Lactobacilli are intestinal bacteria with known immunomodulatory competence. Numerous strains of this genus have been implicated in both the prevention and treatment of intestinal inflammation as well as in maintenance of immunological homeostasis. The frequent inclusion of lactobacilli in probiotic products attests to this ability. These lactic acid bacteria colonise the murine forestomach and burgeon in other environments similarly rich in carbohydrate-containing substrates. Accordingly, lactobacilli may utilise fermentable carbohydrates to synthesise exopolysaccharides (EPS). These polymers are secreted into the cellular milieu and, while the ecological function of EPS is yet to be defined, evidence points towards a protective role. This function may include bacterial protection from immunological attack, via EPS recognition by immune cells, resulting in modulation of immunological activity.
Dendritic cells (DC) are potent antigen presenting cells, providing an essential link between the innate and adaptive arms of the intestinal immune system. DC efficiently sample intestinal antigens and present peptides to cognate naive CD4⁺ T cells in secondary lymphatic tissue. Under the influence of secreted cytokines, DC direct the differentiation of naive CD4⁺ T cells and therefore, instruct the resultant immune response. Anti-inflammatory Th2 and regulatory T cells can down-regulate the destructive Th1 pro-inflammatory effects associated with inflammatory bowel disease (IBD). As such, bioactives with the aptitude to direct DC activity and T cell differentiation have the potential to prevent or reduce intestinal inflammation. Therefore, this study aimed to determine whether heat-killed EPS-producing strains of lactobacilli, and their secreted EPS, exert an immunomodulatory effect on the murine gut which may down-regulate the immune reactions associated with IBD.
Lactobacilli were screened for their ability to produce EPS when grown in the presence of glucose, sucrose or lactose. Heat-killed EPS-producing strains were then used to stimulate bone marrow-derived DC (BMDC) and the resultant cytokine profile was analysed. Nine Lactobacillus strains were found to produce EPS when grown in the presence of sucrose. Of these, L. reuteri 100-23 and L. johnsonii 100-33 exhibited potential anti-inflammatory effects. Therefore, these strains, as well as L. johnsonii 100-5 and L. johnsonii #21, with relatively weak BMDC stimulatory effect, were selected for further investigation.
EPS of the potentially anti-inflammatory strain L. reuteri 100-23 was analysed. This sample contained approximately 85% carbohydrate and was composed of a (2[to]6)-β-fructofuranan (levan) and a mannan. The fructan, with an estimated molecular weight of 7 kDa, comprised at least 50% of the EPS, while the mannan made up at least 22%. The mannan component was likely linked to a protein and may have originated from the culture medium.
The immunostimulatory capacity of heat-killed Lactobacillus bacterial cells and their EPS was determined in vitro. Firstly, the effect of lactobacilli and EPS on BMDC cytokine secretion, particularly levels of anti-inflammatory IL-10 and pro-inflammatory IL-12, as well as the expression of cell surface activation markers, was determined. L. reuteri 100-23 stimulated relatively high IL-10 secretion but low IL-12, while L. johnsonii 100-33-stimulated BMDC produced elevated levels of both IL-10 and IL-12. All bacterial cells up-regulated co-stimulatory molecules CD40 and CD80 on BMDC. The effect of these stimulated BMDC on T cell proliferation and cytokine production was then assessed, employing the ovalbuminDO11.10 T cell model. L. reuteri 100-23-stimulated BMDC down-regulated T cell production of the proliferation-stimulating cytokine, IL-2, up-regulated regulatory TGF-β secretion, but did not affect pro-inflammatory IFN-γ levels. The EPS of all strains did not stimulate significant BMDC cytokine production and failed to alter BMDC activation marker expression. However, BMDC stimulated with L. reuteri 100-23 and L. johnsonii 100-33 EPS significantly enhanced T cell IL-2 secretion, but did not alter TGF-β or IFN-γ levels.
The effect of in vivo L. reuteri 100-23 and EPS intestinal stimulation on the reactivity of immune cells was subsequently investigated. Mesenteric lymph node (MLN) cells and splenic T cells from reconstituted Lactobacillus-free mice fed stimulant or PBS on two occasions were co-cultured with stimulated or unstimulated donor CD11c⁺ splenic DC ex vivo. Cellular proliferation as well as TGF-β and IFN-γ secretion was analysed, and IL-10 neutralisation assays were carried out to ascertain the involvement of this cytokine. Primary exposure of MLN cells to L. reuteri 100-23 resulted in suppressed cell proliferation in the presence of enhanced TGF-β levels, which may have also involved IL-10. Primed splenic T cells exhibited increased proliferation in the presence of elevated TGF-β levels following re-exposure to L. reuteri 100-23, and IL-10 may be involved in limiting this proliferation. L. reuteri 100-23 EPS did not alter MLN cell proliferation, possibly due to the suppressive activity of IL-10, but did enhance that of naive and primed splenic T cells.
The effect of ingested L. reuteri 100-23 and EPS on intestinal sIgA concentration was assessed by quantifying IgA levels in the faecal supernatant of RLF mice previously ingesting L. reuteri 100-23 and EPS. L. reuteri 100-23 EPS-fed female mice exhibited significantly elevated levels of sIgA, while heat-killed bacteria did not affect antibody levels.
The present study demonstrated that oral administration of heat-killed L. reuteri 100-23 and EPS exerts immunomodulatory effects on the murine intestine. These bioactives may promote a suppressive environment by conditioning DC to secrete a cytokine profile conducive to regulatory T cell induction and memory generation. Additionally, mucosal protection may be favoured by the stimulation of elevated sIgA levels. Therefore, a therapeutic composite is possibly obtained to preserve the intestinal barrier by defending against pathogen-induced injury and buffering inflammatory events. In these ways, L. reuteri 100-23 and EPS may confer long-lasting protection against, and down-regulate the immune reactions associated with, IBD.
| Identifer | oai:union.ndltd.org:ADTP/208596 |
| Date | January 2008 |
| Creators | Livingston, Megan M, n/a |
| Publisher | University of Otago. Department of Microbiology & Immunology |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://policy01.otago.ac.nz/policies/FMPro?-db=policies.fm&-format=viewpolicy.html&-lay=viewpolicy&-sortfield=Title&Type=Academic&-recid=33025&-find), Copyright Megan M Livingston |
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