Tavernini MM, and Fedorak RN. Lactobacillus species prevents colitis in interleukin 10 gene-deficient mice. Gastroenterology 116: 1107-1114, 1999. 19. McCarthy J, O'Mahony L, O'Callaghan L, Sheil B, Vaughan EE, Fitzsimons N, Fitzgibbon J, O'Sullivan GC, Kiely B, Collins JK, and Shanahan F. Double blind, placebo controlled trial of two probiotic strains in interleukin 10 knockout mice and mechanistic link with cytokine balance. Gut 52: 975-980, 2003. 20. Montalto MC, Hart ML, Jordan JE, Wada K, and Stahl GL. Role for complement in mediating intestinal nitric oxide synthase-2 and superoxide dismutase expression. Am J Physiol Gastrointest Liver Physiol 285: G197-206, 2003. 21. Mulder TP, Verspaget HW, Janssens AR, de Bruin PA, Pena AS, and Lamers CB. Decrease in two intestinal copper/zinc containing proteins with antioxidant function in inflammatory bowel disease. Gut 32: 1146-1150, 1991. 22. Murphy L, Dunne, C., Kiely, B., Shanahan, F., O'Sullivan, G.C. and Collins, J.K. In vivo assessment of potential probiotic Lactobacillus salivarius: evaluation of their establishment, persistence, and localisation in the murine gastrointestinal tract. Microbial Ecology in Health and Disease 11: 149-157, 1999. 23. O'Mahony L, Feeney M, O'Halloran S, Murphy L, Kiely B, Fitzgibbon J, Lee G, O'Sullivan G, Shanahan F, and Collins JK. Probiotic impact on microbial flora, inflammation and tumour development in IL-10 knockout mice. Aliment Pharmacol Ther 15: 1219-1225, 2001. 24. Osman N, Adawi D, Ahrne S, Jeppsson B, and Molin G. Modulation of the effect of dextran sulfate sodium-induced acute colitis by the administration of different probiotic strains of Lactobacillus and Bifidobacterium. Dig Dis Sci 49: 320-327, 2004. 25. Pavan S, Desreumaux P, and Mercenier A. Use of mouse models to evaluate the persistence, safety, and immune modulation capacities of lactic acid bacteria. Clin Diagn Lab Immunol 10: 696-701, 2003. 26. Pedrosa MC, Golner BB, Goldin BR, Barakat S, Dallal GE, and Russell RM. Survival of yogurt-containing organisms and Lactobacillus gasseri (ADH) and their effect on bacterial enzyme activity in the gastrointestinal tract of healthy and hypochlorhydric elderly subjects. Am J Clin Nutr 61: 353-359, 1995. 27. Pena JA, Rogers AB, Ge Z, Ng V, Li SY, Fox JG, and Versalovic J. Probiotic Lactobacillus spp. diminish Helicobacter hepaticus-induced inflammatory bowel disease in interleukin-10-deficient mice. Infect Immun 73: 912-920, 2005. 28. Peran L, Camuesco D, Comalada M, Nieto A, Concha A, Diaz-Ropero MP, Olivares M, Xaus J, Zarzuelo A, and Galvez J. Preventative effects of a probiotic, Lactobacillus salivarius ssp. salivarius, in the TNBS model of rat colitis. World J Gastroenterol 11: 51855192, 2005.
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Fig. 1. Persistence of parental Emr L. gasseri NC1500 (solid line) an NC1501 (MnSOD, dashed line) in the gut of C57BL/6 mice. Mice were orally challenged with 1 × 109 CFU of either strain daily from day 1-5. 70x49mm (600 x 600 DPI)
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Fig. 2. Schematic of experimental design using Il10-deficient mice. Mice were challenged with parental L. gasseri NC1500, L. gasseri NC1501 (MnSOD) or PBS from week three (W3) to week seven (W7). Colitis begins after four weeks (black arrow) in Il10-deficient mice with an SPF flora housed at UNC-CH.
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Fig. 3. (A) Macroscopic findings of colons extracted from Il10-deficient mice challenged with either PBS (top) L. gasseri NC1500 (middle) or L. gasseri NC1501 (MnSOD, bottom). (B) Inflammatory scores of Haematoxylin and Eosin (H&E) stained colonic tissue after L. gasseri or PBS challenge (* = P < 0.05, nonparametric one-way ANOVA). H&E staining of (C) normal colonic tissue from a wild type C57BL/6 mouse; (D) inflamed colonic tissue from an Il10-deficient control (PBS) mouse; (E) colonic tissue from an Il10-deficient mouse treated with wild type L. gasseri (NC1500); (F) colonic tissue from an Il10deficient mouse treated with L. gasseri expressing SodA (NC1501), all histological images are at 20 × magnification. (G) Proximal and distal inflammatory scores of H&E stained colonic tissue after probiotic or PBS challenge.
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Fig. 4. Inflammatory scores of H&E stained colonic tissue after L. gasseri or PBS treatment in male (A, * = P < 0.05, ** = P < 0.01, nonparametric one-way ANOVA) or female (B) mice. Gender bias displayed by probiotic or control treatments (C). Average inflammatory scores for male and female Il10-deficient mice are plotted on the x-axis and y-axis, respectively. The vertically lined box represents the scores of mice in the control (PBS) group, the horizontally lined box represents mice administered the native form of L. gasseri and the checked box represents mice administered L. gasseri (MnSOD).
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Fig. 5. Myeloperoxidase (MPO) immunohistochemistry of normal colonic tissue from a wild type C57BL/6 mouse (A), a PBS-treated Il10-deficient mouse (B), an L. gasseri NC1500-treated Il10-deficient mouse (C) and an L. gasseri NC1501 (MnSOD)-treated Il10-deficient mouse (D). Tissue sections represent the average inflammation H&E score for each experimental group (Fig. 3 B). All histological images are at 20 × magnification.
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Fig. 6. Cycloxygenase-2 (Cox-2) immunohistochemistry of normal colonic tissue from a wild type C57BL/6 mouse (A), a PBS-treated Il10-deficient mouse (B), an L. gasseri NC1500-treated Il10-deficient mouse (C) and an L. gasseri NC1501 (MnSOD)-treated Il10-deficient mouse (D). Tissue sections represent the average inflammation H&E score for each experimental group (Fig. 3 B). All histological images are at 20 × magnification.
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Fig. 7. Relative transcript levels of mouse antioxidant genes in wild type mice (C57BL/6), Il10-deficient mice and Il10-deficient mice administered either the native L. gasseri or L. gasseri MnSOD (* = P < 0.05, ** = P < 0.01, nonparametric one-way ANOVA). Antioxidant genes neutralize the superoxide radical (A = sod2, manganese superoxide dismutase; B = sod3, extracellular superoxide dismutase) or hydrogen peroxide (C = cat, catalase; D = gpx1, glutathione peroxidase).
