Generation of mucosal dendritic cells from bone marrow reveals a critical role of retinoic acid

It is unknown how dendritic cells (DCs) become specialized as mucosal DCs and maintain intestinal homeostasis. We report that a subset of bone marrow cells freshly isolated from C57BL/6 mice express the retinoic acid (RA)-synthesizing enzyme aldehyde dehydrogenase family 1, subfamily A2 (ALDH1a2) and are capable of providing RA to DC precursors in the bone marrow microenvironment. RA induced bone marrow-derived DCs to express CCR9 and ALDH1a2 and conferred upon them mucosal DC functions, including induction of Foxp3(+) regulatory T cells, IgA-secreting B cells, and gut-homing molecules. This response of DCs to RA was dependent on a narrow time window and stringent dose effect. RA promoted bone marrow-derived DC production of bioactive TGF-β by inhibiting suppressor of cytokine signaling 3 expression and thereby enhancing STAT3 activation. These RA effects were evident in vivo, in that mucosal DCs from vitamin A-deficient mice had reduced mucosal DC function, namely failure to induce Foxp3(+) regulatory T cells. Furthermore, MyD88 signaling enhanced RA-educated DC ALDH1a2 expression and was required for optimal TGF-β production. These data indicate that RA plays a critical role in the generation of mucosal DCs from bone marrow and in their functional activity.     

Outer membrane protein A expression in Escherichia coli K1 is required to prevent the maturation of myeloid dendritic cells and the induction of IL-10 and TGF-beta

Dendritic cells (DCs) are professional APCs that direct both cellular and humoral immune responses. Escherichia coli K1 causes meningitis in neonates; however, the interactions between this pathogen and DCs have not been previously explored. In the present study, we observed that E. coli K1, expressing outer membrane protein A (OmpA), was able to enter, survive, and replicate inside DCs, whereas OmpA(-) E. coli was killed within a short period. Opsonization of OmpA(+) E. coli either with adult or cord serum did not affect its survival inside DCs. Exposure of DCs to live OmpA(+) E. coli K1 prevented DCs from progressing in their maturation process as indicated by failure to up-regulate costimulatory molecules, CD40, HLA-DR, and CD86. The distinct DC phenotype requires direct contact between live bacteria and DCs. The expression of costimulatory molecules was suppressed even after pretreatment of DCs with LPS or peptidoglycan. Furthermore, the suppressive effects of OmpA(+) E. coli on DCs were abrogated when the bacteria were incubated with anti-OmpA Ab. The inhibitory effect on DC maturation was associated with increased production of IL-10 as well as TGF-beta and decreased production of IL-6, TNF-alpha, IL-1beta, and IL-12p70 by DCs, a phenotype associated with tolerogenic DCs. These results suggest that the subversion of DC functions may be a novel strategy deployed by this pathogen to escape immune defense and persist in the infected host to reach a high degree of bacteremia, which is crucial for E. coli to cross the blood-brain barrier.     

Chronic helminth infection promotes immune regulation in vivo through dominance of CD11cloCD103- dendritic cells

Gastrointestinal helminth infections are extremely prevalent in many human populations and are associated with downmodulated immune responsiveness. In the experimental model system of Heligmosomoides polygyrus, a chronic infection establishes in mice, accompanied by a modulated Th2 response and increased regulatory T cell (Treg) activity. To determine if dendritic cell (DC) populations in the lymph nodes draining the intestine are responsible for the regulatory effects of chronic infection, we first identified a population of CD11c(lo) nonplasmacytoid DCs that expand after chronic H. polygyrus infection. The CD11c(lo) DCs are underrepresented in magnetic bead-sorted preparations and spared from deletion in CD11c-diptheria toxin receptor mice. After infection, CD11c(lo) DCs did not express CD8, CD103, PDCA, or Siglec-H and were poorly responsive to TLR stimuli. In DC/T cell cocultures, CD11c(lo) DCs from naive and H. polygyrus-infected mice could process and present protein Ag, but induced lower levels of Ag-specific CD4(+) T cell proliferation and effector cytokine production, and generated higher percentages of Foxp3(+) T cells in the presence of TGF-β. Treg generation was also dependent on retinoic acid receptor signaling. In vivo, depletion of CD11c(hi) DCs further favored the dominance of the CD11c(lo) DC phenotype. After CD11c(hi) DC depletion, effector responses were inhibited dramatically, but the expansion in Treg numbers after H. polygyrus infection was barely compromised, showing a significantly higher regulatory/effector CD4(+) T cell ratio compared with that of CD11c(hi) DC-intact animals. Thus, the proregulatory environment of chronic intestinal helminth infection is associated with the in vivo predominance of a newly defined phenotype of CD11c(lo) tolerogenic DCs.     

