Dendritic Cells Generated From Mops condylurus, a Likely Filovirus Reservoir Host, Are Susceptible to and Activated by Zaire Ebolavirus Infection.
ABSTRACT: Ebola virus infection of human dendritic cells (DCs) induces atypical adaptive immune responses and thereby exacerbates Ebola virus disease (EVD). Human DCs, infected with Ebola virus aberrantly express low levels of the DC activation markers CD80, CD86, and MHC class II. The T cell responses ensuing are commonly anergic rather than protective against EVD. We hypothesize that DCs derived from potential reservoir hosts such as bats, which do not develop disease signs in response to Ebola virus infection, would exhibit features associated with activation. In this study, we have examined Zaire ebolavirus (EBOV) infection of DCs derived from the Angolan free-tailed bat species, Mops condylurus. This species was previously identified as permissive to EBOV infection in vivo, in the absence of disease signs. M. condylurus has also been recently implicated as the reservoir host for Bombali ebolavirus, a virus species that is closely related to EBOV. Due to the absence of pre-existing M. condylurus species-specific reagents, we characterized its de novo assembled transcriptome and defined its phylogenetic similarity to other mammals, which enabled the identification of cross-reactive reagents for M. condylurus bone marrow-derived DC (bat-BMDC) differentiation and immune cell phenotyping. Our results reveal that bat-BMDCs are susceptible to EBOV infection as determined by detection of EBOV specific viral RNA (vRNA). vRNA increased significantly 72 h after EBOV-infection and was detected in both cells and in culture supernatants. Bat-BMDC infection was further confirmed by the observation of GFP expression in DC cultures infected with a recombinant GFP-EBOV. Bat-BMDCs upregulated CD80 and chemokine ligand 3 (CCL3) transcripts in response to EBOV infection, which positively correlated with the expression levels of EBOV vRNA. In contrast to the aberrant responses to EBOV infection that are typical for human-DC, our findings from bat-BMDCs provide evidence for an immunological basis of asymptomatic EBOV infection outcomes.
Project description:The plasticity of dendritic cells (DCs) permits phenotypic modulation ex vivo by gene expression or pharmacologic agents, and these modified DCs can exert therapeutic immunosuppressive effects in vivo through direct interactions with T cells, either inducing T regulatory cells (T(REG)s) or causing anergy. Sphingosine 1-phosphate (S1P) is a sphingolipid and the natural ligand for five G protein-coupled receptors (S1P1, S1P2, S1P3, S1P4, and S1P5), and S1PR agonists reduce kidney ischemia-reperfusion injury (IRI) in mice. S1pr3(-/-)mice are protected from kidney IRI, because DCs do not mature. We tested the therapeutic advantage of S1pr3(-/-) bone marrow-derived dendritic cell (BMDC) transfers in kidney IRI. IRI produced a rise in plasma creatinine (PCr) levels in mice receiving no cells (NCs) and mice pretreated with wild-type (WT) BMDCs. However, S1pr3(-/-) BMDC-pretreated mice were protected from kidney IRI. S1pr3(-/-) BMDC-pretreated mice had significantly higher numbers of splenic T(REG)s compared with NC and WT BMDC-pretreated mice. S1pr3(-/-) BMDCs did not attenuate IRI in splenectomized, Rag-1(-/-), or CD11c(+) DC-depleted mice. Additionally, S1pr3(-/-) BMDC-dependent protection required CD169(+)marginal zone macrophages and the macrophage-derived chemokine CCL22 to increase splenic CD4(+)Foxp3(+) T(REG)s. Pretreatment with S1pr3(-/-) BMDCs also induced T(REG)-dependent protection against IRI in an allogeneic mouse model. In summary, adoptively transferred S1pr3(-/-) BMDCs prevent kidney IRI through interactions within the spleen and expansion of splenic CD4(+)Foxp3(+) T(REG)s. We conclude that genetically induced deficiency of S1pr3 in allogenic BMDCs could serve as a therapeutic approach to prevent IRI-induced AKI.
