Characterization of a Large Panel of Lactic Acid Bacteria Derived from the Human Gut for their Capacity to Polarize Dendritic Cell

Dendritic cells (DCs) are the principal stimulators of naïve T helper (Th) cells and play a pivotal regulatory role in the Th1, Th2 and Treg cell balance. DCs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. Here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate DC. A large panel of human gut-derived Lactobacillus and Bifidobacterium spp. was screened for DC-polarizing capacity by exposing bone marrow-derived murine DC to lethally irradiated bacteria. Cytokines in culture supernatants and DC-surface maturation markers were analysed. Substantial differences were found among strains in the capacity to induce interleukin-12 (IL-12) and tumour necrosis factor (TNF)-α, while the differences for IL-10 and IL-6 were less pronounced. Bifidobacteria tended to be weak IL-12 and TNF-α inducers, while both strong and weak IL-12 inducers were found among the strains of Lactobacillus . Remarkably, strains weak in IL-12 induction inhibited IL-12 and TNF-α production induced by an otherwise strong cytokine-inducing strain of Lactobacillus casei , while IL-10 production remained unaltered. Selected strains were tested for induction of DC maturation markers. Those lactobacilli with greatest capacity to induce IL-12 were most effective in upregulating surface MHC class II and CD86. Moreover, L. casei -induced upregulation of CD86 was reduced in the presence of a weak IL-12-inducing L. reuteri . In conclusion, human Lactobacillus and Bifidobacterium spp. polarize differentially DC maturation. Thus, the potential exists for Th1/Th2/Treg-driving capacities of the gut DC to be modulated according to composition of gut flora including ingested probiotics.     

Streptococcus pyogenes and Lactobacillus rhamnosus differentially induce maturation and production of Th1-type cytokines and chemokines in human monocyte-derived dendritic cells

Dendritic cells (DCs) are the most efficient antigen-presenting cells and thus, have a major role in regulating host immune responses. In the present study, we have analyzed the ability of Gram-positive, pathogenic Streptococcus pyogenes and nonpathogenic Lactobacillus rhamnosus to induce the maturation of human monocyte-derived DCs. Stimulation of DCs with S. pyogenes resulted in strong expression of DC costimulatory molecules CD80, CD83, and CD86 accompanied with a T helper cell type 1 (Th1) cytokine and chemokine response. S. pyogenes also induced interleukin (IL)-2 and IL-12 production at mRNA and protein levels. In addition, IL-23 and IL-27 subunits p40, p19, p28, and EBI3 were induced at mRNA level. In contrast, L. rhamnosus-stimulated DCs showed only moderate expression of costimulatory molecules and produced low levels of cytokines and chemokines. Furthermore, no production of IL-2 or IL-12 family cytokines was detected. Bacteria-induced DC maturation and especially cytokine and chemokine production were reduced when bacteria were heat-inactivated. Our results show that human monocyte-derived DCs respond differently to different Gram-positive bacteria. Although pathogenic S. pyogenes induced a strong Th1-type response, stimulation with nonpathogenic L. rhamnosus resulted in development of semi-mature DCs characterized by moderate expression of costimulatory molecules and low cytokine production.     

Lactobacilli Modulate Proliferation and Cytokine Production of Human Peripheral Blood Natural Killer Cells In Vitro

The intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. Evidence indicates that lactic acid bacteria (LAB), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. Established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. The objective of this study is to evaluate the immunomodulating properties of a range of LAB of human origin. As dendritic cells (DCs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro -generated immature DCs serve as a suitable model for studying the immunomodulating effects of lab. Human immature DCs were generated in vitro from monocytes and exposed to lethally UV-irradiated LAB. The effect of various species of LAB on DCs in direct contact was evaluated. Furthermore, the maturation pattern of DCs separated from the bacteria by an epithelial cell layer (CaCo-2 cells), which should mimic the intestinal environment, was studied. Cytokine secretion (IL-12, IL-10 and TNF-α) and upregulation of maturation surface markers on DCs (CD83 and CD86) was measured. Different LAB induced diverse cytokine responses. Some strains were strong IL-12 and TNF-α inducers and others weak. All strains induced IL-10. Different LAB also differentially modulated expression of CD83 and CD86 on DCs. Although some variation in the response to LAB of DCs generated from different blood donors was observed, general differences in the effect of the various LAB was revealed. Experiments with the DC CaCo-2 coculture system are ongoing. Different species of LAB differentially affect DC maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response.     

