Probiotic Lactobacillus spp. Diminish Helicobacter hepaticus-Induced Inflammatory Bowel Disease in Interleukin-10-Deficient Mice
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感染与免疫杂志 2005年第2期
Departments of Molecular Virology & Microbiology
Pathology, Baylor College of Medicine
Department of Pathology, Texas Children's Hospital, Houston, Texas
Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts
ABSTRACT
Clinical and experimental evidence has demonstrated the potential role of probiotics in the prevention or treatment of inflammatory bowel disease. Probiotic clones with direct immunomodulatory activity may have anti-inflammatory effects in the intestine. We investigated the roles of tumor necrosis factor alpha (TNF-)-inhibitory Lactobacillus clones with a pathogen-induced murine colitis model. Murine-derived probiotic lactobacilli were selected in vitro for their ability to inhibit TNF- secretion by Helicobacter hepaticus-stimulated macrophages. Interleukin-10 (IL-10)-deficient mice were treated with probiotic Lactobacillus reuteri in combination with Lactobacillus paracasei and then challenged with H. hepaticus. Ten weeks postinoculation, the severity of typhlocolitis was assessed by histologic examination of the cecocolic region. Intestinal proinflammatory cytokine responses were evaluated by real-time quantitative reverse transcriptase PCR and immunoassays, and the quantities of intestinal H. hepaticus were evaluated by real-time PCR. Intestinal colonization by TNF--inhibitory lactobacilli reduced intestinal inflammation in H. hepaticus-challenged IL-10-deficient mice despite similar quantities of H. hepaticus in cocolonized animals. Proinflammatory colonic cytokine (TNF- and IL-12) levels were lowered in Lactobacillus-treated animals. In this H. hepaticus-challenged IL-10-deficient murine colitis model, lactobacilli demonstrated probiotic effects by direct modulation of mucosal inflammatory responses.
INTRODUCTION
The rational selection of probiotic bacteria provides opportunities for the prevention or treatment of inflammatory bowel disease (IBD) based on accumulated evidence from animal and human studies (12, 40). Selective deficiencies of intestinal lactobacilli and bifidobacteria have been observed in patients with Crohn's disease (9). Supplementation with probiotic Lactobacillus species has been effective at ameliorating intestinal inflammation in human patients with IBD. Administration of Lactobacillus rhamnosus GG (LGG) to children with Crohn's disease resulted in significant reduction of the Crohn's disease activity index 4 weeks after initiation of therapy (14). Probiotic formulations that included four Lactobacillus species have been effective for the prevention (10) or treatment (11) of IBD-related pouchitis.
The murine interleukin-10 (IL-10)-deficient mouse colitis model has provided additional insights into the roles of probiotic Lactobacillus spp. as potential prophylactic or treatment modalities in IBD. Introduction of a single Lactobacillus reuteri strain into the lower gastrointestinal tracts of IL-10-deficient mice (Crohn's disease model) by intracolonic application restored mucosa-adherent Lactobacillus populations and prevented development of colitis in these animals (27). Similarly, the introduction of Lactobacillus plantarum strain 299V into IL-10-deficient mice ameliorated colitis in these animals (38). Lactobacillus salivarius inhibited proinflammatory cytokine production and attenuated colitis in placebo-controlled trials with IL-10-deficient mice (28, 32). The replenishment of intestinal IL-10 by recombinant lactic acid bacteria ameliorated disease in IL-10-deficient mice, indicating the importance of IL-10 in controlling intestinal inflammation (42). Alternatively, these studies suggest that the absence of IL-10 in the intestine may be compensated by anti-inflammatory activities of probiotic bacteria.
In this study, mechanisms of probiosis were explored in a pathogen-induced mouse IBD model. Mice lacking IL-10 rapidly develop robust IBD-like lesions following infection by Helicobacter hepaticus (20), whereas germ- or pathogen-free IL-10–/– mice fail to develop colitis or manifest delayed onset of disease, respectively (39). Intestinal Lactobacillus spp. were administered orally to IL-10-deficient mice in order to examine probiotic effects in colitis-susceptible mice challenged with pathogenic H. hepaticus. We selected mouse-derived Lactobacillus paracasei and L. reuteri strains on the basis of in vitro tumor necrosis factor-alpha (TNF-)-inhibitory activity toward macrophages (33) and investigated probiotic anti-inflammatory effects in IL-10-deficient mice. The effects of probiotic lactobacilli on intestinal quantities of H. hepaticus were evaluated in parallel with studies of mucosal proinflammatory cytokine responses.
MATERIALS AND METHODS
Bacterial strains. Lactobacillus spp. were grown anaerobically in deMan, Rogosa, Sharpe (MRS) broth (Becton Dickinson, Sparks, Md.) overnight at 37°C. L. paracasei 1602 was isolated from a fecal sample of a mouse in our facility, and L. reuteri 6798 was isolated from jejunal sample of a different mouse in the same facility (33). H. hepaticus type strain 3B1 (ATCC 51449) was grown in Brucella broth (Becton Dickinson) supplemented with 5% heat-inactivated fetal bovine serum (FBS) under microaerobic conditions for 48 to 60 h at 37°C. Cultures were assessed for purity by Gram staining prior to animal inoculation.
Lactobacillus-macrophage bioassays and evaluation of probiotic antagonism. Overnight cultures of lactobacilli were diluted to an optical density at 600 nm of 1.0 (representing approximately 109 cells/ml), further diluted 1:10, and grown in MRS for an additional 24 h. H. hepaticus 3B1 was cultured for 48 h in Brucella broth supplemented with 5% FBS. Cultures were diluted 1:10 and grown for another 24 and 48 h. Bacterial cell-free conditioned medium was collected by centrifugation at 8,500 RCF for 10 min at 4°C. Conditioned medium was separated from cell pellet and filtered through a 0.22-μm-pore-size filter unit (Millipore, Bedford, Mass.).
The mouse monocyte/macrophage cell line RAW 264.7 gamma NO (–) (ATCC CRL-2278) was used as the reporter cell line for studying probiotic antagonism of TNF- production. RAW 264.7 cells were grown in RPMI medium 1640 (Gibco-Invitrogen, Carlsbad, Calif.) supplemented with 10% FBS and 2% antibiotic (5,000 U of penicillin/ml and 5 mg of streptomycin/ml; Sigma, St. Louis, Mo.) at 5% CO2, 37°C until 80 to 90% confluent. Approximately 5 x 104 cells were seeded into 96-well cell culture clusters and allowed to adhere for 2 h prior to addition of conditioned medium. Naive RAW 264.7 cells were exposed to a mixture of lactobacillus-conditioned medium and cell-free Helicobacter-conditioned medium (1:1 ratio). Cell viability was assessed by the trypan blue (Invitrogen) exclusion assay. Production of TNF- in macrophage cell culture supernatants was measured with a mouse TNF--specific sandwich enzyme immunoassay (Biosource, Camarillo, Calif.).
