Plasmodium falciparum-free merozoites and infected RBCs distinctly affect soluble CD40 ligand-mediated maturation of immature monocyte-derived dendritic cells

Free plasmodium merozoites released from the parasitized hepatocytes and erythrocytes represent a transitory, extracellular stage in its mammalian host. In this study, we compared the effect of Plasmodium falciparum-free merozoites with infected RBCs (iRBCs) on the maturation of human monocyte-derived dendritic cells (DCs) in vitro. Phagocytosed-free merozoites prevented soluble CD40 ligand (sCD40L)-induced, phenotypic maturation of DCs and secretion of IL-12p70 but enhanced IL-10 production and primed, naive CD4+ cells to produce a high level of IL-10 compared with IFN-gamma. Free merozoites augmented sCD40L-induced ERK1/2 activation, and inhibition of ERK1/2 with its inhibitor PD98059 markedly abrogated IL-10 production and rescued IL-12 production. Therefore, the molecular mechanisms by which free merozoites antagonized sCD40L-induced DC maturation appeared to involve the activation of the ERK pathway. In contrast, phagocytosed iRBCs by itself induced DCs to semi-maturation, responded to CD40 signaling by maturing and secreting increased levels of TNF-alpha, IL-6, and also IL-12p70, and led to a pronounced, proinflammatory response by the allogenic CD4+ T cells. iRBCs regulate CD40-induced p38MAPK. Studies using inhibitors selective for p38MAPK (SB203580) showed that p38MAPK played an essential role in the maturation and function of DCs. Our results reveal the ability of free merozoites and iRBCs to distinctly alter the sCD40L-induced DC functioning by regulating the activation of the MAPK pathway that can inactivate or exacerbate immune responses to promote their survival and the development of parasite-specific pathologies.     

Whole-body imaging of sequestration of Plasmodium falciparum in the rat

The occlusion of vessels by packed Plasmodium falciparum-infected (iRBC) and uninfected erythrocytes is a characteristic postmortem finding in the microvasculature of patients with severe malaria. Here we have employed immunocompetent Sprague-Dawley rats to establish sequestration in vivo. Human iRBC cultivated in vitro and purified in a single step over a magnet were labeled with 99mtechnetium, injected into the tail vein of the rat, and monitored dynamically for adhesion in the microvasculature using whole-body imaging or imaging of the lungs subsequent to surgical removal. iRBC of different lines and clones sequester avidly in vivo while uninfected erythrocytes did not. Histological examination revealed that a multiadhesive parasite adhered in the larger microvasculature, inducing extensive intravascular changes while CD36- and chondroitin sulfate A-specific parasites predominantly sequester in capillaries, inducing no or minor pathology. Removal of the adhesive ligand Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), preincubation of the iRBC with sera to PfEMP1 or preincubation with soluble PfEMP1-receptors prior to injection significantly reduced the sequestration. The specificity of iRBC binding to the heterologous murine receptors was confirmed in vitro, using primary rat lung endothelial cells and rat lung cryosections. In offering flow dynamics, nonmanipulated endothelial cells, and an intact immune system, we believe this syngeneic animal model to be an important complement to existing in vitro systems for the screening of vaccines and adjunct therapies aiming at the prevention and treatment of severe malaria.     

Dendritic Cell and NK Cell Reciprocal Cross Talk Promotes Gamma Interferon-Dependent Immunity to Blood-Stage Plasmodium chabaudi AS Infection in Mice

Dendritic cells (DCs) are important accessory cells for promoting NK cell gamma interferon (IFN-γ) production in vitro in response to Plasmodium falciparum-infected red blood cells (iRBC). We investigated the requirements for reciprocal activation of DCs and NK cells leading to Th1-type innate and adaptive immunity to P. chabaudi AS infection. During the first week of infection, the uptake of iRBC by splenic CD11c⁺ DCs in resistant wild-type (WT) C57BL/6 mice was similar to that in interleukin 15^−/− (IL-15^−/−) and IL-12p40^−/− mice, which differ in the severity of P. chabaudi AS infection. DCs from infected IL-15^−/− mice expressed costimulatory molecules, produced IL-12, and promoted IFN-γ secretion by WT NK cells in vitro as efficiently as WT DCs. In contrast, DCs from infected IL-12p40^−/− mice exhibited alterations in maturation and cytokine production and were unable to induce NK cell IFN-γ production. Coculture of DCs and NK cells demonstrated that DC-mediated NK cell activation required IL-12 and, to a lesser extent, IL-2, as well as cell-cell contact. In turn, NK cells from infected WT mice enhanced DC maturation, IL-12 production, and priming of CD4⁺ T-cell proliferation and IFN-γ secretion. Infected WT mice depleted of NK cells, which exhibit increased parasitemia, had impaired DC maturation and DC-induced CD4⁺ Th1 cell priming. These findings indicate that DC-NK cell reciprocal cross talk is critical for control and rapid resolution of P. chabaudi AS infection and provide in vivo evidence for the importance of this interaction in IFN-γ-dependent immunity to malaria.     

