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Lactobacilli- and Staphylococcus aureus mediated modulation of immune responses in vitro
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. (Eva Sverremark-Ekström)
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The human gut harbors a vast number of microbes. These microbes are not passive bystanders. They are important in modulating the immune system. We have previously shown that early colonization with lactobacilli and Staphylococcus (S.) aureus differentially associates with allergy development and/or immune profile at early ages. Here we focus on understanding how these microbes modulate the response of intestinal epithelial cells and immune cells in vitro. In paper I, we investigated the impact of UV-killed and/or cell free supernatant (CFS) of different Lactobacillus (L.) species and S. aureus strains on cytokine production from intestinal epithelial cells (IEC) and immune cells. Enterotoxin-expressng S. aureus 161:2-CFS triggered CXCL-1/GRO-α and CXCL-8/IL-8 production by IEC. S. aureus-induced CXCL-8/IL-8 production was hampered by MyD88 gene silencing of IEC, indicating the importance of TLR signaling. Further, lactobacilli-CFS and S. aureus-CFS were able to induce the production of a number of cytokines by peripheral blood mononuclear cells (PBMC) from healthy donors, but only S. aureus triggered T-cell associated cytokines: IL-2, IL-17, IFN-γ and TNF-α; which were dampened by the co-treatment with S. aureus and any of the different Lactobacillus strains. Flow cytometry of the stimulated PBMC further verified IFN-γ and IL-17 production by T cells upon treatment with S. aureus-CFS, which also induced CTLA-4 expression and IL-10 production by Treg cells. In paper II, we investigated the influence of CFS of L. reuteri and S. aureus on the differentiation of monocyte to DC and subsequently how the generated DC influence T cell response. DC generated in the presence of L. reuteri exhibited an increase in expression of surface markers (HL-DR, CD86, CD83, CCR7) and cytokine production (IL-6, IL-10 and IL-23), but had a decreased phagocytic capacity compared with conventional Mo-DC, showing a more mature phenotype. However, upon LPS stimulation, DC generated in the presence of L. reuteri-CFS displayed a more regulatory phenotype, with a reduced cytokine response both at mRNA and protein levels. On the contrary, DC generated in the presence of S. aureus-CFS resembled the control Mo-DC both at mRNA and protein expression, but SA-DC was more efficient in inducing cytokine production in autologous T cells. In paper III, we studied the influence of L. reuteri-CFS on the retinoic acid (RA)-driven mucosal-like DCs’ phenotype and function to modulate T regulatory cells (Treg) in vitro. DC generated in the presence of RA showed a mucosal-like regulatory-DC phenotype with its CD103 expression, high IL10 production and decreased expression of genes associated with inflammation (NFκB1, RELB and TNF). Further, treatment with L. reuteri-CFS enhanced the regulatory phenotype of RA-DC by increasing the production of several chemokines, such as CXCL1, CXCL5, CCL3, CCL15 and CCL20, which are involved in gut homeostasis, while dampening the expression of most chemokine receptor genes. L. reuteri-CFS also increased CCR7 expression on RA-DC.  RA-DC co-cultured with T cell increased IL10 and FOXP3 expression in Treg. However L. reuteri-CFS pre-conditioning of the RA-DC did not improve the Treg phenotype. In conclusion, bacteria-CFS can have an impact on the response of IEC, differentiation and function of DC and, subsequently the T cell response, when taken together in the context of gut; these can have an impact on the health and disease of the host.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University , 2016. , 70 p.
Keyword [en]
lactobacilli, staphylococcus aureus, dendritic cells, retinoic acid, epithelial cells, T cells
National Category
Immunology
Research subject
Immunology
Identifiers
URN: urn:nbn:se:su:diva-127399ISBN: 978-91-7649-365-6 (print)OAI: oai:DiVA.org:su-127399DiVA: diva2:909042
Public defence
2016-04-29, Vivi Täckholmsalen (Q-salen) NPQ-huset, Svante Arrhenius väg 20, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Submitted.