Animals and experimental infection. IL-10-deficient C57BL/6 mice were housed in an Association for Assessment and Accreditation of Laboratory Animal Care-approved facility at the Division of Comparative Medicine, Massachusetts Institute of Technology (Cambridge, Mass.), under specific-pathogen-free conditions in microisolator cages. Animals were provided standard chow and water and allowed to feed ad libitum under a 12-h daylight cycle. Mice were kept free of known murine viruses, Salmonella spp., Citrobacter rodentium, ecto- and endoparasites, and known murine Helicobacter spp.
Research was conducted following protocols approved by the Committee on Animal Care at Massachusetts Institute of Technology. Six- to 13-week-old mice were matched by age and sex and assigned to one of four infection groups. Animals received 109 CFU of lactobacilli or 107 CFU of H. hepaticus per dose by gastric gavage. Lactobacillus was administered twice, two days in a row; each inoculum included equivalent cell counts of L. paracasei 1602 and L. reuteri 6798. Forty-eight hours after the second lactobacillus dose, H. hepaticus was administered thrice (once per day, every other day). A final dose of lactobacilli was given 48 h after the third H. hepaticus dose. Control animals received sterile MRS or Brucella broth (for Lactobacillus spp. or H. hepaticus, respectively). Ten weeks postinfection, animals were euthanized by CO2 asphyxiation. A schematic timeline of the infection study is depicted (Fig. 1).
Histologic examination. At necropsy, tissue was collected and, after expression of intestinal contents, fixed in 10% neutral pH-buffered formalin and processed by routine histologic methods. A comparative pathologist (A.B.R.), blinded to sample identity, evaluated hematoxylin-and-eosin-stained sections of the cecocolic junction. Sections were graded on an ascending scale from 0 to 4 for inflammation, hyperplasia, and dysplasia, using previously defined criteria (6).
Determination of bacterial colonization. Total DNA, RNA, and protein were extracted from the mid-cecum, using Trizol LS (Invitrogen) reagent for subsequent quantitation. Quantities of H. hepaticus in the mid-cecum were determined by real-time PCR amplification of the cdtB gene, as previously described (8). TaqMan probe-based detection was performed with an ABI Prism 7700 sequence detection system (Applied Biosystems), and murine 18S rRNA gene was used as a correction factor (8).
Cytokine quantitation. IL-4, IL-12 (p40), gamma interferon (IFN-), and TNF- mRNA were measured by real-time reverse transcription-PCR of RNA isolated from cecal tissue samples. Briefly, 1 μg of total RNA was reverse transcribed by using oligo(dT) primers and the Superscript III reverse transcriptase system (Invitrogen) according to the manufacturer's recommendations. All cDNA preparations were normalized by endogenous glyceraldehyde-3-phosphate dehydrogenase, using commercial primer-probe sets (Applied Biosystems, Foster City, Calif.) in an ABI Prism 7700 sequence detection system (Applied Biosystems) according to the manufacturer's recommendations.
Immediately after euthanasia, a 10-mm-long section of proximal colon was harvested and the contents were removed by vigorous agitation in sterile saline. Colons were divided into four sections, weighed, and cultured (mucosa side up) in RPMI 1640 supplemented with 10% heat-inactivated FBS and antimicrobial agents (50 U of penicillin/ml, 50 μg of streptomycin/ml, and 125 ng of amphotericin B/ml) (Invitrogen) for 24 h in transwell inserts (Millicell; Millipore). Two sections from each colon served as duplicates and were cultured with or without purified Escherichia coli lipopolysaccharide (LPS) serotype O127:B8 (Sigma) (250 ng/well). TNF- in colon explant supernatants was measured by quantitative enzyme-linked immunosorbent assay (Biosource). Mouse DNA was extracted from entire explants for 18S rRNA gene quantitation and used to standardize TNF- output.
Statistical analyses. Statistics were performed by using SPSS for Windows, version 11.0.1 (SPSS Inc., Chicago, Ill.). Histologic, bacterial colonization and cytokine data were analyzed using the Kruskal-Wallis test followed by the Mann-Whitney U test for two independent samples. Results are presented as median and interquartile range. Correlations were assessed by using Spearman's method and reported as the coefficient, rho. Comparisons yielding P values of 0.05 were considered significant. Nonparametric statistics were used because histology was the primary endpoint, and cytokine data distribution did not meet the requirements for parametric tests.
RESULTS
Probiotic lactobacilli inhibit Helicobacter-mediated stimulation of TNF- production by macrophages. Since dysregulated inflammatory responses appear to be central to the pathogenesis of IBD in mouse models, we identified mouse-derived Lactobacillus strains with anti-inflammatory properties. We screened intestinal murine Lactobacillus isolates (33) for probiotic activity as defined by the inhibition of TNF- production by LPS-stimulated macrophages (34). Approximately 10% of Lactobacillus isolates inhibited TNF- production by LPS-stimulated mouse macrophages (33). L. paracasei 1602 and L. reuteri 6798 represented two TNF--inhibitory strains recovered from mice lacking a predisposition to colitis. Murine macrophages were exposed to cell-free H. hepaticus-conditioned medium in order to assess the ability of selected lactobacilli to antagonize innate immune responses to H. hepaticus. In the presence of conditioned medium from lactobacilli, TNF- production was significantly diminished in H. hepaticus-conditioned medium-stimulated macrophages. Selected Lactobacillus clones inhibited H. hepaticus-mediated stimulation of macrophage TNF- production (Fig. 2).
Probiotic lactobacilli reduce intestinal inflammation in Helicobacter-infected female IL-10-deficient mice. Lactobacillus clones with TNF--inhibitory activity in vitro were evaluated in vivo by studying mucosal protection in a Helicobacter-exacerbated colitis model. We performed two infection studies with IL-10–/– mice. In a pilot study, both male (n = 21) and female (n = 18) mice were precolonized with a combination of two murine lactobacilli, L. paracasei 1602 and L. reuteri 6798, that had displayed anti-inflammatory activity with cultured macrophages. Lactobacillus colonization was followed by infection with the mouse pathogen H. hepaticus. Sham-dosed mice and mice colonized with a combination of L. paracasei 1602 and L. reuteri 6798 in the absence of H. hepaticus were studied as controls. After 10 weeks, we semiquantitatively graded the cecocolic junction for inflammation, hyperplasia, and dysplasia by histopathologic examination. Median histologic scores of uninfected sham-dosed controls and Lactobacillus-colonized animals were similar (Fig. 3a). In contrast, mice infected with H. hepaticus developed moderately severe typhlocolitis. Animals treated with a combination of L. paracasei 1602 and L. reuteri 6798 prior to infection with H. hepaticus did not exhibit significant diminution of IBD-like lesions.