Project description:Mucosal DCs play a critical role in tissue homeostasis. Several stimuli can induce a mucosal phenotype; however, molecular pathways that regulate development of mucosal DC function are relatively unknown. This study sought to determine whether PPAR? contributes to the development of the "mucosal" phenotype in mouse DCs. Experiments demonstrated that PPAR? activation in BMDCs induced an immunosuppressive phenotype in which BMDCs had reduced expression of MHC class II and costimulatory molecules, increased IL-10 secretion, and reduced the ability to induce CD4 T cell proliferation. Activation of PPAR? enhanced the ability of BMDC to polarize CD4 T cells toward iTregs and to induce T cell expression of the mucosal homing receptor, CCR9. Activation of PPAR? increased the ability of BMDCs to induce T cell-independent IgA production in B cells. BMDCs from PPAR?(?DC) mice displayed enhanced expression of costimulatory molecules, enhanced proinflammatory cytokine production, and decreased IL-10 synthesis. Contrary to the inflammatory BMDC phenotype in vitro, PPAR?(?DC) mice showed no change in the frequency or phenotype of mDC in the colon. In contrast, mDCs in the lungs were increased significantly in PPAR?(?DC) mice. A modest increase in colitis severity was observed in DSS-treated PPAR?(?DC) mice compared with control. These results indicate that PPAR? activation induces a mucosal phenotype in mDCs and that loss of PPAR? promotes an inflammatory phenotype. However, the intestinal microenvironment in vivo can maintain the mucosal DC phenotype of via PPAR?-independent mechanisms.
Project description:Ebola viruses (EBOV) can cause severe hemorrhagic disease with high case fatality rates. Currently, no vaccines or therapeutics are approved for use in humans. Ebola virus-like particles (eVLP) comprising of virus protein (VP40), glycoprotein, and nucleoprotein protect rodents and nonhuman primates from lethal EBOV infection, representing as a candidate vaccine for EBOV infection. Previous reports have shown that eVLP stimulate the expression of proinflammatory cytokines in dendritic cells (DCs) and macrophages (M?s) in vitro. However, the molecular mechanisms and signaling pathways through which eVLP induce innate immune responses remain obscure. In this study, we show that eVLP stimulate not only the expression of proinflammatory cytokines but also the expression of type I interferons (IFNs) and IFN-stimulated genes (ISGs) in murine bone marrow-derived DCs (BMDCs) and M?s. Our data indicate that eVLP trigger host responses through toll-like receptor (TLR) pathway utilizing 2 distinct adaptors, MyD88 and TRIF. More interestingly, eVLP activated the IFN signaling pathway by inducing a set of potent antiviral ISGs. Last, eVLP and synthetic adjuvants, Poly I:C and CpG DNA, cooperatively increased the expression of cytokines and ISGs. Further supporting this synergy, eVLP when administered together with Poly I:C conferred mice enhanced protection against EBOV infection. These results indicate that eVLP stimulate early innate immune responses through TLR and type I IFN signaling pathways to protect the host from EBOV infection.
Project description:Helicobacter pylori (H. pylori) infects the gastric mucosa and persists for the life of the host. Bacterial persistence may be due to the induction of regulatory T cells (Tregs) whichmay have protective effects against other diseases such as asthma. It has been shown that H. pylori modulates the T cell response through dendritic cell reprogramming but the molecular pathways involved are relatively unknown. The goal of this study was to identify critical elements of dendritic cell (DC) activation and evaluate potential influence on immune activation. Microarray analysis was used to demonstrate limited gene expression changes in H. pylori stimulated bone marrow derived DCs (BMDCs) compared to the BMDCs stimulated with E. coli. IRAK-M, a negative regulator of TLR signaling, was upregulated and we selectedit for investigation of its role in modulating the DC and T cell responses. IRAK-M(-/-) and wild type BMDC were compared for their response to H. pylori. Cells lacking IRAK-M produced significantly greater amounts of proinflammatory MIP-2 and reduced amounts of immunomodulatory IL-10 than wild type BMDC. IRAK-M(-/-) cells also demonstrated increased MHC II expression upon activation. However, IRAK-M(-/-) BMDCs were comparable to wild type BMDCs in inducing T-helper 17 (TH17) and Treg responses as demonstrated in vitro using BMDC CD4+ T cells co-culture assays,and in vivo though the adoptive transfer of CD4(+) FoxP3-GFP T cells into H. pylori infected IRAK-M(-/-) mice. These results suggest that H. pylori infection leads to the upregulation of anti-inflammatory molecules like IRAK-M and that IRAK-M has a direct impact on innate functions in DCs such as cytokine and costimulation molecule upregulation but may not affect T cell skewing.