Interplay between CD8α+ dendritic cells and monocytes in response to Listeria monocytogenes infection attenuates T cell responses

During the course of a microbial infection, different antigen presenting cells (APCs) are exposed and contribute to the ensuing immune response. CD8α(+) dendritic cells (DCs) are an important coordinator of early immune responses to the intracellular bacteria Listeria monocytogenes (Lm) and are crucial for CD8(+) T cell immunity. In this study, we examine the contribution of different primary APCs to inducing immune responses against Lm. We find that CD8α(+) DCs are the most susceptible to infection while plasmacytoid DCs are not infected. Moreover, CD8α(+) DCs are the only DC subset capable of priming an immune response to Lm in vitro and are also the only APC studied that do so when transferred into β2 microglobulin deficient mice which lack endogenous cross-presentation. Upon infection, CD11b(+) DCs primarily secrete low levels of TNFα while CD8α(+) DCs secrete IL-12 p70. Infected monocytes secrete high levels of TNFα and IL-12p70, cytokines associated with activated inflammatory macrophages. Furthermore, co-culture of infected CD8α(+) DCs and CD11b+ DCs with monocytes enhances production of IL-12 p70 and TNFα. However, the presence of monocytes in DC/T cell co-cultures attenuates T cell priming against Lm-derived antigens in vitro and in vivo. This suppressive activity of spleen-derived monocytes is mediated in part by both TNFα and inducible nitric oxide synthase (iNOS). Thus these monocytes enhance IL-12 production to Lm infection, but concurrently abrogate DC-mediated T cell priming.     

Neutrophil elastase treated dendritic cells promote the generation of CD4(+)FOXP3(+) regulatory T cells in vitro

We have previously shown that neutrophilic elastase converts human immature dendritic cells (DCs) into TGF-β secreting cells and reduces its allostimulatory ability. Since TGF-β has been involved in regulatory T cells (Tregs) induction we analyzed whether elastase or neutrophil-derived culture supernatant treated DCs induce CD4⁺FOXP3⁺ Tregs in a mixed lymphocyte reaction (MLR). We found that elastase or neutrophil-derived culture supernatant treated DCs increased TGF-β and decreased IL-6 production. Together with this pattern of cytokines, we observed a higher number of CD4⁺FOXP3⁺ cells in the MLR cultures induced by elastase or neutrophil-derived culture supernatant treated DCs but not with untreated DCs. The higher number of CD4⁺FOXP3⁺ T cell population was not observed when the enzymatic activity of elastase was inhibited with an elastase specific inhibitor and also when a TGF-β1 blocking antibody was added during the MLR culture. The increased number of CD4⁺ that express FOXP3 was also seen when CD4⁺CD25^- purified T cells were cocultured with the TGF-β producing DCs. Furthermore, these FOXP3⁺ T cells showed suppressive activity in vitro.These results identify a novel mechanism by which the tolerogenic DCs generated by elastase exposure contribute to the immune regulation and may be relevant in the pathogenesis of several lung diseases where the inflammatory infiltrate contains high numbers of neutrophils and high elastase concentrations.     