When animals were stratified by gender, we observed significant reductions in cecocolic lesion scores (inflammation, hyperplasia, and dysplasia; P = 0.032, 0.032, and 0.016, respectively) in female mice cocolonized with Lactobacillus and H. hepaticus versus animals that were monoinfected with H. hepaticus (Fig. 3b). In contrast, male mice cocolonized with murine lactobacilli displayed only a slight and statistically nonsignificant reduction in intestinal lesion grades. Real-time PCR confirmed that intestinal Lactobacillus DNA quantities were significantly elevated only in Lactobacillus-treated animals and were maintained for the duration of the study (data not shown). Because female mice appeared to derive the greatest benefit from probiotic therapy, we performed a follow-up study with female mice only (n = 36), using the same cocolonization and infection protocol. A summary of histologic scores for the follow-up study is provided (Table 1).
As in the pilot study, uninfected animals exhibited normal cecocolic histology (Fig. 4a), while those monoinfected with H. hepaticus (Fig. 4b) developed moderate to severe typhlocolitis characterized by infiltration of lymphocytes and macrophages, with fewer granulocytes, in the mucosa and submucosa. Reactive epithelial changes included hyperplasia with crypt elongation as well as goblet cell loss and mild dysplasia with distortion of glandular architecture, cell crowding (piling), and early aberrant crypt foci including slit and back-to-back forms. Lesions were partially eliminated in mice treated with L. paracasei and L. reuteri (Fig. 4c). Subsequent molecular data presented in this study utilized samples from the follow-up population only.
Lactobacillus-mediated probiosis functions independently of quantities of intestinal H. hepaticus. In order to correlate intestinal inflammation with the presence of H. hepaticus, cecal DNA was extracted and analyzed by real-time quantitative PCR. As expected, H. hepaticus DNA was detectable only in animals deliberately infected with the pathogen. Unexpectedly, we identified no correlation between H. hepaticus colonization levels and lower bowel median lesion scores (inflammation [ = 0.253; P = 0.082] or hyperplasia [ = 0.225; P = 0.108]).
Animals receiving the L. paracasei 1602, L. reuteri 6798 combination (L. paracasei 1602/L. reuteri 6798) yielded quantities of H. hepaticus (P = 1.0) comparable to those for H. hepaticus-monoinfected controls (Fig. 5). L. paracasei 1602/L. reuteri 6798 provided partial protection against H. hepaticus-induced IBD-like lesions (Fig. 4c).
Reductions in proinflammatory cytokines correlate with probiosis. Since cytokine transcript levels have correlated with disease activity in IBD patients (41), we measured mRNA levels of selected cytokines in the ceca of mice (Fig. 6). Cecal levels of IL-12 (p40) and TNF- were significantly elevated in H. hepaticus-infected mice compared to levels in uninfected controls (P = 0.048 and P = 0.005, respectively), while IFN- was not significantly altered. IL-4 levels were similar between uninfected and H. hepaticus-infected mice. In contrast, introduction of L. paracasei 1602/L. reuteri 6798 in the absence of H. hepaticus did not alter IL-12, TNF-, IFN-, or IL-4 transcript levels compared to those for uninfected controls (P < 0.05 for all).
A significant reduction in mucosal IL-12p40 mRNA levels (P = 0.016) was observed when animals were cocolonized with L. paracasei 1602/L. reuteri 6798 prior to H. hepaticus challenge, while transcript levels for IFN-, IL-4, and TNF- were not significantly altered. This difference may be biologically important considering the partial anti-inflammatory effect in the intestine without concomitant reductions in H. hepaticus levels. The data trends indicate reductions in overall proinflammatory cytokine profiles with the L. paracasei 1602/L. reuteri 6798 probiotic combination (Fig. 7).
We determined whether the presence of anti-inflammatory Lactobacillus clones rendered mucosal explants tolerant to E. coli LPS challenge with TNF- as a biomarker. TNF- production by LPS-stimulated colonic explants was blunted in mucosal tissue from mice cocolonized with L. paracasei 1602/L. reuteri 6798 and H. hepaticus compared to results for uninfected animals or animals infected only with H. hepaticus. Explants from mice treated with the L. paracasei 1602/L. reuteri 6798 combination yielded a trend toward reduced amounts of intestinal TNF- when challenged with E. coli LPS (Fig. 8).
DISCUSSION
The IL-10–/– mouse is a well-established model for the study of colitis and IBD, including Helicobacter-induced disease (20). IL-10-deficient mice receiving lactic acid bacteria expressing recombinant murine IL-10 were protected from colitis (42), demonstrating the singular importance of this cytokine in regulating intestinal inflammation. Intestinal colonization with probiotic Lactobacillus clones, selected on the basis of TNF--inhibitory activity, diminished inflammation in IL-10-deficient mice infected with H. hepaticus. In the L. paracasei/L. reuteri-treated group, Lactobacillus-mediated protection was due to direct immunomodulatory activity and not reductions in the levels of H. hepaticus. The combination of L. paracasei and L. reuteri was associated with reductions in mucosal proinflammatory cytokines despite similar quantities of intestinal H. hepaticus. Interestingly, the anti-inflammatory effect was restricted to female mice in this model.
H. hepaticus induces IBD-like typhlocolitis in specific-pathogen-free IL-10-deficient mouse models (20). Chronic intestinal inflammation in this model has been presumably driven by a Th-1-predominant immune response. Elevated levels of mucosal TNF- and nitric oxide are present in the intestines of these animals, and the colitis is associated with increased levels of IL-12 and IFN- (19, 20). In the present study, intestinal inflammation was partially eliminated by a combination of L. paracasei and L. reuteri. L. paracasei/L. reuteri-treated animals demonstrated a partial elimination of intestinal inflammation and significantly reduced mucosal IL-12 mRNA levels. The present findings support the role of intestinal IL-12 in mediating mucosal inflammation in this mouse colitis model independently of reductions of H. hepaticus quantities. Colitis in H. hepaticus-treated IL-10-deficient mice is dependent on mucosal IL-12 (20), and reduced intestinal inflammation was correlated with decreased mucosal IL-12 levels in L. plantarum-treated IL-10-deficient mice (38). Possibly, lactobacilli inhibit NF-B activation in the intestinal mucosa, resulting in diminished expression of IL-12. The lack of an effect on intestinal IFN-, despite reductions in mucosal IL-12 mRNA, may be due to the absence of IL-10 in the intestine. IL-10 directly suppressed T- and NK cell-derived IFN- independently of TNF-, IL-12, or IL-18 (37).
In this study, we selected Lactobacillus strains on the basis of macrophage TNF--inhibitory activity in vitro. Animal studies support the primary role of TNF- in the pathogenesis of chronic colitis. Intraperitoneal injection of rat anti-mouse TNF- monoclonal antibodies markedly reduced morbidity in IL-10-deficient mice with colitis (13). Murine macrophages represent important sources of mucosal TNF- in murine colitis models. In vivo depletion of mucosal and lymphoid follicle-associated macrophages in mice deficient in IL-10 diminished colitis in these animals, suggesting that macrophages are important mediators of intestinal inflammation (44). Data obtained in this study indicate that the L. paracasei/L. reuteri combination may have reduced the mucosal TNF- response to bacterial LPS. Although cell populations from the intestinal mucosa were not separated, both enterocytes and macrophages were likely affected in these explant studies. Colons from mice precolonized with L. paracasei 1602/L. reuteri 6798 yielded diminished responses to LPS stimulation. L. reuteri and L. paracasei may contribute to the development of LPS tolerance in intestinal epithelial cells or macrophages. LPS tolerance in macrophages includes hallmark molecular features, such as the up-regulation of NF-B subunits p50 and p52 (22). Although induction of tolerance is a possibility, we have no evidence to support such a mechanism in this model.
