Barbara Sheil

Double blind, placebo controlled trial of two probiotic strains in interleukin 10 knockout mice and mechanistic link with cytokine balance

Background: Prophylactic efficacy against colitis following lactobacillus consumption in interleukin 10 (IL-10) knockout (KO) mice has been reported. Whether this applies equally to other probiotic strains is unknown, and the mechanism is unclear. Aims: (1) To compare the effect of feeding Lactobacillus salivarius subspecies salivarius 433118 and Bifidobacterium infantis 35624 against placebo on enterocolitis, the intestinal microflora, and (2) to compare the systemic immunological response to in vitro microbial challenge in probiotic fed and control IL-10 KO mice. Methods: Three groups of 10 IL-10 KO mice were fed fermented milk products containing Lb salivarius 433118 at 109 CFU/ml, B infantis 35624 at 108 CFU/ml, and unmodified milk, respectively, for 19 weeks. Faecal samples were taken at regular intervals to confirm gut transit, recovery of fed probiotics, and to assess the impact on the microflora. At sacrifice, the bowels were histologically scored. Cytokine production from Peyers’ patches and splenocytes was measured in vitro by ELISA. Results: Faecal recovery of probiotics was confirmed in all probiotic fed mice but not in controls. Colonic and caecal inflammatory scores were significantly decreased in both groups of probiotic fed mice (p<0.05). Proinflammatory cytokine production by Peyers’ patches and splenocytes was significantly reduced in probiotic fed animals whereas transforming growth factor β (TGF-β) levels were maintained. Conclusion: Both Lactobacillus salivarius 433118 and Bifidobacterium infantis 35624 significantly attenuate colitis in this murine model. Attenuation of colitis is associated with a reduced ability to produce Th1-type cytokines systemically and mucosally, while levels of TGF-β are maintained.

Is the mucosal route of administration essential for probiotic function? Subcutaneous administration is associated with attenuation of murine colitis and arthritis

Background: We and others have reported the prophylactic efficacy of oral consumption of probiotic lactobacilli in the interleukin 10 knockout (IL-10 KO) model of colitis. It has not been demonstrated that the oral route is essential for probiotic efficacy. Aims: (i) To determine the effect of parenteral administration (subcutaneous) of Lactobacillus salivarius 118 on colitis of IL-10 KO mice; and (ii) to determine if observed responses are disease specific. Methods: (i) IL-10 KO mice were injected subcutaneously with L salivarius 118 or saline over 19 weeks. At sacrifice, the bowels were histologically scored. Isolated splenocytes were cultured in vitro and cytokine levels measured. (ii) In the collagen induced arthritis model, DBA/1 mice were injected subcutaneously with the probiotic or saline. At sacrifice, paw thickness was measured and joints were histologically scored. Results: (i) Colonic inflammatory scores were significantly decreased in IL-10 KO mice injected with L salivarius 118 compared with controls (p<0.05). Proinflammatory cytokine production from stimulated splenocytes was significantly lower for the probiotic group whereas stimulated transforming growth factor β (TGF-β) levels were significantly increased (p<0.05). (ii) Scoring of arthritis and paw thickness were significantly improved in the group of mice injected with L salivarius 118 compared with controls. Conclusions: (1) Subcutaneous administration of L salivarius 118 significantly attenuated colitis in the IL-10 KO model and suppressed collagen induced arthritis, suggesting that the oral route may not be essential for probiotic anti-inflammatory effects and that responses are not disease specific. (2) The probiotic effect was associated with reduced production of proinflammatory (T helper 1) cytokines and maintained production of anti-TGF-β.

Bacterial strain-specific induction of Foxp3+ T regulatory cells is protective in murine allergy models

Background The incidence of atopic disease has increased dramatically during recent decades and the potential immunoregulatory influence of the microbiota in these individuals is under investigation.