Immune evasion by Yersinia enterocolitica: differential targeting of dendritic cell subpopulations in vivo

CD4(+) T cells are essential for the control of Yersinia enterocolitica (Ye) infection in mice. Ye can inhibit dendritic cell (DC) antigen uptake and degradation, maturation and subsequently T-cell activation in vitro. Here we investigated the effects of Ye infection on splenic DCs and T-cell proliferation in an experimental mouse infection model. We found that OVA-specific CD4(+) T cells had a reduced potential to proliferate when stimulated with OVA after infection with Ye compared to control mice. Additionally, proliferation of OVA-specific CD4(+) T cells was markedly reduced when cultured with splenic CD8α(+) DCs from Ye infected mice in the presence of OVA. In contrast, T-cell proliferation was not impaired in cultures with CD4(+) or CD4(-)CD8α(-) DCs isolated from Ye infected mice. However, OVA uptake and degradation as well as cytokine production were impaired in CD8α(+) DCs, but not in CD4(+) and CD4(-)CD8α(-) DCs after Ye infection. Pathogenicity factors (Yops) from Ye were most frequently injected into CD8α(+) DCs, resulting in less MHC class II and CD86 expression than on non-injected CD8α(+) DCs. Three days post infection with Ye the number of splenic CD8α(+) and CD4(+) DCs was reduced by 50% and 90%, respectively. The decreased number of DC subsets, which was dependent on TLR4 and TRIF signaling, was the result of a faster proliferation and suppressed de novo DC generation. Together, we show that Ye infection negatively regulates the stimulatory capacity of some but not all splenic DC subpopulations in vivo. This leads to differential antigen uptake and degradation, cytokine production, cell loss, and cell death rates in various DC subpopulations. The data suggest that these effects might be caused directly by injection of Yops into DCs and indirectly by affecting the homeostasis of CD4(+) and CD8α(+) DCs. These events may contribute to reduced T-cell proliferation and immune evasion of Ye.     

Involvement of intestinal epithelial cells in dendritic cell recruitment during C. parvum infection

Dendritic cells (DCs) play a key role in activating and orientating immune responses. Little is currently known about DC recruitment during Cryptosporidium parvum infection. In the intestine, epithelial cells act as sensors, providing the first signals in response to infection by enteric pathogens. We analyzed the contribution of these cells to the recruitment of DCs during cryptosporidiosis. We found that intestinal epithelial cells produced a broad range of DC-attracting chemokines in vitro in response to C. parvum infection. The supernatant of the infected cells induced the migration of both bone marrow-derived DCs (BMDC) and the SRDC lymphoid dendritic cell line. Chemokine neutralization abolished DC migration in these assays. We next analyzed chemokine mRNA expression in the mucosa of C. parvum-infected neonatal mice and recruitment of the various subsets of DCs. Myeloid (CD11c+ CD11b+) and double-negative DCs (CD11c+ CD11b- CD8alpha-) were the main subsets recruited in the ileum during C. parvum infection, via a mechanism involving IFNgamma. DCs were also recruited and activated in the draining lymph nodes during C. parvum infection, as shown by the upregulation of expression of MHC II and of the costimulation molecules CD40 and CD86.     

Interplay between modified vaccinia virus Ankara and dendritic cells: phenotypic and functional maturation of bystander dendritic cells

Modified vaccinia virus Ankara (MVA) is an attenuated poxvirus strain, currently under evaluation as a vaccine vector in various clinical settings. It has been reported that human dendritic cells (DCs) mature after infection with MVA, but reports on the functionality of DCs have so far been controversial. In this work, we studied the phenotype and functionality of MVA-infected DCs. As previously reported, we found that human monocyte-derived DCs upregulated CD86 and HLA-DR in response to MVA infection. Moreover, infected DCs produced a broad array of chemokines and cytokines and were able to activate and induce gamma interferon (IFN-γ) production both in CD4(+) and in CD8(+) allogeneic T cells and in specific autologous peripheral blood lymphocytes (PBLs). Analysis of DC maturation following infection with a recombinant green fluorescent protein (GFP)-expressing MVA revealed that upregulation of CD86 expression was mainly observed in GFP(neg) (bystander) cells. While GFP(pos) (infected) DCs produced tumor necrosis factor alpha (TNF-α), they were unable to produce CXCL10 and were less efficient at inducing IFN-γ production in CEF-specific autologous PBLs. Maturation of bystander DCs could be achieved by incubation with supernatant from infected cultures or with apoptotic infected cells. Type I IFNs were partially responsible for the induction of CXCL10 on bystander DCs. Our findings demonstrate for the first time that, in MVA-infected DC cultures, the leading role with respect to functionality and maturation characteristics is achieved by the bystander DCs.     