Probiosis ultimately has a clonal basis and depends on the functional characterization of specific bacterial strains. Contrasting results have been obtained because investigators use a variety of different strains, cross species barriers, and work with different primary and cultured cell types. Some investigators have described the up-regulation of proinflammatory cytokine responses and NF-B activation by intact lactobacilli (29-31). Cytokine-modulatory effects are strain dependent (3, 34) and vary with growth phase and cell preparation (29-31, 34). Recent data indicate that viable L. reuteri organisms were required for anti-inflammatory effects (25). Lactobacillus species differed in their abilities to modulate proinflammatory cytokine production in bone marrow-derived dendritic cells, and cytokine-inhibitory L. reuteri strains antagonized effects of cytokine-inducing L. casei clones (3). Interactions between cells in the intestinal mucosa are important, since leukocyte-sensitized intestinal epithelial cells (IECs) were differentially modulated by Lactobacillus clones, in contrast to human IECs cultured in the absence of leukocytes (15). The proinflammatory cytokines TNF- and IL-1 were up-regulated in human IECs in the presence of Lactobacillus sakei but were not affected by Lactobacillus johnsonii. Furthermore, only L. johnsonii up-regulated the anti-inflammatory cytokine transforming growth factor 1 (15). In our experimental studies, anti-inflammatory effects in mouse models were obtained only with mouse-derived lactobacilli and raise the concern of using commensal bacteria derived from different species (including humans) in animal models. In our studies, IL-10-deficient mice precolonized with human-derived LGG developed more severe colitis after challenge with H. hepaticus (data not shown). In support of the potential importance of animal species origin for probiotic strategies, rat-derived L. reuteri was more effective than LGG at ameliorating intestinal inflammation in an acetic acid-induced rat colitis model (16).
Only female mice benefited significantly from the anti-inflammatory activity of probiotic Lactobacillus therapy in our model. Histologically, male and female IL-10-deficient mice challenged with H. hepaticus exhibited similar patterns of colitis (20), indicating that development of H. hepaticus-triggered colitis is not influenced by gender in C57BL mice. As expected, our present study shows that male and female H. hepaticus-infected C57BL mice yielded similar disease phenotypes in the absence of Lactobacillus. However, responsiveness to anti-inflammatory effects of probiotic bacteria appears to be affected by gender status in the present model. In contrast, A/JCr mice with an intact IL-10 gene demonstrated increased severity of colitis in females when challenged with H. hepaticus (23). Perhaps probiotic Lactobacillus strains modulate intestinal inflammation by an estrogen-dependent mechanism potentiated in an IL-10-deficient animal. Gender differences in immunologic responses have been well documented for humans and rodents (23, 43). Recent molecular studies of gender-specific responses point towards the role of sex hormones as possible determinants of immune responses and disease (23, 43). Specifically, estrogen has been implicated in T-cell-mediated autoimmune diseases (5). Of particular interest is that murine myeloid cells (dendritic cells or macrophages) lacking the estrogen receptor alpha produce elevated TNF- after challenge with E. coli LPS or Mycobacterium avium (21). Other studies revealed that estrogen receptor agonists suppressed inducible nitric oxide synthase and TNF- production in LPS-stimulated macrophages (18, 45). Estrogen down-modulates monocyte chemoattractant protein-1 production by murine macrophages (7). The estrogen, 17- estradiol, protects mice from experimental autoimmune encephalomyelitis in an estrogen receptor-dependent fashion (35). Differences in estrogen receptor subunit levels may partially account for the inability of Lactobacillus-derived immunomodulins to protect male mice from H. hepaticus-induced IBD. In data not shown, we also found that conditioned medium from probiotic lactobacilli decreased TNF- production in LPS-stimulated primary peritoneal macrophages from female mice but not from male mice. Since probiosis appears to be a balancing act, with anti-inflammatory effectors (i.e., IL-10) competing with proinflammatory mediators (e.g., TNF- and LPS), relative deficiencies of estrogen receptor alpha and circulating estrogens in male mice may tip the balance towards an inflammatory phenotype.
Probiosis may function by interference with enteric pathogens or proinflammatory components of the intestinal microbiota. L. paracasei 1602/L. reuteri 6798 may diminish inflammation by inhibition of H. hepaticus virulence gene expression or virulence factor activities without necessarily affecting quantities of intestinal H. hepaticus. Lactobacillus gasseri prevents gastritis by suppression of Helicobacter pylori type IV secretion system-dependent virulence factors, leading to decreased production of IL-8 by gastric epithelial cells (Y. Koga, personal communication). Commensal organisms may also enhance the production of antibacterial factors in the intestine. The commensal bacterium Bacteroides thetaiotaomicron induces the expression of bactericidal angiogenins (17) and prodefensin-processing matrilysin (24) by intestinal Paneth cells in the mouse. In our model, L. paracasei 1602/L. reuteri 6798 reduced mucosal IL-12 expression and LPS-stimulated colonic TNF- production, possibly facilitating H. hepaticus colonization. Probiotics likely regulate inflammatory responses by direct interactions with the intestinal mucosa or antagonism of pathogenic or proinflammatory bacteria.
Data from the present study and prior studies suggest that probiotics exert their beneficial effects by one or a combination of mechanisms, including the following: (i) direct modulation of immune function (1, 2, 28, 34), (ii) competition with or exclusion of pathogen(s) (4), (iii) interference with virulence of enteric pathogens (36), or (iv) increasing epithelial/mucosal barrier function (26). The optimal application of probiotics for prevention or treatment of IBD and enteric infections may ultimately require the characterization of bacterial clones with specific probiotic properties. Individuals with enteric infections may benefit from probiotic strains with antagonistic properties towards a specific infectious agent. Patients with IBD may require probiotic strains with specific anti-inflammatory features that complement the immunomodulatory activities of other drugs or dietary changes in multicomponent treatment regimens. Additionally, the functional importance of host- or patient-specific characteristics (e.g., gender) should also be considered when designing clinical probiotic trials. The studies presented in this report stress the importance of rational selection of probiotic bacterial clones for therapy of intestinal inflammatory diseases, including IBD.
ACKNOWLEDGMENTS
We thank Rao Varada, Michele DeMarco, Philip Lee, Jeffrey Bajko, Erinn Stefanich, Ellen Buckley, Kathleen Cormier, and Ernie Smith for technical assistance. We also thank M. J. Finegold for critical review and comments.
This work was supported by grants from the Crohn's & Colitis Foundation of America First Award (J.V.), National Institutes of Health grant award K08 DK02705 (J.V.), R01 CA67529 (J.G.F.), and R01 AI50592 (J.G.F.). J.V. was also supported by U.S. Public Health Service grant DK56338, which funds the Texas Gulf Coast Digestive Diseases Center.
