Functional interaction between helicobacter-stimulated B (Hstim-B) cells and dendritic cells

Helicobacter pylori (H. pylori) is a microaerophilic gram negative and spiral-shaped bacterium which is firstly identified by Robbin Warren and Barry Marshall. About more than 50% of the people in the world have been infected with this bacterium; however, the bacteria cause serious gastrointestinal diseases only in 10-20% of infected people, whereas the remaining infected people has only asymptomatic gastritis. H. pylori can lead to development of gastric cancer and categorized as a group-I carcinogen by World Health Organization. Due to lack of efficient immune response against H.pylori in murines; mice has been infected with Helicobacter felis (H. felis) to create mouse model of Helicobacter infection. Dendritic cells (DCs) which are firstly identified by Steinmann, are the member of antigen presenting cells (APCs) and provide a connection between innate and adaptive immune response due to having a crucial role for activation of specific immune response against foreign antigens. Regulatory DCs express low levels of IL-12 and co-stimulatory molecules such as CD40, CD80 and CD86. Instead, expression of inhibitory molecules such as IL-10, PD-L1, CD95L and indoleamine 2,3 deoxygenase (IDO) are upregulated in regulatory DCs. Moreover, in addition to immature DCs, also semi-mature DCs characterized with MHC-IIhiCD80/CD86hiIL-12-TNF-α-IL-10+ phentotype are thought as tolerogenic DCs and inducer of type-1 regulatory T (Tr1) cell-mediated immune suppression. B cells have an important role for regulation of immune response in addition to their effector roles such as antibody production and antigen presentation. Regulatory role of B cells was shown with the use of mice lacking B cells which was not able to overcome autoimmune encephalitis. Helicobacter felis activates B cells via TLR2 and Myd88 and suppress immune responses in vivo and in vitro by triggering IL-10-producing Tr1-like cells. This suppression results in persistency of bacteria in gastric mucosa and decrease in pathology score. Therefore, balance between regulatory and effector response could determine the fate of infection. As demonstrated by our previous laboratory studies, H. felis-mediated activation of B cells induce two different B cell subgroups: IL-10-producing IL-10+ B cells and TGF-β-producing IL-10- B cells. Our first aim was to understand the effects of H. felis on bone marrow-derived dendritic cells (BM-DCs). Second aim was to determine effects of Helicobacter-stimulated B (Hfstim-B) cells on bone marrow-derived dendritic cells (BM-DCs). To perform these experiments; C57BL/6 mouse bone marrow cells were differentiated into immature dendritic cells in the presence of IL-4 and GM-CSF. B cells were magnetically isolated from spleens of C57BL/6 mice and treated with H. felis sonicate in order to obtain Helicobacter-stimulated IL-10+ B and IL-10- B cells through magnetic separation. Afterwards, these B cell subgroups were treated with H.felis sonicate, their supernatant were collected and applied onto immature BM-DCs. Following incubation and washing of the cells, BM-DCs were treated with LPS or H. felis sonicate. Cytokine profile and activation status of DCs were determined on protein and expression level. Moreover; for functional analysis, these DCs were co-cultured with naive CD4+ T cells at a ratio of 1:2 (DC:T) in the presence of 10 ng/ml IL-2 and 1 µg/ml anti-CD3Ԑ. Then, frequency of IL-10 secreting CD4+ T cells in different groups were analyzed by flow cytometry. H. felis-induced BM-DCs secrete IL-12/IL-23 (p40), IL-1β, TNF- α and IL-10 into the culture medium. Consistently, expression of IL-12/IL-23 (p40), TNF-α and IL-10 in addition to IL-6 was also induced. Flow cytometric analysis showed that H. felis activates DCs and causes upregulation of CD86 expression. All Hfstim-B cell groups including IL-10+ B and IL-10- B cells, induced DCs to express CD86 molecules in the absence of additional antigenic stimulation. However, there was no difference between DC groups stimulated with supernatant of IL-10- B and IL-10+ B in terms of expression of CD86 in untreated and LPS- or H. felis-treated groups. Moreover, IL-10 secretion was not induced upon stimulation of DCs with supernatant of unstimulated B, Helicobacter felis-stimulated B (Hfstim) or IL-10+ B cell followed by LPS or H. felis sonicate treatment. Nevertheless, DCs secreted significantly more IL-10 upon stimulation with IL-10+ B cell followed by LPS or H.felis treatment compared to IL-10- B cell supernatant stimulation. IL-12 secretion from DCs was induced by supernatant of all Helicobacter-stimulated B cell types. Strikingly, TNF-α secretion from DCs was suppressed upon stimulation of DCs with Hfstim-B cell supernatant prior to LPS or H. felis treatment. Interaction of BM-DCs with supernatant of Hfstim-IL-10+ B cells in the absence of subsequent LPS or H. felis treatment retained the frequency of IL-10-producing CD4+ T cells generated by immature DCs. Moreover, LPS treatment of DCs which had been previously stimulated with supernatant of unstimulated B cells, Hfstim-B cells or Hfstim-IL-10- B cells, but not Hfstim-IL-10+ B cells, resulted in the decreased frequency of IL-10-producing CD4+ T cells compared to LPS-treated control group. However, H. felis treatment of DCs following stimulation with supernatant of B cells have no effect on DCs to induce IL-10-producing CD4+ population. Therefore, it is possible that in addition to IL-10, also some other factors may be involved in differentiation of IL-10-producing CD4+ T cells by DCs stimulated with supernatant of different Hfstim-B cells subgroups. Furthermore, immature BM-DCs were co-cocultured with Hfstim-B cells at a ratio of 1:1 and 1:2 (DC:B) to evaluate the role of cell-to cell contact on this interaction. Accordingly, direct contact between Hfstim-B cells and immature DCs activates DCs and upregulates CD86 expression. B cells alone did not induce any IL-10 and TNF-α secretion regardless of the ratio between DC and B cell. However LPS or H. felis treatment of DCs after interaction with B cells resulted in decreased IL-10 production when the ratio between these cells was 1:1. Importantly, interaction of immature BM-DCs with Hfstim-B cells prior to LPS treatment induced production of IL-10 by DCs; whereas, unstimulated B cells exhibited inhibitory effect on IL-10 production when the ratio between these cells was 1:2 (DC:B). Moreover, TNF-α production by DCs was inhibited efficiently by Hfstim-B cells and unstimulated B cells when BM-DCs were co-cultured with these B cells at a ratio of 1:2 (DC:B) prior to LPS or H. felis treatment. It seems that the number of B cells co-cultured with immature DCs could be a critical factor to exhibit their regulatory function on DCs. Interestingly, Hfstim-IL-10+ B cells and Hfstim-IL-10- B cells did not affect the production of TNF-α by DCs. Collectively; some molecules secreted from Hfstim-B cells in addition to IL-10 synergistically with cell-to-cell contact have a regulatory role on activation and function of DCs. Consequently; first, DCs were activated and secreted both pro-inflammatory and anti-infammatory cytokines in response to H. felis antigens in vitro. Second, Helicobacter felis-stimulated B cells converts immature BM-DCs into IL-10-secreting tolerogenic form. Therefore, this study includes comprehensive analysis for the effects of H. felis on mouse BM-DCs and the first investigation that clarifies the ex vivo interaction of Hfstim-B cells subgroups with BM-DCs.