Role of TGF-β and IL-6 in dendritic cells,Treg and Th17 mediated immune response during experimental cerebral malaria

The role of cytokines in Plasmodium infection have been extensively investigated, but pro and anti inflammatory cytokines mediated imbalance during malaria immune-pathogenesis is still unrevealed. Malaria is associated with the circulating levels of Interleukin-6 (IL-6) and transforming growth factor β (TGF-β), but association between these two cytokines in immune response remains largely obscured. Using mouse model, we proposed that IL-6 and TGF-β are involved in immune regulation of dendritic cells (DC), regulatory T cells (Treg), T-helper cells (Th17) during P. berghei ANKA (PbA) infection. Association between the cytokines and the severity of malaria was established with anti-TGF-β treatment resulting in increased parasitemia and increased immunopathology, whereas; anti-IL-6 treatment delays immunopathology during PbA infection. Further, splenocytes revealed differential alteration of myeloid DC (mDC), plasmocytoid DC (pDC), Treg, Th17 cells following TGF-β and IL-6 neutralization. Interestingly anti-TGF-β reduces CD11c⁺CD8⁺ DC expression, whereas anti-IL-6 administration causes a profound increase during PbA infection in Swiss mice. We observed down regulation of TGF-β, IL-10, NFAT, Foxp3, STAT-5 SMAD-3 and upregulation of IL-6, IL-23, IL-17 and STAT-3 in splenocytes during PbA infection. The STAT activity probably plays differential role in induction of Th17 and Treg cells. Interestingly we found increase in STAT-3 and decrease in STAT-5 expression during PbA infection. This pattern of STAT indicates that possibly TGF-β and IL-6 play a crucial role in differentiation of DCs subsets and Treg/Th17 imbalance during experimental cerebral malaria (ECM).     

In Vitro HIV Infection Impairs the Capacity of Myeloid Dendritic Cells to Induce Regulatory T Cells

Myeloid dendritic cells (mDCs) are the antigen-presenting cells best capable of promoting peripheral induction of regulatory T cells (Tregs), and are among the first targets of HIV. It is thus important to understand whether HIV alters their capacity to promote Treg conversion. Monocyte-derived DCs (moDCs) from uninfected donors induced a Treg phenotype (CD25(+)FOXP3(+)) in autologous conventional T cells. These converted FOXP3(+) cells suppressed the proliferation of responder T cells similarly to circulating Tregs. In contrast, the capacity of moDCs to induce CD25 or FOXP3 was severely impaired by their in vitro infection with CCR5-utilizing virus. MoDC exposure to inactivated HIV was sufficient to impair FOXP3 induction. This DC defect was not dependent on IL-10, TGF-β or other soluble factors, but was due to preferential killing of Tregs by HIV-exposed/infected moDCs, through a caspase-dependent pathway. Importantly, similar results were obtained with circulating primary myeloid DCs. Upon infection in vitro, these mDCs also killed Treg through mechanisms at least partially caspase-dependent, leading to a significantly lower proportion of induced Tregs. Taken together, our data suggest that Treg induction may be defective when DCs are exposed to high levels of virus, such as during the acute phase of infection or in AIDS patients.     