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Pathology, Baylor College of Medicine
Department of Pathology, Texas Children's Hospital, Houston, Texas
Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts
ABSTRACT
Clinical and experimental evidence has demonstrated the potential role of probiotics in the prevention or treatment of inflammatory bowel disease. Probiotic clones with direct immunomodulatory activity may have anti-inflammatory effects in the intestine. We investigated the roles of tumor necrosis factor alpha (TNF-)-inhibitory Lactobacillus clones with a pathogen-induced murine colitis model. Murine-derived probiotic lactobacilli were selected in vitro for their ability to inhibit TNF- secretion by Helicobacter hepaticus-stimulated macrophages. Interleukin-10 (IL-10)-deficient mice were treated with probiotic Lactobacillus reuteri in combination with Lactobacillus paracasei and then challenged with H. hepaticus. Ten weeks postinoculation, the severity of typhlocolitis was assessed by histologic examination of the cecocolic region. Intestinal proinflammatory cytokine responses were evaluated by real-time quantitative reverse transcriptase PCR and immunoassays, and the quantities of intestinal H. hepaticus were evaluated by real-time PCR. Intestinal colonization by TNF--inhibitory lactobacilli reduced intestinal inflammation in H. hepaticus-challenged IL-10-deficient mice despite similar quantities of H. hepaticus in cocolonized animals. Proinflammatory colonic cytokine (TNF- and IL-12) levels were lowered in Lactobacillus-treated animals. In this H. hepaticus-challenged IL-10-deficient murine colitis model, lactobacilli demonstrated probiotic effects by direct modulation of mucosal inflammatory responses.
INTRODUCTION
The rational selection of probiotic bacteria provides opportunities for the prevention or treatment of inflammatory bowel disease (IBD) based on accumulated evidence from animal and human studies (12, 40). Selective deficiencies of intestinal lactobacilli and bifidobacteria have been observed in patients with Crohn's disease (9). Supplementation with probiotic Lactobacillus species has been effective at ameliorating intestinal inflammation in human patients with IBD. Administration of Lactobacillus rhamnosus GG (LGG) to children with Crohn's disease resulted in significant reduction of the Crohn's disease activity index 4 weeks after initiation of therapy (14). Probiotic formulations that included four Lactobacillus species have been effective for the prevention (10) or treatment (11) of IBD-related pouchitis.
The murine interleukin-10 (IL-10)-deficient mouse colitis model has provided additional insights into the roles of probiotic Lactobacillus spp. as potential prophylactic or treatment modalities in IBD. Introduction of a single Lactobacillus reuteri strain into the lower gastrointestinal tracts of IL-10-deficient mice (Crohn's disease model) by intracolonic application restored mucosa-adherent Lactobacillus populations and prevented development of colitis in these animals (27). Similarly, the introduction of Lactobacillus plantarum strain 299V into IL-10-deficient mice ameliorated colitis in these animals (38). Lactobacillus salivarius inhibited proinflammatory cytokine production and attenuated colitis in placebo-controlled trials with IL-10-deficient mice (28, 32). The replenishment of intestinal IL-10 by recombinant lactic acid bacteria ameliorated disease in IL-10-deficient mice, indicating the importance of IL-10 in controlling intestinal inflammation (42). Alternatively, these studies suggest that the absence of IL-10 in the intestine may be compensated by anti-inflammatory activities of probiotic bacteria.
In this study, mechanisms of probiosis were explored in a pathogen-induced mouse IBD model. Mice lacking IL-10 rapidly develop robust IBD-like lesions following infection by Helicobacter hepaticus (20), whereas germ- or pathogen-free IL-10–/– mice fail to develop colitis or manifest delayed onset of disease, respectively (39). Intestinal Lactobacillus spp. were administered orally to IL-10-deficient mice in order to examine probiotic effects in colitis-susceptible mice challenged with pathogenic H. hepaticus. We selected mouse-derived Lactobacillus paracasei and L. reuteri strains on the basis of in vitro tumor necrosis factor-alpha (TNF-)-inhibitory activity toward macrophages (33) and investigated probiotic anti-inflammatory effects in IL-10-deficient mice. The effects of probiotic lactobacilli on intestinal quantities of H. hepaticus were evaluated in parallel with studies of mucosal proinflammatory cytokine responses.
MATERIALS AND METHODS
Bacterial strains. Lactobacillus spp. were grown anaerobically in deMan, Rogosa, Sharpe (MRS) broth (Becton Dickinson, Sparks, Md.) overnight at 37°C. L. paracasei 1602 was isolated from a fecal sample of a mouse in our facility, and L. reuteri 6798 was isolated from jejunal sample of a different mouse in the same facility (33). H. hepaticus type strain 3B1 (ATCC 51449) was grown in Brucella broth (Becton Dickinson) supplemented with 5% heat-inactivated fetal bovine serum (FBS) under microaerobic conditions for 48 to 60 h at 37°C. Cultures were assessed for purity by Gram staining prior to animal inoculation.
Lactobacillus-macrophage bioassays and evaluation of probiotic antagonism. Overnight cultures of lactobacilli were diluted to an optical density at 600 nm of 1.0 (representing approximately 109 cells/ml), further diluted 1:10, and grown in MRS for an additional 24 h. H. hepaticus 3B1 was cultured for 48 h in Brucella broth supplemented with 5% FBS. Cultures were diluted 1:10 and grown for another 24 and 48 h. Bacterial cell-free conditioned medium was collected by centrifugation at 8,500 RCF for 10 min at 4°C. Conditioned medium was separated from cell pellet and filtered through a 0.22-μm-pore-size filter unit (Millipore, Bedford, Mass.).
The mouse monocyte/macrophage cell line RAW 264.7 gamma NO (–) (ATCC CRL-2278) was used as the reporter cell line for studying probiotic antagonism of TNF- production. RAW 264.7 cells were grown in RPMI medium 1640 (Gibco-Invitrogen, Carlsbad, Calif.) supplemented with 10% FBS and 2% antibiotic (5,000 U of penicillin/ml and 5 mg of streptomycin/ml; Sigma, St. Louis, Mo.) at 5% CO2, 37°C until 80 to 90% confluent. Approximately 5 x 104 cells were seeded into 96-well cell culture clusters and allowed to adhere for 2 h prior to addition of conditioned medium. Naive RAW 264.7 cells were exposed to a mixture of lactobacillus-conditioned medium and cell-free Helicobacter-conditioned medium (1:1 ratio). Cell viability was assessed by the trypan blue (Invitrogen) exclusion assay. Production of TNF- in macrophage cell culture supernatants was measured with a mouse TNF--specific sandwich enzyme immunoassay (Biosource, Camarillo, Calif.).