Available from: 2016-04-06 Created: 2016-03-03 Last updated: 2017-02-17Bibliographically approved
List of papers
1. 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
Show others...
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
2. Lactobacillus reuteri and Staphylococcus aureus differentially influence the generation of monocyte-derived dendritic cells and subsequent autologous T cell responses
Open this publication in new window or tab >>Lactobacillus reuteri and Staphylococcus aureus differentially influence the generation of monocyte-derived dendritic cells and subsequent autologous T cell responses
Show others...
2016 (English)In: Immunity, Inflammation and Disease, ISSN 2050-4527, Vol. 4, no 3, 315-326 p.Article in journal (Refereed) Published
Abstract [en]

Introduction: In early-life, the immature mucosal barrier allows contact between the gut microbiota and the developing immune system. Due to their strategic location and their ability to sample luminal antigen, dendritic cells (DC) play a central role in the interaction of microbes and immune cells in the gut. Here, we investigated how two bacteria associated with opposite immune profiles in children, that is, Lactobacillus (L.) reuteri and Staphylococcus (S.) aureus, influenced the differentiation of monocytes in vitro as well how the generated DC impacted T cell responses.

Methods: We exposed monocyte cultures to cell-free supernatants (CFS) from these bacteria during their differentiation to DC.

Results: The presence of L. reuteri-CFS during DC differentiation resulted in DC with a more mature phenotype, in terms of up-regulated surface markers (HLA-DR, CD86, CD83, CCR7) and enhanced cytokine production (IL6, IL10, and IL23), but had a reduced phagocytic capacity compared with non-treated monocyte-derived DC (Mo-DC). However, upon LPS activation, L. reuteri-CFS-generated DC displayed a more regulated phenotype than control Mo-DC with notable reduction of cytokine responses both at mRNA and protein levels. In contrast, S. aureus-CFS-generated DC were more similar to control Mo-DC both without and after LPS stimulation, but they were still able to induce responses in autologous T cells, in the absence of further T cell stimulation.

Conclusions: We show that bacterial signals during DC differentiation have a profound impact on DC function and possibly also for shaping the T cell pool.

Keyword
Cytokine, dendritic cells, Lactobacillus reuteri, monocytes, PCR array, Staphylococcus aureus, T cell
National Category
Biological Sciences
Research subject
Immunology
Identifiers
urn:nbn:se:su:diva-128263 (URN)10.1002/iid3.115 (DOI)000383521400006 ()
Available from: 2016-03-22 Created: 2016-03-22 Last updated: 2016-10-31Bibliographically approved
3. Postbiotic Modulation of retinoic acid imprinted Mucosal-like Dendritic cells by Probiotic Lactobacillus reuteri 17938 In Vitro
Open this publication in new window or tab >>Postbiotic Modulation of retinoic acid imprinted Mucosal-like Dendritic cells by Probiotic Lactobacillus reuteri 17938 In Vitro
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2016 (English)In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 7, 1-11 p., 96Article in journal (Refereed) Published
Abstract [en]

Lactobacilli are widely used as probiotics with beneficial effects on infection-associated diarrhea, but also used in clinical trials of e.g., necrotizing enterocolitis and inflammatory bowel diseases. The possibility of using probiotic metabolic products, so-called postbiotics, is desirable as it could prevent possible side effects of live bacteria in individuals with a disturbed gut epithelial barrier. Here, we studied how Lactobacillus reuteri DSM 17938 cell-free supernatant (L. reuteri-CFS) influenced retinoic acid (RA)-driven mucosal-like dendritic cells (DC) and their subsequent effect on T regulatory cells (Treg) in vitro. RA clearly imprinted a mucosal-like DC phenotype with higher IL10 production, increased CD103 and CD1d expression, and a downregulated mRNA expression of several inflammatory-associated genes (NFκB1, RELB, and TNF). Treatment with L. reuteri-CFS further influenced the tolerogenic phenotype of RA-DC by downregulating most genes involved in antigen uptake, antigen presentation, and signal transduction as well as several chemokine receptors, while upregulating IL10 production. L. reuteri-CFS also augmented CCR7 expression on RA-DC. In cocultures, RA-DC increased IL10 and FOXP3 expression in Treg, but pre-treatment with L. reuteri-CFS did not further influence the Treg phenotype. In conclusion, L. reuteri-CFS modulates the phenotype and function of mucosal-like DC, implicating its potential application as postbiotic.

Keyword
retinoic acid, postbiotics, Lactobacillus reuteri, dendritic cells, probiotics
National Category
Immunology
Research subject
Immunology
Identifiers
urn:nbn:se:su:diva-126959 (URN)10.3389/fimmu.2016.00096 (DOI)000372147600001 ()
Available from: 2016-02-18 Created: 2016-02-18 Last updated: 2017-11-30Bibliographically approved

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