Recognition of enteroinvasive Escherichia coli and Shigella flexneri by dendritic cells: distinct dendritic cell activation states

The innate and adaptive immune responses of dendritic cells (DCs) to enteroinvasive Escherichia coli (EIEC) infection were compared with DC responses to Shigella flexneri infection. EIEC triggered DCs to produce interleukin (IL)-10, IL-12 and tumour necrosis factor (TNF)-α, whereas S. flexneri induced only the production of TNF-α. Unlike S. flexneri, EIEC strongly increased the expression of toll like receptor (TLR)-4 and TLR-5 in DCs and diminished the expression of co-stimulatory molecules that may cooperate to inhibit CD4(+) T-lymphocyte proliferation. The inflammation elicited by EIEC seems to be related to innate immunity both because of the aforementioned results and because only EIEC were able to stimulate DC transmigration across polarised Caco-2 cell monolayers, a mechanism likely to be associated with the secretion of CC chemokine ligands (CCL)20 and TNF-α. Understanding intestinal DC biology is critical to unravelling the infection strategies of EIEC and may aid in the design of treatments for infectious diseases.     

Cutting edge: mTORC1 in intestinal CD11c+ CD11b+ dendritic cells regulates intestinal homeostasis by promoting IL-10 production

The mammalian target of rapamycin (mTOR) controls cell growth and survival through two distinct complexes called mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Although several reports have suggested the involvement of mTORC1 in development and function of dendritic cells (DCs), its physiological roles remain obscure. We therefore established mTORC1 signal-deficient mice lacking Raptor, an essential component of mTORC1 signal, specifically in DC lineage (referred to here as Raptor(DC-/-)). Raptor(DC-/-) mice exhibited cell expansion in specific subsets of DCs such as splenic CD8(+) DCs and intestinal CD11c(+)CD11b(+) DCs. We also found that impaired mTORC1 signal resulted in the suppression of IL-10 production along with enhanced CD86 expression in intestinal CD11c(+)CD11b(+) DCs and that Raptor(DC-/-) mice were highly susceptible to dextran sodium sulfate-induced colitis. Our results uncover mTORC1-mediated anti-inflammatory programs in intestinal CD11c(+)CD11b(+) DCs to limit the intestinal inflammation.     

Cutting edge: in the absence of TGF-β signaling in T cells, fewer CD103+ regulatory T cells develop, but exuberant IFN-γ production renders mice more susceptible to helminth infection

Multiple factors control susceptibility of C57BL/6 mice to infection with the helminth Heligmosomoides polygyrus, including TGF-β signaling, which inhibits immunity in vivo. However, mice expressing a T cell-specific dominant-negative TGF-β receptor II (TGF-βRII DN) show dampened Th2 immunity and diminished resistance to infection. Interestingly, H. polygyrus-infected TGF-βRII DN mice show greater frequencies of CD4(+)Foxp3(+)Helios(+) Tregs than infected wild-type mice, but levels of CD103 are greatly reduced on both these cells and on the CD4(+)Foxp3(+)Helios(-) population. Although Th9 and Th17 levels are comparable between infected TGF-βRII DN and wild-type mice, the former develop exaggerated CD4(+) and CD8(+) T cell IFN-γ responses. Increased susceptibility conferred by TGF-βRII DN expression was lost in IFN-γ-deficient mice, although they remained unable to completely clear infection. Hence, overexpression of IFN-γ negatively modulates immunity, and the presence of Helios(+) Tregs may maintain susceptibility on the C57BL/6 background.     