Animals and experimental infection. IL-10-deficient C57BL/6 mice were housed in an Association for Assessment and Accreditation of Laboratory Animal Care-approved facility at the Division of Comparative Medicine, Massachusetts Institute of Technology (Cambridge, Mass.), under specific-pathogen-free conditions in microisolator cages. Animals were provided standard chow and water and allowed to feed ad libitum under a 12-h daylight cycle. Mice were kept free of known murine viruses, Salmonella spp., Citrobacter rodentium, ecto- and endoparasites, and known murine Helicobacter spp.
Research was conducted following protocols approved by the Committee on Animal Care at Massachusetts Institute of Technology. Six- to 13-week-old mice were matched by age and sex and assigned to one of four infection groups. Animals received 109 CFU of lactobacilli or 107 CFU of H. hepaticus per dose by gastric gavage. Lactobacillus was administered twice, two days in a row; each inoculum included equivalent cell counts of L. paracasei 1602 and L. reuteri 6798. Forty-eight hours after the second lactobacillus dose, H. hepaticus was administered thrice (once per day, every other day). A final dose of lactobacilli was given 48 h after the third H. hepaticus dose. Control animals received sterile MRS or Brucella broth (for Lactobacillus spp. or H. hepaticus, respectively). Ten weeks postinfection, animals were euthanized by CO2 asphyxiation. A schematic timeline of the infection study is depicted (Fig. 1).
Histologic examination. At necropsy, tissue was collected and, after expression of intestinal contents, fixed in 10% neutral pH-buffered formalin and processed by routine histologic methods. A comparative pathologist (A.B.R.), blinded to sample identity, evaluated hematoxylin-and-eosin-stained sections of the cecocolic junction. Sections were graded on an ascending scale from 0 to 4 for inflammation, hyperplasia, and dysplasia, using previously defined criteria (6).
Determination of bacterial colonization. Total DNA, RNA, and protein were extracted from the mid-cecum, using Trizol LS (Invitrogen) reagent for subsequent quantitation. Quantities of H. hepaticus in the mid-cecum were determined by real-time PCR amplification of the cdtB gene, as previously described (8). TaqMan probe-based detection was performed with an ABI Prism 7700 sequence detection system (Applied Biosystems), and murine 18S rRNA gene was used as a correction factor (8).
Cytokine quantitation. IL-4, IL-12 (p40), gamma interferon (IFN-), and TNF- mRNA were measured by real-time reverse transcription-PCR of RNA isolated from cecal tissue samples. Briefly, 1 μg of total RNA was reverse transcribed by using oligo(dT) primers and the Superscript III reverse transcriptase system (Invitrogen) according to the manufacturer's recommendations. All cDNA preparations were normalized by endogenous glyceraldehyde-3-phosphate dehydrogenase, using commercial primer-probe sets (Applied Biosystems, Foster City, Calif.) in an ABI Prism 7700 sequence detection system (Applied Biosystems) according to the manufacturer's recommendations.
Immediately after euthanasia, a 10-mm-long section of proximal colon was harvested and the contents were removed by vigorous agitation in sterile saline. Colons were divided into four sections, weighed, and cultured (mucosa side up) in RPMI 1640 supplemented with 10% heat-inactivated FBS and antimicrobial agents (50 U of penicillin/ml, 50 μg of streptomycin/ml, and 125 ng of amphotericin B/ml) (Invitrogen) for 24 h in transwell inserts (Millicell; Millipore). Two sections from each colon served as duplicates and were cultured with or without purified Escherichia coli lipopolysaccharide (LPS) serotype O127:B8 (Sigma) (250 ng/well). TNF- in colon explant supernatants was measured by quantitative enzyme-linked immunosorbent assay (Biosource). Mouse DNA was extracted from entire explants for 18S rRNA gene quantitation and used to standardize TNF- output.
Statistical analyses. Statistics were performed by using SPSS for Windows, version 11.0.1 (SPSS Inc., Chicago, Ill.). Histologic, bacterial colonization and cytokine data were analyzed using the Kruskal-Wallis test followed by the Mann-Whitney U test for two independent samples. Results are presented as median and interquartile range. Correlations were assessed by using Spearman's method and reported as the coefficient, rho. Comparisons yielding P values of 0.05 were considered significant. Nonparametric statistics were used because histology was the primary endpoint, and cytokine data distribution did not meet the requirements for parametric tests.
RESULTS
Probiotic lactobacilli inhibit Helicobacter-mediated stimulation of TNF- production by macrophages. Since dysregulated inflammatory responses appear to be central to the pathogenesis of IBD in mouse models, we identified mouse-derived Lactobacillus strains with anti-inflammatory properties. We screened intestinal murine Lactobacillus isolates (33) for probiotic activity as defined by the inhibition of TNF- production by LPS-stimulated macrophages (34). Approximately 10% of Lactobacillus isolates inhibited TNF- production by LPS-stimulated mouse macrophages (33). L. paracasei 1602 and L. reuteri 6798 represented two TNF--inhibitory strains recovered from mice lacking a predisposition to colitis. Murine macrophages were exposed to cell-free H. hepaticus-conditioned medium in order to assess the ability of selected lactobacilli to antagonize innate immune responses to H. hepaticus. In the presence of conditioned medium from lactobacilli, TNF- production was significantly diminished in H. hepaticus-conditioned medium-stimulated macrophages. Selected Lactobacillus clones inhibited H. hepaticus-mediated stimulation of macrophage TNF- production (Fig. 2).
Probiotic lactobacilli reduce intestinal inflammation in Helicobacter-infected female IL-10-deficient mice. Lactobacillus clones with TNF--inhibitory activity in vitro were evaluated in vivo by studying mucosal protection in a Helicobacter-exacerbated colitis model. We performed two infection studies with IL-10–/– mice. In a pilot study, both male (n = 21) and female (n = 18) mice were precolonized with a combination of two murine lactobacilli, L. paracasei 1602 and L. reuteri 6798, that had displayed anti-inflammatory activity with cultured macrophages. Lactobacillus colonization was followed by infection with the mouse pathogen H. hepaticus. Sham-dosed mice and mice colonized with a combination of L. paracasei 1602 and L. reuteri 6798 in the absence of H. hepaticus were studied as controls. After 10 weeks, we semiquantitatively graded the cecocolic junction for inflammation, hyperplasia, and dysplasia by histopathologic examination. Median histologic scores of uninfected sham-dosed controls and Lactobacillus-colonized animals were similar (Fig. 3a). In contrast, mice infected with H. hepaticus developed moderately severe typhlocolitis. Animals treated with a combination of L. paracasei 1602 and L. reuteri 6798 prior to infection with H. hepaticus did not exhibit significant diminution of IBD-like lesions.
When animals were stratified by gender, we observed significant reductions in cecocolic lesion scores (inflammation, hyperplasia, and dysplasia; P = 0.032, 0.032, and 0.016, respectively) in female mice cocolonized with Lactobacillus and H. hepaticus versus animals that were monoinfected with H. hepaticus (Fig. 3b). In contrast, male mice cocolonized with murine lactobacilli displayed only a slight and statistically nonsignificant reduction in intestinal lesion grades. Real-time PCR confirmed that intestinal Lactobacillus DNA quantities were significantly elevated only in Lactobacillus-treated animals and were maintained for the duration of the study (data not shown). Because female mice appeared to derive the greatest benefit from probiotic therapy, we performed a follow-up study with female mice only (n = 36), using the same cocolonization and infection protocol. A summary of histologic scores for the follow-up study is provided (Table 1).