CD8α+ and CD8α- DC subsets from BCG-infected mice inhibit allergic Th2-cell responses by enhancing Th1-cell and Treg-cell activity respectively

The hygiene hypothesis has suggested an inhibitory effect of infections on allergic diseases, but the related mechanism remains unclear. We recently reported that DCs played a critical role in Mycobacterium bovis Bacille Calmette-Guérin (BCG)-mediated inhibition of allergy, which depended on IL-12 and IL-10-related mechanisms. Here, we tested the hypothesis that BCG infection could modulate the function of DC subsets, which might in turn inhibit allergic responses through different mechanisms. We sorted CD8α(+) and CD8α(-) DCs from BCG-infected mice and tested their ability to modulate Th2-cell responses to ovalbumin (OVA) using in vitro and in vivo approaches. We found that both DC subsets could inhibit the allergic Th2-cell response in both a DC:T-cell co-culture system and after adoptive transfer. These subsets exhibited different co-stimulatory marker expression and cytokine production patterns and were different in inducing Th1 and Treg cells. Specifically, we found that CD8α(+) DCs produced higher IL-12, inducing higher Th1 cell response, while CD8α(-) DCs expressed higher ICOS-L and produced higher IL-10, inducing CD4(+) CD25(+) FoxP3(+) Treg cells with IL-10 production and membrane-bound TGF-β expression. The finding suggests that one infection may inhibit allergy by both immune deviation and regulation mechanisms through modulation of DC subsets.     

Induction by a lactic acid bacterium of a population of CD4(+) T cells with low proliferative capacity that produce transforming growth factor beta and interleukin-10.

We investigated whether certain strains of lactic acid bacteria (LAB) could antagonize specific T-helper functions in vitro and thus have the potential to prevent inflammatory intestinal immunopathologies. All strains tested induced various levels of both interleukin-12 (IL-12) and IL-10 in murine splenocytes. In particular, Lactobacillus paracasei (strain NCC2461) induced the highest levels of these cytokines. Since IL-12 and IL-10 have the potential to induce and suppress Th1 functions, respectively, we addressed the impact of this bacterium on the outcome of CD4(+) T-cell differentiation. For this purpose, bacteria were added to mixed lymphocyte cultures where CD4(+) T-cells from naive BALB/c mice were stimulated weekly in the presence of irradiated allogeneic splenocytes. In these cultures, L. paracasei NCC2461 strongly inhibited the proliferative activity of CD4(+) T cells in a dose-dependent fashion. This was accompanied by a marked decrease of both Th1 and Th2 effector cytokines, including gamma interferon, IL-4, and IL-5. In contrast, IL-10 was maintained and transforming growth factor beta (TGF-beta) was markedly induced in a dose-dependent manner. The bacteria were not cytotoxic, because cell viability was not affected after two rounds of stimulation. Thus, unidentified bacterial components from L. paracasei NCC2461 induced the development of a population of CD4(+) T cells with low proliferative capacity that produced TGF-beta and IL-10, reminiscent of previously described subsets of regulatory cells implicated in oral tolerance and gut homeostasis.     