TGF-beta-exposed plasmacytoid dendritic cells participate in Th17 commitment

TGF-β is required for both Foxp3(+) regulatory T cell (Treg) and Th17 commitment. Plasmacytoid DCs (pDC) have been shown to participate to both Treg and Th17 commitment as well. However, few studies have evaluated the direct effect of TGF-β on pDC, and to our knowledge, no study has assessed the capacity of TGF-β-exposed pDC to polarize naive CD4(+) T cells. In this paper, we show that TGF-β-treated pDC favor Th17 but not Treg commitment. This process involves a TGF-β/Smad signal, because TGF-β treatment induced Smad2 phosphorylation in pDC and blockade of TGF-β signaling with the SD208 TGF-βRI kinase inhibitor abrogated Th17 commitment induced by TGF-β-treated pDC. Moreover, TGF-β mRNA synthesis and active TGF-β release were induced in TGF-β-treated pDC and anti-TGF-β Ab blocked Th17 commitment. Unexpectedly, TGF-β treatment also induced increased IL-6 production by pDC, which serves as the other arm for Th17 commitment driven by TGF-β-exposed pDC, because elimination of IL-6-mediated signal with either IL-6- or IL-6Rα-specific Abs prevented Th17 commitment. The in vivo pathogenic role of TGF-β-treated pDC was further confirmed in the Th17-dependent collagen-induced arthritis model in which TGF-β-treated pDC injection significantly increased arthritis severity and pathogenic Th17 cell accumulation in the draining lymph nodes. Thus, our data reveal a previously unrecognized effect of TGF-β-rich environment on pDC ability to trigger Th17 commitment. Such findings have implications in the pathogenesis of autoimmune diseases or immune responses against mucosal extracellular pathogens.     

Effect of mesenchymal stem cell-derived exosomes on the induction of mouse tolerogenic dendritic cells

Dendritic cells (DCs) orchestrate innate inflammatory responses and adaptive immunity through T-cell activation via direct cell–cell interactions and/or cytokine production. Tolerogenic DCs (tolDCs) help maintain immunological tolerance through the induction of T-cell unresponsiveness or apoptosis, and generation of regulatory T cells. Mesenchymal stromal cells (MSCs) are adult multipotent cells located within the stroma of bone marrow (BM), but they can be isolated from virtually all organs. Extracellular vesicles and exosomes are released from inflammatory cells and act as messengers enabling communication between cells. To investigate the effects of MSC-derived exosomes on the induction of mouse tolDCs, murine adipose-derived MSCs were isolated from C57BL/6 mice and exosomes isolated by ExoQuick-TC kits. BM-derived DCs (BMDCs) were prepared and cocultured with MSCs-derived exosomes (100 μg/ml) for 72 hr. Mature BMDCs were derived by adding lipopolysaccharide (LPS; 0.1μg/ml) at Day 8 for 24 hr. The study groups were divided into (a) immature DC (iDC, Ctrl), (b) iDC + exosome (Exo), (c) iDC + LPS (LPS), and (d) iDC + exosome + LPS (EXO + LPS). Expression of CD11c, CD83, CD86, CD40, and MHCII on DCs was analyzed at Day 9. DC proliferation was assessed by coculture with carboxyfluorescein succinimidyl ester-labeled BALB/C-derived splenocytes p. Interleukin-6 (IL-6), IL-10, and transforming growth factor-β (TGF-β) release were measured by enzyme-linked immunosorbent assay. MSC-derived exosomes decrease DC surface marker expression in cells treated with LPS, compared with control cells ( ≤ .05). MSC-derived exosomes decrease IL-6 release but augment IL-10 and TGF-β release (p ≤ .05). Lymphocyte proliferation was decreased (p ≤ .05) in the presence of DCs treated with MSC-derived exosomes. CMSC-derived exosomes suppress the maturation of BMDCs, suggesting that they may be important modulators of DC-induced immune responses.     

Control of nitric oxide synthase expression by transforming growth factor-β: Implications for homeostasis

Production of nitric oxide (NO) can be stimulated by inflammatory cytokines and bacterial lipopolysaccharide (LPS) in mammalian cells via an inducible nitric oxide synthase (iNOS). Conversely, the transforming growth factor-βs (TGF-βs) suppress NO production by reducing iNOS expression. Production of NO leads to disparate consequences, some beneficial and some damaging to the host, depending on the cell and context in which iNOS is induced. The TGF-βs counter these NO-mediated processes in macrophages, cardiac myocytes, smooth muscle cells, bone marrow cells, and retinal pigment epithelial cells. Autocrine or paracrine production of TGF-β may thus serve as a physiological counterbalance for iNOS expression, a mechanism which may be subverted by pathogens and tumors for their own survival. A greater understanding of the mechanisms and consequences of NO and TGF-β production may lead to effective therapeutic strategies in various diseases.