As in the pilot study, uninfected animals exhibited normal cecocolic histology (Fig. 4a), while those monoinfected with H. hepaticus (Fig. 4b) developed moderate to severe typhlocolitis characterized by infiltration of lymphocytes and macrophages, with fewer granulocytes, in the mucosa and submucosa. Reactive epithelial changes included hyperplasia with crypt elongation as well as goblet cell loss and mild dysplasia with distortion of glandular architecture, cell crowding (piling), and early aberrant crypt foci including slit and back-to-back forms. Lesions were partially eliminated in mice treated with L. paracasei and L. reuteri (Fig. 4c). Subsequent molecular data presented in this study utilized samples from the follow-up population only.
Lactobacillus-mediated probiosis functions independently of quantities of intestinal H. hepaticus. In order to correlate intestinal inflammation with the presence of H. hepaticus, cecal DNA was extracted and analyzed by real-time quantitative PCR. As expected, H. hepaticus DNA was detectable only in animals deliberately infected with the pathogen. Unexpectedly, we identified no correlation between H. hepaticus colonization levels and lower bowel median lesion scores (inflammation [ = 0.253; P = 0.082] or hyperplasia [ = 0.225; P = 0.108]).
Animals receiving the L. paracasei 1602, L. reuteri 6798 combination (L. paracasei 1602/L. reuteri 6798) yielded quantities of H. hepaticus (P = 1.0) comparable to those for H. hepaticus-monoinfected controls (Fig. 5). L. paracasei 1602/L. reuteri 6798 provided partial protection against H. hepaticus-induced IBD-like lesions (Fig. 4c).
Reductions in proinflammatory cytokines correlate with probiosis. Since cytokine transcript levels have correlated with disease activity in IBD patients (41), we measured mRNA levels of selected cytokines in the ceca of mice (Fig. 6). Cecal levels of IL-12 (p40) and TNF- were significantly elevated in H. hepaticus-infected mice compared to levels in uninfected controls (P = 0.048 and P = 0.005, respectively), while IFN- was not significantly altered. IL-4 levels were similar between uninfected and H. hepaticus-infected mice. In contrast, introduction of L. paracasei 1602/L. reuteri 6798 in the absence of H. hepaticus did not alter IL-12, TNF-, IFN-, or IL-4 transcript levels compared to those for uninfected controls (P < 0.05 for all).
A significant reduction in mucosal IL-12p40 mRNA levels (P = 0.016) was observed when animals were cocolonized with L. paracasei 1602/L. reuteri 6798 prior to H. hepaticus challenge, while transcript levels for IFN-, IL-4, and TNF- were not significantly altered. This difference may be biologically important considering the partial anti-inflammatory effect in the intestine without concomitant reductions in H. hepaticus levels. The data trends indicate reductions in overall proinflammatory cytokine profiles with the L. paracasei 1602/L. reuteri 6798 probiotic combination (Fig. 7).
We determined whether the presence of anti-inflammatory Lactobacillus clones rendered mucosal explants tolerant to E. coli LPS challenge with TNF- as a biomarker. TNF- production by LPS-stimulated colonic explants was blunted in mucosal tissue from mice cocolonized with L. paracasei 1602/L. reuteri 6798 and H. hepaticus compared to results for uninfected animals or animals infected only with H. hepaticus. Explants from mice treated with the L. paracasei 1602/L. reuteri 6798 combination yielded a trend toward reduced amounts of intestinal TNF- when challenged with E. coli LPS (Fig. 8).
DISCUSSION
The IL-10–/– mouse is a well-established model for the study of colitis and IBD, including Helicobacter-induced disease (20). IL-10-deficient mice receiving lactic acid bacteria expressing recombinant murine IL-10 were protected from colitis (42), demonstrating the singular importance of this cytokine in regulating intestinal inflammation. Intestinal colonization with probiotic Lactobacillus clones, selected on the basis of TNF--inhibitory activity, diminished inflammation in IL-10-deficient mice infected with H. hepaticus. In the L. paracasei/L. reuteri-treated group, Lactobacillus-mediated protection was due to direct immunomodulatory activity and not reductions in the levels of H. hepaticus. The combination of L. paracasei and L. reuteri was associated with reductions in mucosal proinflammatory cytokines despite similar quantities of intestinal H. hepaticus. Interestingly, the anti-inflammatory effect was restricted to female mice in this model.
H. hepaticus induces IBD-like typhlocolitis in specific-pathogen-free IL-10-deficient mouse models (20). Chronic intestinal inflammation in this model has been presumably driven by a Th-1-predominant immune response. Elevated levels of mucosal TNF- and nitric oxide are present in the intestines of these animals, and the colitis is associated with increased levels of IL-12 and IFN- (19, 20). In the present study, intestinal inflammation was partially eliminated by a combination of L. paracasei and L. reuteri. L. paracasei/L. reuteri-treated animals demonstrated a partial elimination of intestinal inflammation and significantly reduced mucosal IL-12 mRNA levels. The present findings support the role of intestinal IL-12 in mediating mucosal inflammation in this mouse colitis model independently of reductions of H. hepaticus quantities. Colitis in H. hepaticus-treated IL-10-deficient mice is dependent on mucosal IL-12 (20), and reduced intestinal inflammation was correlated with decreased mucosal IL-12 levels in L. plantarum-treated IL-10-deficient mice (38). Possibly, lactobacilli inhibit NF-B activation in the intestinal mucosa, resulting in diminished expression of IL-12. The lack of an effect on intestinal IFN-, despite reductions in mucosal IL-12 mRNA, may be due to the absence of IL-10 in the intestine. IL-10 directly suppressed T- and NK cell-derived IFN- independently of TNF-, IL-12, or IL-18 (37).
In this study, we selected Lactobacillus strains on the basis of macrophage TNF--inhibitory activity in vitro. Animal studies support the primary role of TNF- in the pathogenesis of chronic colitis. Intraperitoneal injection of rat anti-mouse TNF- monoclonal antibodies markedly reduced morbidity in IL-10-deficient mice with colitis (13). Murine macrophages represent important sources of mucosal TNF- in murine colitis models. In vivo depletion of mucosal and lymphoid follicle-associated macrophages in mice deficient in IL-10 diminished colitis in these animals, suggesting that macrophages are important mediators of intestinal inflammation (44). Data obtained in this study indicate that the L. paracasei/L. reuteri combination may have reduced the mucosal TNF- response to bacterial LPS. Although cell populations from the intestinal mucosa were not separated, both enterocytes and macrophages were likely affected in these explant studies. Colons from mice precolonized with L. paracasei 1602/L. reuteri 6798 yielded diminished responses to LPS stimulation. L. reuteri and L. paracasei may contribute to the development of LPS tolerance in intestinal epithelial cells or macrophages. LPS tolerance in macrophages includes hallmark molecular features, such as the up-regulation of NF-B subunits p50 and p52 (22). Although induction of tolerance is a possibility, we have no evidence to support such a mechanism in this model.