Epstein–Barr virus induces the differentiation of semi-mature dendritic cells from cord blood monocytes

Background

Epstein–Barr virus (EBV) is a tumorigenic virus which has effectively infected nearly all human beings with over 95% adult being seropositive. The persistence of latent EBV infection is not fully understood. Recent studies point towards a hypothesis of immune suppression and immune evasion involving regulatory T cells (Tregs) and dendritic cells (DCs). We sought to explore the mechanism of EBV suppression and immune evasion.

Methods

We compared the effects of EBV on cord blood (CB) and adult DCs differentiation and maturation including phenotype by flow cytometry, cytokine by ELISA and RT-PCR. And we evaluated the function of DC by co-culture DC and Treg by detection the expression of Foxp3, the phenotype and the cytokine profile of Tregs by flow cytometry.

Results

CB DCs derived from EBV-infected CB monocytes or from EBV-infected CB immature DCs (iDCs) displayed distinct phenotypes of “semi-mature” DCs with high expression of co-stimulatory molecules, such as CD40, CD80 and CD86 but low cytokine production, related to immune tolerance and homeostasis. While the EBV-infected adult iDCs resemble that of “pathogen-driven regulatory mature DCs” with high expression of co-stimulatory molecules, down-regulation of IL-12 secretion and up-regulation of IL-10 secretion, related to protection of host and immune evasion of pathogens. EBV infected cord blood monocytes-derived DCs drived Tregs development by driving the expression of Foxp3, increasing the expression of CTLA-4, decreasing the expression of GITR and promoted the generation of intracellular IL-2 and IL-10 by Tregs.

Conclusion

Epstein–Barr virus induces the differentiation of semi-mature dendritic cells from cord blood monocytes. The differences between CB and adult DCs suggested that the developmental maturity of the cells may affect their immune responses to EBV infection.     

Adhesive substrate-modulation of adaptive immune responses

While it is well-known that adsorbed proteins on implanted biomaterials modulate inflammatory responses, modulation of dendritic cells (DCs) via adhesion-dependent signaling has only been begun to be characterized. In this work, we demonstrate that adhesive substrates elicit differential DC maturation and adaptive immune responses. We find that adhesive substrates support similar levels of DC adhesion and expression of stimulatory and co-stimulatory molecules. Conversely, DC morphology and differential production of pro- and anti-inflammatory cytokines (IL-12p40 and IL-10, respectively) is adhesive substrate-dependent. For example, DCs cultured on collagen and vitronectin substrates generate higher levels of IL-12p40, whereas DCs cultured on albumin and serum-coated tissue culture-treated substrates produce the higher levels of IL-10 compared to other substrates. Additionally, our results suggest substrate-dependent trends in DC-mediated allogeneic CD4(+) T-cell proliferation and T-helper cell type responses. Specifically, we show that substrate-dependent modulation of DC IL-12p40 cytokine production correlates with CD4(+) T-cell proliferation and T(h)1 type response in terms of IFN-gamma producing T-helper cells. Furthermore, our results suggest substrate-dependent trends in DC-mediated stimulation of IL-4 producing T-cells, but this T(h)2 type response is not dependent on DC production of IL-10 cytokine. This work has impact in the rational design of biomaterials for diverse applications such as tissue-engineered constructs, synthetic particle-based vaccines and the ex vivo culture of DCs for immunotherapies.     

Supernatant of Bifidobacterium breve induces dendritic cell maturation, activation, and survival through a Toll-like receptor 2 pathway