Probiotic Effects on Inflammatory Bowel Disease

Components of the commensal flora, including Bifidobacteria and Lactobacilli, have been associated with beneficial effects on the host. These beneficial effects include maintenance of intestinal homeostasis, competitive exclusion of pathogens, production of antimicrobial compounds, promotion of gut barrier function, and immune modulation. Probiotics currently can be administered in dairy yogurts and drinks and also in the form of sachets or capsules. Although preliminary studies are clearly promising, placebo-controlled, randomized, double-blind clinical trials are required to clarify the role of probiotic bacteria in the treatment of inflammatory bowel disease. The choice of probiotic bacteria, the optimal dose, mode of administration, and duration of therapy still need to be established. Detailed strain characterization is also required for all potential probiotic strains. As evidence accumulates to suggest a breakdown in tolerance toward ubiquitous intestinal bacteria, it appears logical to intervene by modulating the enteric flora. Increasingly, research suggests that probiotics may offer an alternative or adjuvant approach to conventional therapy by altering the intestinal microflora and modulating the host immune system.

Comparative and Functional Analysis of Sortase-Dependent Proteins in the Predicted Secretome of Lactobacillus salivarius UCC118

ABSTRACT Surface proteins are important factors in the interaction of probiotic and pathogenic bacteria with their environment or host. We performed a comparative bioinformatic analysis of four publicly available Lactobacillus genomes and the genome of Lactobacillus salivarius subsp. salivarius strain UCC118 to identify secreted proteins and those linked to the cell wall. Proteins were identified which were predicted to be anchored by WXL-binding domains, N- or C-terminal anchors, GW repeats, lipoprotein anchors, or LysM-binding domains. We identified 10 sortase-dependent surface proteins in L. salivarius UCC118, including three which are homologous to mucus-binding proteins (LSL_0152, LSL_0311, and LSL_1335), a collagen-binding protein homologue (LSL_2020b), two hypothetical proteins (LSL_1838 and LSL_1902b), an enterococcal surface protein homologue (LSL_1085), a salivary agglutinin-binding homologue (LSL_1832b), an epithelial binding protein homologue (LSL_1319), and a proteinase homologue (LSL_1774b). However, two of the genes are gene fragments and four are pseudogenes, suggesting a lack of selection for their function. Two of the 10 genes were not transcribed in vitro, and 1 gene showed a 10-fold increase in transcript level in stationary phase compared to logarithmic phase. The sortase gene was deleted, and three genes encoding sortase-dependent proteins were disrupted. The sortase mutant and one sortase-dependent protein (mucus-binding homologue) mutant showed a significant reduction in adherence to human epithelial cell lines. The genome-wide investigation of surface proteins can thus help our understanding of their roles in host interaction.

Modulation of pathogen-induced CCL20 secretion from HT-29 human intestinal epithelial cells by commensal bacteria

BackgroundHuman intestinal epithelial cells (IECs) secrete the chemokine CCL20 in response to infection by various enteropathogenic bacteria or exposure to bacterial flagellin. CCL20 recruits immature dendritic cells and lymphocytes to target sites. Here we investigated IEC responses to various pathogenic and commensal bacteria as well as the modulatory effects of commensal bacteria on pathogen-induced CCL20 secretion. HT-29 human IECs were incubated with commensal bacteria (Bifidobacterium infantis or Lactobacillus salivarius), or with Salmonella typhimurium, its flagellin, Clostridium difficile, Mycobacterium paratuberculosis, or Mycobacterium smegmatis for varying times. In some studies, HT-29 cells were pre-treated with a commensal strain for 2 hr prior to infection or flagellin stimulation. CCL20 and interleukin (IL)-8 secretion and nuclear factor (NF)-κB activation were measured using enzyme-linked immunosorbent assays.ResultsCompared to untreated cells, S. typhimurium, C. difficile, M. paratuberculosis, and flagellin activated NF-κB and stimulated significant secretion of CCL20 and IL-8 by HT-29 cells. Conversely, B. infantis, L. salivarius or M. smegmatis did not activate NF-κB or augment CCL20 or IL-8 production. Treatment with B. infantis, but not L. salivarius, dose-dependently inhibited the baseline secretion of CCL20. In cells pre-treated with B. infantis, C. difficile-, S. typhimurium-, and flagellin-induced CCL20 were significantly attenuated. B. infantis did not limit M. Paratuberculosis- induced CCL20 secretion.ConclusionThis study is the first to demonstrate that a commensal strain can attenuate CCL20 secretion in HT-29 IECs. Collectively, the data indicate that M. paratuberculosis may mediate mucosal damage and that B. infantis can exert immunomodulatory effects on IECs that mediate host responses to flagellin and flagellated enteric pathogens.