Plasmacytoid and myeloid dendritic cells with a partial activation phenotype accumulate in lymphoid tissue during asymptomatic chronic HIV-1 infection

Dendritic cells (DCs) from HIV-1-infected individuals display numeric and functional defects, and recent evidence suggests that HIV-1 can directly and indirectly activate DCs in vitro. The in vivo activation state and compartmentalization of DC subsets during HIV-1 infection remain poorly understood, however. We evaluated phenotypic and functional characteristics of myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) directly ex vivo in peripheral blood and lymphoid tissue from HIV-1-infected and HIV-seronegative individuals. Analysis of a wide range of chemokine receptors and activation/maturation markers on circulating DCs from viremic HIV-1-infected donors revealed a phenotype indicative of partial activation. Yet, blood DCs from viremic subjects still achieved full maturation when stimulated in vitro. In addition, blood pDCs from viremic individuals had a reduced capacity to migrate to CXCL12 in vitro. Total numbers of both DC subsets were increased in lymph nodes of asymptomatic untreated HIV-1-infected subjects, consistent with DC accumulation in the lymphoid compartment. Lymph node DCs also expressed high levels of CD40 in the absence of increases of other typical activation/maturation markers. Activation and depletion of DCs in blood with accumulation in lymphoid tissue may contribute to HIV-associated chronic immune activation and T-cell dysfunction.     

Plasmodium falciparum-infected erythrocytes and beta-hematin induce partial maturation of human dendritic cells and increase their migratory ability in response to lymphoid chemokines

Acute and chronic Plasmodium falciparum infections alter the immune competence of the host possibly through changes in dendritic cell (DC) functionality. DCs are the most potent activators of T cells, and migration is integral to their function. Mature DCs express lymphoid chemokine receptors (CCRs), expression of which enables them to migrate to the lymph nodes, where they encounter naïve T cells. The present study aimed to investigate the impact of the synthetic analog to malaria parasite pigment hemozoin, i.e., β-hematin, or infected erythrocytes (iRBCs) on the activation status of human monocyte-derived DCs and on their expression of CCRs. Human monocyte-derived DCs partially matured upon incubation with β-hematin as indicated by an increased expression of CD80 and CD83. Both β-hematin and iRBCs provoked the release of proinflammatory and anti-inflammatory cytokines, such as interleukin-6 (IL-6), IL-10, and tumor necrosis factor alpha, but not IL-12, and induced upregulation of the lymphoid chemokine receptor CXCR4, which was coupled to an increased migration to lymphoid ligands. Taken together, these results suggest that the partial and transient maturation of human myeloid DCs upon stimulation with malaria parasite-derived products and the increased IL-10 but lack of IL-12 secretion may lead to suboptimal activation of T cells. This may in turn lead to impaired adaptive immune responses and therefore insufficient clearance of the parasites.     

Ebola and Marburg virus-like particles activate human myeloid dendritic cells

The filoviruses, Ebola (EBOV) and Marburg (MARV), are potential global health threats, which cause deadly hemorrhagic fevers. Although both EBOV and MARV logarithmically replicate in dendritic cells (DCs), these viruses do not elicit DC cytokine secretion and fail to activate and mature infected DCs. Here, we employed virus-like particles (VLPs) of EBOV and MARV to investigate whether these genome-free particles maintain similar immune evasive properties as authentic filoviruses. Confocal microscopy indicated that human myeloid-derived DCs readily took up VLPs. However, unlike EBOV and MARV, VLPs induced maturation of DCs including upregulation of costimulatory molecules (CD40, CD80, CD86), major histocompatibility complex (MHC) class I and II surface antigens, and the late DC maturation marker CD83. The chemokine receptors CCR5 and CCR7 were also modulated on VLP-stimulated DCs, indicating that DC could migrate following VLP exposure. Furthermore, VLPs also elicited DC secretion of the pro-inflammatory cytokines TNF-alpha, IL-8, IL-6, and MIP-1alpha. Most significantly, in stark contrast to DC treated with intact EBOV or MARV, DC stimulated with EBOV or MARV VLPs showed enhanced ability to support human T-cell proliferation in an allogenic mixed lymphocyte response (MLR). Thus, our findings suggest that unlike EBOV and MARV, VLPs are effective stimulators of DCs and have potential in enhancing innate and adaptive immune responses.     