BACKGROUND: Commensal gut bacteria are essential for the development and maintenance of the gut's immune system. Some bacteria strains, such as Lactobacillus and Bifidobacterium species, have been reported to provide protection from allergic and inflammatory bowel diseases. However, the interactions between these commensal bacteria and the immune system are largely unknown. OBJECTIVE: We studied the effects of a supernatant from the culture of B breve C50 (BbC50) on the maturation, activation, and survival of human dendritic cells (DCs). METHODS: DCs were differentiated from human monocytes with IL-4 and GM-CSF for 5 days and cultured with BbC50 supernatant (BbC50SN) or LPS for 2 days. RESULTS: BbC50SN induced DC maturation, with increase in CD83, CD86, and HLA-DR expression. We also showed, for the first time, that BbC50SN prolonged DC survival, with high IL-10 and low IL-12 production compared with that seen in LPS-DCs. Moreover, BbC50SN inhibited the effects of LPS on DCs, both in terms of IL-12 production and in terms of survival. The prolonged DC survival was independent of IL-10 production and nuclear factor kappaB pathway but was associated with an upregulation of Bcl-xL and Phospho-Bad. Finally, BbC50SN induced activation of Toll-like receptor 2 (TLR2)-transfected cells in contrast to TLR4-, TLR7-, and TLR9-transfected cells. CONCLUSION: The supernatant of B breve C50 can induce DC maturation and prolonged DC survival through TLR2, with high IL-10 production. These properties might correspond to a regulatory DC profile, which could limit the excessive TH1 response and control the excessive TH2 polarization observed in atopic newborns.     

Dendritic cells differentiated in the presence of a single-stranded viral RNA sequence conserve their ability to activate CD4 T lymphocytes but lose their capacity for Th1 polarization

Monocyte-derived dendritic cells (DCs) differentiate in the presence of Toll-like-receptor (TLR) ligands in the course of ongoing infections. A single-stranded RNA (ssRNA) sequence, corresponding to the sequence of the U5 region of human immunodeficiency virus type 1 RNA, was used to mimic viral activation of TLR7 in human DCs. We determined the effector potential of DCs differentiated in the presence of this ssRNA molecule (ssRNA-DCs). ssRNA-DCs phenotypically resembled mature DCs. In contrast, their capacity to allostimulate naive CD4(+) T cells resembled that of conventional immature DCs and could be increased by TLR4 stimulation. Th1 polarization of CD4(+) T cells and production of interleukin 12p70 (IL-12p70) by ssRNA-DCs were selectively abrogated in response to a late TLR4, but not in response to a CD40, maturation signal. Inhibition of p38 mitogen-activated protein kinase partially restored IL-12p70 secretion but did not restore Th1 polarization, whereas addition of exogenous IL-12 led to recovery of Th1 polarization. In contrast to lipopolysaccharide, ssRNA induced IL-12p70 production at the very earliest stages of DC differentiation, indicating a particular role for TLR7 in monocyte-derived DCs recently engaged in differentiation. These data demonstrate generation of phenotypically mature DCs with the ability to expand CD4(+) T lymphocytes lacking Th1/2-polarizing capacity.     

Dexamethasone turns tumor antigen-presenting cells into tolerogenic dendritic cells with T cell inhibitory functions

BackgroundDendritic cells (DCs) are usually immunogenic, but they are also capable of inducing tolerance under anti-inflammatory conditions. Immunotherapy based on autologous DCs loaded with an allogeneic melanoma cell lysate (TRIMEL/DCs) induces immunological responses and increases melanoma patient survival. Glucocorticoids can suppress DC maturation and function, leading to a DC-mediated inhibition of T cell responses.MethodsThe effect of dexamethasone, a glucocorticoid extensively used in cancer therapies, on TRIMEL/DCs phenotype and immunogenicity was examined.ResultsDexamethasone induced a semi-mature phenotype on TRIMEL/DC with low maturation surface marker expressions, decreased pro-inflammatory cytokine induction (IL-1β and IL-12) and increased release of regulatory cytokines (IL-10 and TGF-β). Dexamethasone-treated TRIMEL/DCs inhibited allogeneic CD4⁺ T cell proliferation and cytokine release (IFNγ, TNF-α and IL-17). Co-culturing melanoma-specific memory tumor-infiltrating lymphocytes with dexamethasone-treated TRIMEL/DC inhibited proliferation and effector T cell activities, including cytokine secretion and anti-melanoma cytotoxicity.ConclusionsThese findings suggest that dexamethasone repressed melanoma cell lysate-mediated DC maturation, generating a potent tolerogenic-like DC phenotype that inhibited melanoma-specific effector T cell activities. These results suggest that dexamethasone-induced immunosuppression may interfere with the clinical efficacy of DC-based melanoma vaccines, and must be taken into account for optimal design of cellular therapy against cancer.     