Project description:Ebola virus (EBOV) causes severe systemic disease in humans and non-human primates characterized by high levels of viremia and virus titers in peripheral organs. The natural portals of virus entry are the mucosal surfaces and the skin where macrophages and dendritic cells (DCs) are primary EBOV targets. Due to the migratory properties of DCs, EBOV infection of these cells has been proposed as a necessary step for virus dissemination via draining lymph nodes and blood. Here we utilize chimeric mice with competent hematopoietic-driven immunity, to show that EBOV primarily infects CD11b+ DCs in non-lymphoid and lymphoid tissues, but spares the main cross-presenting CD103+ DC subset. Furthermore, depletion of CD8 and CD4 T cells resulted in loss of early control of virus replication, viremia and fatal Ebola virus disease (EVD). Thus, our findings point out at T cell function as a key determinant of EVD progress and outcome.
Project description:In vitro generation of dendritic cells (DCs) is advantageous for overcoming the low frequency of primary DCs and the difficulty of applying isolation techniques for studying DC immunobiology. The culture of bone marrow cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) has been used extensively to generate bone marrow-derived dendritic cells (BMDCs). Studies have reported the heterogeneity of cells grown in murine GM-CSF culture based on the levels of MHCII expression. Although porcine DCs are generated by this classical method, the exact characteristics of the BMDC population have not yet been defined. In this study, we discriminated GM-CSF-grown BMDCs from gnotobiotic miniature pigs according to several criteria including morphology, phenotype, gene expression pattern and function. We showed that porcine BMDCs were heterogeneous cells that differentially expressed MHCII. MHCIIhigh cells displayed more representative of DC-like morphology and phenotype, including costimulatory molecules, as well as they showed a superior T cell priming capacity as compared to MHCIIlow cell. Our data showed that the difference in MHCIIhigh and MHCIIlow cell populations involved distinct maturation states rather than the presence of different cell types. Overall, characterization of porcine BMDC cultures provides important information about this widely used cellular model.
Project description:Defining and loading of immunogenic and safe cancer antigens remain a major challenge for designing dendritic cell (DC)-based cancer vaccines. In this study, we defined a prototype strategy of using DC-based vaccines pulsed with placenta-derived heat shock protein gp96 to induces anti-tumor T cell responses. Placental gp96 was efficiently taken up by CD11c+ bone marrow-derived DCs (BMDCs) and resulted in moderate BMDC maturation. Splenocytes and cytotoxic T cells (CTLs) generated with mouse BMDCs pulsed with placental gp96 specifically lysed B16 melanoma and LLC lung carcinoma cells. In both transplantable melanoma and lung carcinoma mice models, immunization with placental gp96-stimulated BMDCs led to a significant decrease in tumor growth and mouse mortality with respect to mice treated with liver gp96-pulsed BMDCs or placental gp96 alone. This vaccine induced strong cross-reactive tumor-specific T cell responses. Our results revealed that DCs pulsed with placenta-derived gp96 represent an effective immunotherapy to induce tumor-reactive immune responses, possibly via loading DCs with its associated carcinoembryonic antigens.