Association of CD40 ligand expression on HTLV-I-infected T cells and maturation of dendritic cells

Human T lymphotropic virus type I (HTLV-I) induces HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T-cell leukemia (ATL). The development of HAM/TSP is associated with rapid maturation of dendritic cells (DCs), while ATL is accomplished with their maturation defect. The DC maturation is induced by cell-to-cell contact with CD4+ T cells expressing CD40 ligand (L). We determined the influence of CD40L expressed on various HTLV-I-infected T cells on the DC maturation. Around 60% of CD4+ T cells infected with HTLV-I for 1 week, expressed CD40L molecules involved in DC maturation. DCs matured by the CD40L+ T cells activated autologous CD4+ and CD8+ T cells. HTLV-I-immortalized T-cell lines established from healthy donors consistently expressed CD40L molecules for 3 months, however, some lines lost the expression soon thereafter. Interleukin (IL)-2-independent and transformed lines lacked that expression. Furthermore, T cells obtained from HAM/TSP patients expressed CD40L molecules for at least 3 weeks, whereas T cells from ATL patients did not express that. The CD40L T cells did not induce DC maturation, and required exogenous CD40L molecules for maturation. The CD40L+ T-cell-induced maturation was blocked by anti-CD40L antibody. Therefore, the lack of CD40L expression on HTLV-I-infected T cells may be associated with the development of ATL.     

CD8alpha(-) and CD8alpha(+) subclasses of dendritic cells undergo phenotypic and functional maturation in vitro and in vivo

Dendritic cells (DCs) are essential for the priming of immune responses. This antigen-presenting function of DCs develops in sequence in a process called maturation, during which they become potent sensitizers of na?ve T cells but reduce their ability to capture and process antigens. Some heterogeneity exists in mouse-DC populations, and two distinct subsets of DCs expressing high levels of CD11c can be identified on the basis of CD8alpha expression. We have studied the phenotype and maturation state of mouse splenic CD8alpha(-) and CD8alpha(+) DCs. Both subsets were found to reside in the spleen as immature cells and to undergo a phenotypic maturation upon culture in vitro in GM-CSF-containing medium or in vivo in response to lipopolysaccharide. In vitro and in vivo analyses showed that this maturation process is an absolute requisite for DCs to acquire their T-cell priming capacity, transforming CD8alpha(-) and CD8alpha(+) DCs into potent and equally efficient activators of na?ve CD4(+) and CD8(+) T cells. Furthermore, these results highlight the importance that environmental factors may have on the ability of DC subsets to influence Th responses qualitatively; i.e., the ability to drive Th1 versus Th2 differentiation may not be fixed immutably for each DC subset.     

Recombinant adenovirus-transduced human dendritic cells engineered to secrete interleukin-10 (IL-10) suppress Th1-type responses while selectively activating IL-10-producing CD4+ T cells

Recombinant adenoviruses (rAd) are efficient tools for genetic modification of human dendritic cells (DC) in vitro. Infection of DCs by rAd encoding beta-galactosidase (betagal) results in partial maturation of DCs, as witnessed by the upregulation of major histocompatibility complex and costimulatory molecules. Accordingly, these DCs are more potent stimulators of Th1-type proliferative responses. We now demonstrate that infection of immature DCs with rAd encoding human interleukin (IL)-10 results in the secretion by the DCs of large amounts of IL-10, while not affecting expression of activation markers indicative of partial DC maturation. In contrast to rAd-betagal-infected DCs, rAdIL-10-infected DCs are very poor stimulators of monoclonal and polyclonal Th1-type responses. Instead, stimulation of nonpolarized CD4+ T-cell cultures with rAdIL-10-infected DCs selectively activates and expands an IL-10-producing CD4+ T-cell subset capable of suppressing Th1 responses in vitro. Our data argue that rAd-infected human DCs genetically engineered to produce IL-10 may be exploited for the modulation of harmful Th1-type responses in transplantation and autoimmune diseases.     

Langerhans cells acquire a CD8+ dendritic cell phenotype on maturation by CD40 ligation

Dendritic cell (DC) reconstitution experiments and phenotypic analysis of DC subpopulations have allowed the definition in the mouse of two main DC categories: CD8+ lymphoid DCs and CD8- myeloid DCs. With regard to Langerhans cells (LCs), which represent immature DCs differentiating into mature DCs on migration to the lymph nodes after an antigenic stimulation, although classically considered as myeloid DCs, there is no experimental evidence of their origin. It has been recently shown that mouse LCs, negative for CD8 and LFA-1, undergo CD8/LFA-1 up-regulation on migration, suggesting that LCs belong to the CD8+ lymphoid DC lineage. To further reinforce this hypothesis, we have analyzed the modulation of CD8 expression by LCs on culture with molecules known to induce LC maturation. Our results show that LC acquired a CD8+ lymphoid phenotype on CD40 ligation.     