Galectin-9 induces maturation of human monocyte-derived dendritic cells]

We investigated the role of galectin-9 (Gal-9) in maturation of dendritic cells (DC). Culture of immature DCs with exogenous Gal-9 markedly increased the surface expression of CD40, CD54, CD80, CD83, CD86, and HLA-DR in a concentration-dependent manner, although Gal-9 had no effect on differentiation of human monocytes into immature DCs. Gal-9-treated DCs secreted IL-12 but not IL-10, and they elicited the production of Th1 cytokines (IFN-gamma and IL-2), but not that of the Th2 cytokines (IL-4 and IL-5) by allogeneic CD4(+) T cells. These effects of Gal-9 on immature DCs were not essentially dependent on its lectin properties, given that they were only slightly inhibited by lactose. We further found that a Gal-9 mutant that lacks beta-galactoside binding activity reproduced the above activities, and that an anti-Gal-9 mAb suppressed them. Gal-9 induced phosphorylation of the p38 MAPK and ERK1/2 in DCs, and an inhibitor of p38 signaling, but not inhibitors of signaling by either ERK1/2 or phosphatidylinositol 3-kinase, blocked Gal-9-induced up-regulation of costimulatory molecule expression and IL-12 production. These findings suggest that Gal-9 plays a role not only in innate immunity but also in acquired immunity by inducing DC maturation and promoting Th1 immune responses.     

Enveloped virus but not bacteria block IL-13 responses in human cord blood T cells in vitro

Infections that occur early in life may have a beneficial effect on the immune system and thereby reduce the risk of allergen sensitization and/or allergic disease. It is not yet clear to what extent specific virus and/or bacteria can mediate this effect. The purpose of this study was to assess the role of virus and bacteria in CD4(+) T cell-derived cytokine production in newborns. We compared the effects of five bacteria (Staphlococcus aureus, Escherichia coli, Clostridium difficile, Lactobacillus rhamnosus and Bifidobacterium bifidus) and seven virus (adenovirus, coronavirus, cytomegalovirus, herpes simplex virus, influenza virus, morbillivirus and poliovirus) on the Th1/Th2 cytokine production in mixed lymphocyte reactions using CD4(+) T cells from cord blood cocultured with allogenic myeloid or plasmacytoid dendritic cells. When comparing the baseline cytokine production prior to microbial stimulation, we observed that cord plasmacytoid DC were stronger inducers of Th2 cytokines (IL-5 and IL-13) compared with cord myeloid DC and to adult DC. When adding microbes to these cultures, bacteria and virus differed in two major respects; Firstly, all enveloped viruses, but none of the bacteria, blocked Th2 (IL-13) production by cord CD4(+) cells. Secondly, all Gram-positive bacteria, but none of the virus, induced IL-12p40 responses, but the IL-12p40 responses did not affect Th1 cytokine production (IFN-γ). Instead, Th1 responses were correlated with the capacity to induce IFN-α secretion, which in cord cells were induced by S. aureus and influenza virus alone. These data imply that enveloped virus can deviate Th2 responses in human cord T cells.     