Role of interleukin (IL-10) in probiotic-mediated immune modulation: an assessment in wild-type and IL-10 knock-out mice

While the impact of Bifidobacterium infantis 35624 and other probiotics on cytokines has been shown in established colitis, the effects of B. infantis consumption in pre‐inflammation of interleukin (IL)‐10 knock‐out (KO) mice and on the wild‐type (WT) C57Bl/6 mice have not been well demonstrated. The objective of this study was to examine cytokine responses in mucosal and systemic lymphoid compartments of IL‐10 KO mice early in disease and to compare with control WT mice. Mice were fed B. infantis or placebo for 5 weeks and culled prior to the onset of chronic intestinal inflammation (12–14 weeks). The spleen, Peyers patches and intestinal mucosa were removed and stimulated with various bacterial stimuli. Cytokine levels were measured by enzyme‐linked immunosorbent assay. While basal intestinal and systemic cytokine profiles of WT and IL‐10 KO mice were similar, transforming growth factor (TGF)‐β was reduced in the spleen of IL‐10 KO mice. Following probiotic consumption, interferon (IFN)‐γ was reduced in the Peyers patch of both WT and IL‐10 KO mice. Alterations in IFN‐γ in the Peyers patches of WT mice (enhancement) versus IL‐10 KO (reduction) were observed following in vitro stimulation with salmonella. Differential IL‐12p40, CCL2 and CCL5 responses were also observed in IL‐10 KO mice and WT mice. The cytokine profile of IL‐10 KO mice in early disease was similar to that of WT mice. The most pronounced changes occurred in the Peyers patch of IL‐10 KO mice, suggesting a probiotic mechanism of action independent of IL‐10. This study provides a rationale for the use of B. infantis 35624 for the treatment of gastrointestinal inflammation.

Bacterial signalling overrides cytokine signalling and modifies dendritic cell differentiation

Heterogeneity of dendritic cells (DC) is evident in the gut‐associated lymphoid tissue and determined, in part, by incompletely understood local environmental factors. Bacterial signalling is likely to be a dominant influence on precursor cells when recruited to the mucosa. We assessed the influence of commensal bacteria on DC differentiation and function. Murine bone marrow progenitors were exposed to Lactobacillus salivarius, Bifidobacterium breve or Bifidobacterium infantis. Differences in cell surface phenotype and function were assessed. Myeloid differentiation factor 88−/− (MyD88) cells were used to determine the influence of Toll‐like receptor signalling. While bacterial strains varied in impact, there was a consistent dose‐dependent inhibition of DC differentiation with a shift toward a Gr‐1+ CD11b+ monocyte‐like phenotype. A single bacterium on a per cell basis (1 : 1) was sufficient to alter cell phenotype. The effect was only evident in early precursors. Enhanced interleukin‐10 production correlated with increased Forkhead box P3 expression and reduced T‐cell proliferation. The bacterial effect on DC differentiation was found to be MyD88‐dependent. Signalling by enteric commensals through pattern recognition receptors on precursor cells alters DC differentiation and results in cells that are phenotypically monocyte‐like and functionally suppressive. This may account for some of the features of mucosal immune tolerance to the microbiota.