Project description:How the aryl hydrocarbon receptor (AhR) regulates dendritic-cell (DC) differentiation is unknown. We show that activation of AhR by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) caused enhanced differentiation from immature DCs (IDCs) to mature DCs (MDCs) in the bone-marrow-derived DCs (BMDC) from B6 wild-type mice but not in the BMDCs from AhR-null mice as indicated by the expression of CD11c and class II major histocompatibility complex (MHC). Enhanced maturation of BMDCs was associated with elevated levels of CD86 and an increased AhR-dependent nuclear accumulation of nuclear factor-kappa-light-chain enhancer of activated B cell (NF-?B) member RelB in BMDCs. The expression of interleukin (IL) 10 and chemokine DC-CK1 was suppressed, whereas that of CXCL2, CXCL3 and IL-22 was significantly increased in AhR-activated BMDCs. Furthermore, TCDD induced expression of the regulatory enzymes indoleamine 2,3-dioxygenase (IDO1) and indoleamine 2,3-dioxygenase-like 1 (IDO2). Increased expression of IDO2 was associated with coexpression of the cell-surface marker CCR6. Interestingly, mRNA expression of the chemokine receptor CCR6 was drastically decreased in AhR-null IDCs and MDCs. Overall, these data demonstrate that AhR modifies the maturation of BMDCs associated with the induction of the regulatory enzyme IDO and altered expression of cytokine, chemokines and DC-specific surface markers and receptors.
Project description:The significance of the integral membrane protein Niemann-Pick C1 (NPC1) in the ebolavirus entry process has been determined using various cell lines derived from humans, non-human primates and fruit bats. Fruit bats have long been purported as the potential reservoir host for ebolaviruses, however several studies provide evidence that Mops condylurus, an insectivorous microbat, is also an ebolavirus reservoir. NPC1 receptor expression in the context of ebolavirus replication in microbat cells remains unstudied. In order to study Ebola virus (EBOV) cellular entry and replication in M. condylurus, we derived primary and immortalized cell cultures from 12 different organs. The NPC1 receptor expression was characterized by confocal microscopy and flow cytometry comparing the expression levels of M. condylurus primary and immortalized cells, HeLa cells, human embryonic kidney cells and cells from a European microbat species. EBOV replication kinetics was studied for four representative cell cultures using qRT-PCR. The aim was to elucidate the suitability of primary and immortalized cells from different tissues for studying NPC1 receptor expression levels and their potential influence on EBOV replication. The NPC1 receptor expression level in M. condylurus primary cells differed depending on the organ they were derived from and was for most cell types significantly lower than in human cell lines. Immortalized cells showed for most cell types higher expression levels than their corresponding primary cells. Concluding from our infection experiments with EBOV we suggest a potential correlation between NPC1 receptor expression level and virus replication rate in vitro.
Project description:A dendritic cell (DC) vaccine strategy has been developed as a new cancer immunotherapy, but the goal of complete tumor eradication has not yet been achieved. We have previously shown that baculoviruses potently infect DCs and induce antitumor immunity against hepatomas in a mouse model. Baculovirus-infected, bone marrow-derived DCs (BMDCs) display increased surface expression of costimulatory molecules, such as CD80, CD86 and major histocompatibility complex (MHC) classes I and II, and secrete interferons and other proinflammatory cytokines. In this study, we evaluated the induction of antitumor immunity in mice by baculovirus-infected BMDCs against lung cancer and melanoma. After treatment with baculovirus-infected BMDCs, murine lung tumors caused by Lewis lung carcinoma (LLC) cells were significantly reduced in size, and the survival of the mice was improved. In addition, experiments using a melanoma mouse model showed that baculovirus-infected BMDCs inhibited tumor growth and improved survival compared with controls. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and creatinine levels remained normal in baculovirus-infected BMDC-treated mice. Our findings show that baculovirus-infected DCs induce antitumor immunity and pave the way for the use of this technique as an effective tool for DC immunotherapy against malignancies.