Probiosis ultimately has a clonal basis and depends on the functional characterization of specific bacterial strains. Contrasting results have been obtained because investigators use a variety of different strains, cross species barriers, and work with different primary and cultured cell types. Some investigators have described the up-regulation of proinflammatory cytokine responses and NF-B activation by intact lactobacilli (29-31). Cytokine-modulatory effects are strain dependent (3, 34) and vary with growth phase and cell preparation (29-31, 34). Recent data indicate that viable L. reuteri organisms were required for anti-inflammatory effects (25). Lactobacillus species differed in their abilities to modulate proinflammatory cytokine production in bone marrow-derived dendritic cells, and cytokine-inhibitory L. reuteri strains antagonized effects of cytokine-inducing L. casei clones (3). Interactions between cells in the intestinal mucosa are important, since leukocyte-sensitized intestinal epithelial cells (IECs) were differentially modulated by Lactobacillus clones, in contrast to human IECs cultured in the absence of leukocytes (15). The proinflammatory cytokines TNF- and IL-1 were up-regulated in human IECs in the presence of Lactobacillus sakei but were not affected by Lactobacillus johnsonii. Furthermore, only L. johnsonii up-regulated the anti-inflammatory cytokine transforming growth factor 1 (15). In our experimental studies, anti-inflammatory effects in mouse models were obtained only with mouse-derived lactobacilli and raise the concern of using commensal bacteria derived from different species (including humans) in animal models. In our studies, IL-10-deficient mice precolonized with human-derived LGG developed more severe colitis after challenge with H. hepaticus (data not shown). In support of the potential importance of animal species origin for probiotic strategies, rat-derived L. reuteri was more effective than LGG at ameliorating intestinal inflammation in an acetic acid-induced rat colitis model (16).
Only female mice benefited significantly from the anti-inflammatory activity of probiotic Lactobacillus therapy in our model. Histologically, male and female IL-10-deficient mice challenged with H. hepaticus exhibited similar patterns of colitis (20), indicating that development of H. hepaticus-triggered colitis is not influenced by gender in C57BL mice. As expected, our present study shows that male and female H. hepaticus-infected C57BL mice yielded similar disease phenotypes in the absence of Lactobacillus. However, responsiveness to anti-inflammatory effects of probiotic bacteria appears to be affected by gender status in the present model. In contrast, A/JCr mice with an intact IL-10 gene demonstrated increased severity of colitis in females when challenged with H. hepaticus (23). Perhaps probiotic Lactobacillus strains modulate intestinal inflammation by an estrogen-dependent mechanism potentiated in an IL-10-deficient animal. Gender differences in immunologic responses have been well documented for humans and rodents (23, 43). Recent molecular studies of gender-specific responses point towards the role of sex hormones as possible determinants of immune responses and disease (23, 43). Specifically, estrogen has been implicated in T-cell-mediated autoimmune diseases (5). Of particular interest is that murine myeloid cells (dendritic cells or macrophages) lacking the estrogen receptor alpha produce elevated TNF- after challenge with E. coli LPS or Mycobacterium avium (21). Other studies revealed that estrogen receptor agonists suppressed inducible nitric oxide synthase and TNF- production in LPS-stimulated macrophages (18, 45). Estrogen down-modulates monocyte chemoattractant protein-1 production by murine macrophages (7). The estrogen, 17- estradiol, protects mice from experimental autoimmune encephalomyelitis in an estrogen receptor-dependent fashion (35). Differences in estrogen receptor subunit levels may partially account for the inability of Lactobacillus-derived immunomodulins to protect male mice from H. hepaticus-induced IBD. In data not shown, we also found that conditioned medium from probiotic lactobacilli decreased TNF- production in LPS-stimulated primary peritoneal macrophages from female mice but not from male mice. Since probiosis appears to be a balancing act, with anti-inflammatory effectors (i.e., IL-10) competing with proinflammatory mediators (e.g., TNF- and LPS), relative deficiencies of estrogen receptor alpha and circulating estrogens in male mice may tip the balance towards an inflammatory phenotype.
Probiosis may function by interference with enteric pathogens or proinflammatory components of the intestinal microbiota. L. paracasei 1602/L. reuteri 6798 may diminish inflammation by inhibition of H. hepaticus virulence gene expression or virulence factor activities without necessarily affecting quantities of intestinal H. hepaticus. Lactobacillus gasseri prevents gastritis by suppression of Helicobacter pylori type IV secretion system-dependent virulence factors, leading to decreased production of IL-8 by gastric epithelial cells (Y. Koga, personal communication). Commensal organisms may also enhance the production of antibacterial factors in the intestine. The commensal bacterium Bacteroides thetaiotaomicron induces the expression of bactericidal angiogenins (17) and prodefensin-processing matrilysin (24) by intestinal Paneth cells in the mouse. In our model, L. paracasei 1602/L. reuteri 6798 reduced mucosal IL-12 expression and LPS-stimulated colonic TNF- production, possibly facilitating H. hepaticus colonization. Probiotics likely regulate inflammatory responses by direct interactions with the intestinal mucosa or antagonism of pathogenic or proinflammatory bacteria.
Data from the present study and prior studies suggest that probiotics exert their beneficial effects by one or a combination of mechanisms, including the following: (i) direct modulation of immune function (1, 2, 28, 34), (ii) competition with or exclusion of pathogen(s) (4), (iii) interference with virulence of enteric pathogens (36), or (iv) increasing epithelial/mucosal barrier function (26). The optimal application of probiotics for prevention or treatment of IBD and enteric infections may ultimately require the characterization of bacterial clones with specific probiotic properties. Individuals with enteric infections may benefit from probiotic strains with antagonistic properties towards a specific infectious agent. Patients with IBD may require probiotic strains with specific anti-inflammatory features that complement the immunomodulatory activities of other drugs or dietary changes in multicomponent treatment regimens. Additionally, the functional importance of host- or patient-specific characteristics (e.g., gender) should also be considered when designing clinical probiotic trials. The studies presented in this report stress the importance of rational selection of probiotic bacterial clones for therapy of intestinal inflammatory diseases, including IBD.
ACKNOWLEDGMENTS
We thank Rao Varada, Michele DeMarco, Philip Lee, Jeffrey Bajko, Erinn Stefanich, Ellen Buckley, Kathleen Cormier, and Ernie Smith for technical assistance. We also thank M. J. Finegold for critical review and comments.
This work was supported by grants from the Crohn's & Colitis Foundation of America First Award (J.V.), National Institutes of Health grant award K08 DK02705 (J.V.), R01 CA67529 (J.G.F.), and R01 AI50592 (J.G.F.). J.V. was also supported by U.S. Public Health Service grant DK56338, which funds the Texas Gulf Coast Digestive Diseases Center.
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