Phosphoantigen Burst upon Plasmodium falciparum Schizont Rupture Can Distantly Activate Vγ9Vδ2 T Cells

Malaria induces potent activation and expansion of the Vγ9Vδ2 subpopulation of γδT cells, which inhibit the Plasmodium falciparum blood cycle through soluble cytotoxic mediators, abrogating merozoite invasion capacity. Intraerythrocytic stages efficiently trigger Vγ9Vδ2 T-cell activation and degranulation through poorly understood mechanisms. P. falciparum blood-stage extracts are known to contain phosphoantigens able to stimulate Vγ9Vδ2 T cells, but how these are presented by intact infected red blood cells (iRBCs) remains elusive. Here we show that, unlike activation by phosphoantigen-expressing cells, Vγ9Vδ2 T-cell activation by intact iRBCs is independent of butyrophilin expression by the iRBC, and contact with an intact iRBC is not required. Moreover, blood-stage culture supernatants proved to be as potent activators of Vγ9Vδ2 T cells as iRBCs. Bioactivity in the microenvironment is attributable to phosphoantigens, as it is dependent on the parasite DOXP pathway, on Vγ9Vδ2 TCR signaling, and on butyrophilin expression by Vγ9Vδ2 T cells. Kinetic studies showed that the phosphoantigens were released at the end of the intraerythrocytic cycle at the time of parasite egress. We document exquisite sensitivity of Vγ9Vδ2 T cells, which respond to a few thousand parasites. These data unravel a novel framework, whereby release of phosphoantigens into the extracellular milieu by sequestered parasites likely promotes activation of distant Vγ9Vδ2 T cells that in turn exert remote antiparasitic functions.     

Neonatal and maternal immunological responses to conserved epitopes within the DBL-gamma3 chondroitin sulfate A-binding domain of Plasmodium falciparum erythrocyte membrane protein 1

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) mediates the adherence of P. falciparum-infected erythrocytes to placental syncytiotrophoblasts via interactions with chondroitin sulfate A (CSA), a characteristic of pregnancy-associated malaria. Pregnancy-associated malaria predicts increased susceptibility of newborns to malaria, and it is postulated that transplacental passage of parasite antigen induces immune regulatory activity in the neonate. We wished to examine the immune responsiveness to a CSA-binding domain of PfEMP1, the DBL-gamma3 domain, in cord and maternal venous blood obtained from pregnancies with various histories of P. falciparum infection. We assessed in vitro T-cell cytokine and plasma immunoglobulin G (IgG) and IgM responses to four peptides corresponding to highly conserved regions of a DBL-gamma3 domain common to central African parasite isolates. The presence of placental P. falciparum infection at delivery was associated with elevated frequencies of DBL-gamma3 peptide-specific CD3+ interleukin-10-positive T cells in cord blood, while treatment and clearance of infection prior to delivery was associated with elevated frequencies of CD3+ gamma interferon-positive T cells. DBL-gamma3 peptide-specific IgM antibodies were detected in 12 of 60 (20%) cord plasma samples from those born to mothers with P. falciparum infection during pregnancy. Consistent with polyclonal anti-PfEMP1 antibody responses that are associated with protection against pregnancy-associated malaria, the presence of maternal IgG antibodies with specificity for one of the DBL-gamma3 peptides showed a parity-dependent profile. These data demonstrate that peptides corresponding to conserved regions of the DBL-gamma3 domain of PfEMP1 are immunogenic in P. falciparum-infected mothers and their offspring.     

Human primary gastric dendritic cells induce a Th1 response to H. pylori

Adaptive CD4 T-cell responses are important in the pathogenesis of chronic Helicobacter pylori gastritis. However, the gastric antigen-presenting cells that induce these responses have not yet been identified. Here we show that dendritic cells (DCs) are present in the gastric mucosa of healthy subjects and are more prevalent and more activated in the gastric mucosa of H. pylori-infected subjects. H. pylori induced gastric DCs isolated from noninfected subjects to express increased levels of CD11c, CD86 and CD83, and to secrete proinflammatory cytokines, particularly interleukin (IL)-6 and IL-8. Importantly, gastric DCs pulsed with live H. pylori, but not control DCs, mediated T-cell secretion of interferon-γ. The ability of H. pylori to induce gastric DC maturation and stimulate gastric DC activation of Th1 cells implicates gastric DCs as initiators of the immune response to H. pylori.     