C1q regulation of dendritic cell development from monocytes with distinct cytokine production and T cell stimulation

The causative association of complement C1q deficiency with systemic lupus erythematosus (SLE), which inevitably involves the breakdown of tolerance, remains poorly explained. Its non-hepatic, macrophage and dendritic cell (DC) origin may be highly relevant. In tissues, C1q is produced by DCs and macrophages which deposits around these cells and we ask whether this pericellular form of C1q regulates DC development from monocytes. DCs cultured on immobilized C1q (C1q-DCs) show similar MHC, CD40, CD80, CD86, CD83 and CCR7 expression as normal DCs, but these cells exhibit increased phagocytosis of apoptotic cells and elevated IL-10 but reduced IL-12 and IL-23 production. Intracellularly, C1q-DCs exhibit increased ERK, p38 and p70S6 kinase activity. By mixed leukocyte reaction, C1q-DCs show reduced Th1 and Th17 induction from allogeneic CD4(+) T cells. LPS and IFNγ, which cause normal DCs to induce increased CD25 expression on CD4(+) T cells, attenuate C1q-DC induction of CD25. These imply that the DC pericellular C1q may induce tolerogenic properties in developing DCs.     

Ethanol extract from birch bark (Betula pubescens) suppresses human dendritic cell mediated Th1 responses and directs it towards a Th17 regulatory response in vitro

Extracts and fractions from birch bark have been used to treat various diseases, such as skin disorders and rheumatism, and for analgesic effects. Results from studies in vitro and in vivo have shown that birch bark extracts can have immunoregulatory effects. These effects have mainly been attributed to the various triterpenes found in birch bark. The effects of birch bark from Betula pubescens on immune responses have not been reported. Ethanol extract was prepared from dry birch bark (DBBEE) and five fractions made using various ratios of dichloromethane and methanol (fractions I-V). Human monocyte-derived dendritic cells (DCs) were matured with or without DBBEE or fractions I-V at several concentrations. The effects of the extract and fractions on DC maturation were determined by measuring cytokine secretion by ELISA and expression of surface molecules by flow cytometry. DBBEE and fractions III and IV reduced DC secretion of IL-6, IL-10 and IL-12p40 and expression of CD83, CD86, CCR7 and DC-SIGN compared with control DCs. Proliferation of allogeneic CD4(+) T cells co-cultured with DCs matured with fraction IV, as measured by (3)H-thymidine incorporation, was similar to proliferation of allogeneic CD4(+) T cells co-cultured with control DCs. However, IFN-γ secretion was reduced and IL-10 and IL-17 secretion was increased, a cytokine profile consistent with a Th17 regulatory phenotype. These results indicate that bark from Betula pubescens contains compound(s) that can modulate DCs so that their interaction with T cells leads to an immunoregulatory response.     

Tim-1 stimulation of dendritic cells regulates the balance between effector and regulatory T cells

We show that the T-cell immunoglobalin mucin, Tim-1, initially reported to be expressed on CD4(+) T cells, is constitutively expressed on dendritic cells (DCs) and that its expression further increases after DC maturation. Tim-1 signaling into DCs upregulates costimulatory molecule expression and proinflammatory cytokine production, thereby promoting effector T-cell responses, while inhibiting Foxp3(+) Treg responses. By contrast, Tim-1 signaling in T cells only regulates Th2 responses. Using a high-avidity/agonistic anti-Tim-1 antibody as a co-adjuvant enhances the immunogenic function of DCs, decreases the suppressive function of Tregs, and substantially increases proinflammatory Th17 responses in vivo. The treatment with high- but not low-avidity anti-Tim-1 not only worsens experimental autoimmune encephalomyelitis (EAE) in susceptible mice but also breaks tolerance and induces EAE in a genetically resistant strain of mice. These findings indicate that Tim-1 has an important role in regulating DC function and thus shifts the balance between effector and regulatory T cells towards an enhanced immune response. By understanding the mechanisms by which Tim-1 regulates DC and T-cell responses, we may clarify the potential utility of Tim-1 as a target of therapy against autoimmunity, cancer, and infectious diseases.     

