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THE INFLUENCE OF LACTOBACILLI AND STAPHYLOCOCCUS AUREUS ON IMMUNE RESPONSIVENESS IN VITRO
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. (Eva Sverremark-Ekström)
2013 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Alteration of gut microbiota has been associated with development of immune mediated diseases, such as allergy. In part, this could be due to the influence of microbes in shaping the immune response. In paper I, we investigated the association of early-life gut colonization with bacteria, and numbers of IL-4, IL-10 and IFN-γ producing cells at two years of age in response to PBMC stimulation with phytohemagglutinin (PHA) in vitro. Early Staphylococcus (S) aureus colonization was directly proportional to increased numbers of IL-4 and IL-10 secreting cells, while early co-colonization with lactobacilli and S. aureus associated with a decrease in IL-4, IL-10 and IFN-γ secreting cells compared to S. aureus alone. This was also confirmed in in vitro stimulations of PBMC with Lactobacillus and/or S. aureus strains, where S. aureus-induced IFN-γ production by Th cells was down regulated by co-stimulation with Lactobacillus. In paper II, we investigated the effects of UV-killed and/or culture supernatant (sn) of Lactobacillus strains and S. aureus strains on IEC and immune cell responses. IEC exposed to S. aureus-sn produced CXCL-1/GRO-α and CXCL-8/IL-8, while UV-killed bacteria had no effect. Further, PBMC from healthy donors exposed to Lactobacillus-sn and S. aureus-sn were able to produce a plethora of cytokines, but only S. aureus induced the T-cell associated cytokines: IL-2, IL-17, IFN-γ and TNF-α; which were down regulated by co-stimulation with any of the different Lactobacillus strains. Intracellular staining verified S. aureus-induced IFN-γ and IL-17 production by Th cells, and increased CTLA-4 expression and IL-10 production by T reg cells.

In conclusion, we show that colonization with gut microbiota at early age modulates the cytokine response in infancy. In addition, bacterial species influence cytokine response in a species-specific manner and we demonstrate that lactobacilli modulate S. aureus-induced immune response away from an inflammatory phenotype.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute , 2013. , 44 p.
Keyword [en]
Cytokine, T cells, immune response, HT-29, Intestinal epithelial cells, Lactobacillus, Staphylococcus aureus, bacteria and infants
National Category
Immunology in the medical area
Research subject
Immunology
Identifiers
URN: urn:nbn:se:su:diva-94326OAI: oai:DiVA.org:su-94326DiVA: diva2:653203
Presentation
2013-10-25, F564, Arrheniuslaboratorierna, Stockholm, 10:00 (English)
Supervisors
Available from: 2013-10-06 Created: 2013-10-03 Last updated: 2013-11-28Bibliographically approved
List of papers
1. Early-Life Gut Bacteria Associate with IL-4-, IL-10- and IFN-γ Production at Two Years of Age
Open this publication in new window or tab >>Early-Life Gut Bacteria Associate with IL-4-, IL-10- and IFN-γ Production at Two Years of Age
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 11, e49315-(9 pp) p.Article in journal (Refereed) Published
Abstract [en]

Microbial exposure early in life influences immune maturation and potentially also the development of immune-mediated disease. Here we studied early-life gut colonization in relation to cytokine responses at two years of age. Fecal samples were collected from infants during the first two months of life. DNA was extracted from the fecal samples and Bifidobacterium (B.) adolescentis, B. breve, B. bifidum, a group of lactobacilli (L. casei, L. paracasei and L. rhamnosus) as well as Staphylococcus (S.) aureus were detected with real time PCR. Peripheral mononuclear cells were stimulated with phytohaemagglutinin (PHA) and numbers of IL-4-, IL-10- and IFN-γ secreting cells were evaluated using ELISpot. We further stimulated peripheral blood mononuclear cells with bacterial supernatants in vitro and assessed the IL-4-, IL-10- and IFN-γ inducing capacity by flow cytometry and ELISA. Early S. aureus colonization associated with higher numbers of IL-4- (p = 0.022) and IL-10 (p = 0.016) producing cells at two years of age. In contrast to colonization with S. aureus alone, co-colonization with lactobacilli associated with suppression of IL-4- (p = 0.004), IL-10- (p = 0.004) and IFN-γ (p = 0.034) secreting cells. In vitro stimulations of mononuclear cells with bacterial supernatants supported a suppressive role of L. rhamnosus GG on S. aureus-induced cytokine responses. We demonstrate that the early gut colonization pattern associates with the PHA-induced cytokine profile at two years of age and our in vitro findings support that specific bacterial species influence the T helper cell subsets. This suggests that dysbiosis in the early microbiota may modulate the risk of developing inflammatory conditions like allergy.

National Category
Immunology
Research subject
Immunology
Identifiers
urn:nbn:se:su:diva-86260 (URN)10.1371/journal.pone.0049315 (DOI)000311535700028 ()23185315 (PubMedID)
Available from: 2013-01-11 Created: 2013-01-11 Last updated: 2017-12-06Bibliographically approved
2. Lactobacilli Regulate Staphylococcus aureus 161:2-Induced Pro-Inflammatory T-Cell Responses In Vitro
Open this publication in new window or tab >>Lactobacilli Regulate Staphylococcus aureus 161:2-Induced Pro-Inflammatory T-Cell Responses In Vitro
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 10Article in journal (Refereed) Published
Abstract [en]

There seems to be a correlation between early gut microbiota composition and postnatal immune development. Alteration in the microbial composition early in life has been associated with immune mediated diseases, such as autoimmunity and allergy. We have previously observed associations between the presence of lactobacilli and Staphylococcus (S.) aureus in the early-life gut microbiota, cytokine responses and allergy development in children. Consistent with the objective to understand how bacteria modulate the cytokine response of intestinal epithelial cell (IEC) lines and immune cells, we exposed IEC lines (HT29, SW480) to UV-killed bacteria and/or culture supernatants (-sn) from seven Lactobacillus strains and three S. aureus strains, while peripheral blood mononuclear cells (PBMC) and cord blood mononuclear cells (CBMC) from healthy donors were stimulated by bacteria-sn or with bacteria conditioned IEC-sn. Although the overall IEC response to bacterial exposure was characterized by limited sets of cytokine and chemokine production, S. aureus 161: 2-sn induced an inflammatory response in the IEC, characterized by CXCL1/GROa and CXCL8/IL-8 production, partly in a MyD88-dependent manner. UV-killed bacteria did not induce a response in the IEC line, and a combination of both UV-killed bacteria and the bacteria-sn had no additive effect to that of the supernatant alone. In PBMC, most of the Lactobacillus-sn and S. aureus-sn strains were able to induce a wide array of cytokines, but only S. aureus-sn induced the T-cell associated cytokines IL-2, IL-17 and IFN-gamma, independently of IEC-produced factors, and induced up regulation of CTLA-4 expression and IL-10 production by T-regulatory cells. Notably, S. aureus-sn-induced T-cell production of IFN-gamma and IL-17 was down regulated by the simultaneous presence of any of the different Lactobacillus strains, while the IEC CXCL8/IL-8 response was unaltered. Thus these studies present a possible role for lactobacilli in induction of immune cell regulation, although the mechanisms need to be further elucidated.

National Category
Biological Sciences Mathematics
Research subject
Immunology
Identifiers
urn:nbn:se:su:diva-96642 (URN)10.1371/journal.pone.0077893 (DOI)000326029300123 ()
Note

AuthorCount:12;

Available from: 2013-11-28 Created: 2013-11-25 Last updated: 2017-12-06Bibliographically approved

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