Potential involvement of dendritic cells in delayed-type hypersensitivity reactions to beta-lactams

BACKGROUND: Although the involvement of T cells in delayed reactions to drugs has been studied, little is known about the interaction between the drug and the antigen-presenting cells. Dendritic cells (DCs) are professional antigen-presenting cells essential for initiating T-cell responses. Their ability is regulated in a process known as maturation, by which they modulate the effector immune response. OBJECTIVES: We studied the role of DCs in subjects who had a delayed-type hypersensitivity reaction to amoxicillin to assess the effect on the pattern of maturation and determine the capacity of DCs to activate T lymphocytes. METHODS: We examined the consequences of the interaction between monocyte-derived DCs, lymphocytes, and amoxicillin by means of phenotypic and functional studies, including endocytosis, proliferation, and cytokine production. RESULTS: Amoxicillin drove DCs from hypersensitive subjects to a phenotypic and functional semimature status, inducing a T-cell proliferation response. CONCLUSIONS: In delayed reactions to amoxicillin, DCs play a relevant role in inducing the T-cell responses. These results are useful not only to understand the mechanism but potentially as a possible approach to diagnosis. CLINICAL IMPLICATIONS: A better understanding of T-cell and DC involvement in delayed-type hypersensitivity reactions is needed. This in vitro assay might provide clues to the diagnostic evaluation of patients allergic to penicillins.     

Contribution of dendritic cells to measles virus induced immunosuppression

Measles virus (MV) remains an important pathogen in children worldwide. The morbidity and mortality of MV is associated with severe immune suppression. Dendritic cells (DCs) were identified as initial target cells in vivo, and DCs were efficiently infected by MV in vitro. MV infection of DCs likely contributes to functional deficiency in these cells; therefore playing a role in MV‐induced immunosuppression. DCs appeared to mature phenotypically; however, the ability of infected cells to stimulate T cells was compromised. Phenotypic maturation of infected immature DCs was partially controlled by IFN production; however, infected DCs also maintained markers of an immature phenotype such as the continued uptake of antigen and lack of expression of chemokine receptor CCR7. Furthermore, mature DCs did not appear to maintain phenotypic maturation following infection demonstrated by decreased MHC and co‐stimulatory molecule expression. Several mechanisms of MV‐induced DC dysfunction have been suggested, each likely contributing to the immunosuppressive effect of MV‐infected DCs. Infected DCs responded aberrantly to secondary maturation stimuli such as CD40L or TLR4 stimulation. MV infection resulted in apoptosis in DC/T‐cell cocultures, which may contribute to a reduced T‐cell response. Additionally, the immunological synapse between infected DCs and T cells was compromised resulting in reduced T‐cell interaction times and activation signaling. The mechanisms of MV contribution to DC dysfunction appear multifaceted and central to MV‐induced immunosuppression. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.     

Characterization of monocyte-derived dendritic cells in recent-onset diabetes mellitus type 1

Dendritic cells (DC) may play an important role in the immunopathogenesis of type 1 diabetes mellitus (DM-1). In this study, we have analyzed phenotypical changes during cytokine-driven maturation from CD14+ monocytes to mature DC and DC-dependent T-cell stimulation in recent-onset pediatric DM-1 patients and healthy controls. DC maturation was monitored by flow cytometric analyses for the expression of surface markers (HLA-DR, CD1a, CD40, CD80, CD86, CD83, CD14, CD32, mannose-receptor, and CD11c). Flow cytometric analysis of isolated peripheral blood monocytes did not reveal apparent differences between patients and controls. During DC maturation no obvious differences in the expression patterns of surface markers over time or evidence for maturation impairments in DM-1 patients could be appreciated. Solely, a marginal, but significant, transient down-regulation of CD1a on Day 3 (mean MDFI 3.82 vs 7.25; P = 0.021), which was accompanied by an increase of IL-6, could be observed. The comparison of mature DCs (Day 10) between patients and controls indicated no significant differences, except for CD83 (mean MDFI 1.7 vs 1.5; P = 0.042) and CD80 (mean MDFI 15.92 vs 12.73; P = 0.042). Moreover, no difference in T-cell stimulatory capacity was seen. In conclusion, our analysis of a cohort of recent-onset DM-1 patients and controls does not support a role for disease-related alterations in cytokine-driven maturation of monocyte-derived DC.