Human CD1c (BDCA-1)+ myeloid dendritic cells secrete IL-10 and display an immuno-regulatory phenotype and function in response to Escherichia coli

Human blood myeloid DCs can be subdivided into CD1c (BDCA-1)(+) and CD141 (BDCA-3)(+) subsets that display unique gene expression profiles, suggesting specialized functions. CD1c(+) DCs express TLR4 while CD141(+) DCs do not, thus predicting that these two subsets have differential capacities to respond to Escherichia coli. We isolated highly purified CD1c(+) and CD141(+) DCs and compared them to in vitro generated monocyte-derived DCs (MoDCs) following stimulation with whole E. coli. As expected, MoDCs produced high levels of the proinflammatory cytokines TNF, IL-6, and IL-12, were potent inducers of Th1 responses, and processed E. coli-derived Ag. In contrast, CD1c(+) DCs produced only low levels of TNF, IL-6, and IL-12 and instead produced high levels of the anti-inflammatory cytokine IL-10 and regulatory molecules IDO and soluble CD25. Moreover, E. coli-activated CD1c(+) DCs suppressed T-cell proliferation in an IL-10-dependent manner. Contrary to their mouse CD8(+) DC counterparts, human CD141(+) DCs did not phagocytose or process E. coli-derived Ag and failed to secrete cytokines in response to E. coli. These data demonstrate substantial differences in the nature of the response of human blood DC subsets to E. coli.     

Secretion of interleukin-10 or interleukin-12 by LPS-activated dendritic cells is critically dependent on time of stimulus relative to initiation of purified DC culture

Dendritic cells (DC) are key regulators of adaptive immunity with the potential to induce T cell activation/immunity or T cell suppression/tolerance. DC are themselves induced by "maturation" signals such as bacterial lipopolysaccharide (LPS). We demonstrate here that LPS can stimulate DC to display similar maturation phenotypes but to differentiate toward an interleukin (IL)-10(high)- or IL-12(high)-secretor profile depending on the timing of maturation signal induction. Immediate/early administration of LPS induced purified bone marrow-derived DC (BMDC) to differentiate as IL-10(high)IL-12(low)-secreting cells, termed early DC (eDC). Conversely, delayed administration of LPS altered the DC cytokine profile to IL-10(low)IL-12(high), termed later DC (lDC). The presence of IL-4 enhanced the yield and maturation of BMDC but inhibited LPS-induced IL-10 production by eDC. In contrast, interferon-gamma reduced the yield of DC but promoted the level of LPS-induced IL-10 production by lDC. Our data provide new evidence that ex vivo manipulation and the cytokine environment regulate DC maturation status and cytokine-secretor phenotype with implications for the control of T cell differentiation and function via DC-based immunotherapeutic strategies.     

Rv0315, a novel immunostimulatory antigen of Mycobacterium tuberculosis, activates dendritic cells and drives Th1 immune responses

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is one of the most deadly infectious diseases, with approximately two million people dying of TB annually. An effective therapeutic method for activating dendritic cells (DCs) and driving Th1 immune responses would improve host defenses and further the development of a TB vaccine. Given the importance of DC maturation in eliciting protective immunity against TB, we investigated whether Rv0315, a newly identified Mtb antigen, can prompt DC maturation. We found that Rv0315 functionally activated DCs by augmenting the expression of the co-stimulatory molecules CD80 and CD86 as well as MHC class I/II molecules. Moreover, it increased DC secretion of the pro-inflammatory cytokines IL-6, IL-1β, and TNF-α. Unlike LPS, however, Rv0315 induced the secretion of IL-12p70, but not IL-10. In addition, Rv0315-treated DCs accelerated the proliferation of CD4(+) and CD8(+) splenic T cells from Mtb-infected mice, with increased levels of IFN-γ, in syngeneic and allogeneic mixed lymphocyte reactions, indicating that Rv0315 contributes to Th1 polarization of the immune response. Importantly, both mitogen-activated protein kinases and nuclear factor κB signaling mediated the expression of DC surface markers and cytokines. Taken together, our results indicate that Rv0315 is a novel DC maturation-inducing antigen that drives T cell immune responses toward Th1 polarization, suggesting that Rv0315 plays a key role in determining the nature of the immune response to TB.     

Role of interleukin-1beta in activating the CD11c(high) CD45RB- dendritic cell subset and priming Leishmania amazonensis-specific CD4+ T cells in vitro and in vivo

Cutaneous leishmaniasis associated with Leishmania amazonensis infection is characterized by uncontrolled parasite replication and profound immunosuppression; however, the underlying mechanisms remain largely unclear. One possibility is that the L. amazonensis parasite modulates antigen-presenting cells, favoring the generation of pathogenic Th cells that are capable of recruiting leukocytes but insufficient to fully activate their microbicidal activities. To test this possibility, we infected bone marrow-derived dendritic cells (DCs) of C57BL/6 mice with L. amazonensis or Leishmania major promastigotes and assessed the activation of DC subsets and their capacity in priming CD4(+) T cells in vitro. In comparison to L. major controls, L. amazonensis-infected DCs secreted lower levels of interleukin-1alpha (IL-1alpha) and IL-1beta, were less potent in activating the IL-12p40-producing CD11c(high) CD45RB(-) CD83(+) CD40(+) DC subset, and preferentially activated CD4(+) T cells with a IFN-gamma(low) IL-10(high) IL-17(high) phenotype. Although the addition of IL-1beta at the time of infection markedly enhanced DC activation and T-cell priming, it did not skew the cytokine profile of DCs and pathogenic Th cells, as local injection of IL-1beta following L. amazonensis infection accelerated Th cell activation and disease progression. This study suggests that intrinsic defects at the level of DC activation are responsible for the susceptible phenotype in L. amazonensis-infected hosts and that this parasite may have evolved unique mechanisms to interfere with innate and adaptive immunity.     

Lactic acid bacteria inducing a weak interleukin-12 and tumor necrosis factor alpha response in human dendritic cells inhibit strongly stimulating lactic acid bacteria but act synergistically with gram-negative bacteria

The development and maintenance of immune homeostasis indispensably depend on signals from the gut flora. Lactic acid bacteria (LAB), which are gram-positive (G+) organisms, are plausible significant players and have received much attention. Gram-negative (G-) commensals, such as members of the family Enterobacteriaceae, may, however, be immunomodulators that are as important as G+ organisms but tend to be overlooked. Dendritic cells (DCs) are crucial immune regulators, and therefore, the present study aimed at investigating differences among human gut flora-derived LAB and G- bacteria in their patterns of DC polarization. Human monocyte-derived DCs were exposed to UV-killed bacteria, and cytokine secretion and surface marker expression were analyzed. Profound differences in the DC polarization patterns were found among the strains. While strains of LAB varied greatly in their capacity to induce interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-alpha), G- strains were consistently weak IL-12 and TNF-alpha inducers. All strains induced significant amounts of IL-10, but G- bacteria were far more potent IL-10 inducers than LAB. Interestingly, we found that when weakly IL-12- and TNF-alpha-inducing LAB and strong IL-12- and TNF-alpha-inducing LAB were mixed, the weakly IL-12- and TNF-alpha-inducing LAB efficiently inhibited otherwise strong IL-12- and TNF-alpha-inducing LAB, yet when weakly IL-12- and TNF-alpha-inducing LAB were mixed with G- bacteria, they synergistically induced IL-12 and TNF-alpha. Furthermore, strong IL-12- and TNF-alpha-inducing LAB efficiently up-regulated surface markers (CD40, CD83, CD86, and HLA-DR), which were inhibited by weakly IL-12- and TNF-alpha-inducing LAB. All G- bacteria potently up-regulated surface markers; however, these markers were not inhibited by weakly IL-12- and TNF-alpha-inducing LAB. These much divergent DC stimulation patterns among intestinal bacteria, which encompass both antagonistic and synergistic relationships, support the growing evidence that the composition of the gut flora affects immune regulation and that compositional imbalances may be involved in disease etiology.