Leishmania amazonensis: multiple receptor-ligand interactions are involved in amastigote infection of human dendritic cells

In their mammalian hosts, Leishmania are obligate intracellular parasites that reside in macrophages and dendritic cells (DCs). In the present study, we have investigated in vitro the mechanisms of entry into human DCs of Leishmania amazonensis amastigotes isolated from lesions in nude mice (Am nude). The DC infection rate with Am nude was approximately 36%, while opsonization of Am nude with normal human serum and infected human serum increased the DC infection rates to 60% and 62%, respectively. Heat inactivation and depletion of antibodies in sera brought the DC infection rate down to 40%. The DC infection rate was inhibited after pre-treatment of Am nude with heparin. We were unable to implicate mannose-fucose receptors in the uptake of Am nude by DCs. Our data suggest that the ability of L. amazonensis amastigotes to infect human DCs involves the participation of at least three multiple receptor-ligand interactions, antibodies/FcR, complement components/CR and proteoglycans/heparin-binding protein.     

Functional dichotomy of dendritic cells following interaction with Leishmania braziliensis: infected cells produce high levels of TNF-alpha, whereas bystander dendritic cells are activated to promote T cell responses

Leishmania braziliensis infections are often associated with exaggerated immune responses that can sometimes lead to severe disease associated with high levels of IFN-gamma and TNF-alpha. To explore the role played by dendritic cells (DCs) in these responses, we characterized DCs that were exposed to L. braziliensis. We found that DCs cultured with L. braziliensis parasites up-regulated DC activation markers and produced IL-12 and TNF-alpha. However, not all DCs in the culture became infected, and an analysis of infected and uninfected DCs demonstrated that the up-regulation of activation markers and IL-12 production was primarily confined to the uninfected (bystander) DCs. Further studies with Transwell chambers and parasite fractions indicated that the activation of bystander DCs was mediated by a soluble parasite product, in a type 1 IFN- and MyD88-independent, but TNF-alpha-dependent fashion, and that the activated DCs were more efficient at presenting Ag than control DCs. In contrast, L. braziliensis-infected DCs failed to up-regulate activation markers, but exhibited a dramatic enhancement in their ability to produce TNF-alpha in response to LPS as compared with uninfected DCs. These findings uncover a dual role for DCs in L. braziliensis infection: T cell activation by bystander DCs due to enhanced Ag-presenting capacity following exposure to soluble parasite products, and increased production of TNF-alpha by infected cells that may contribute to the local control of the parasites, but concomitantly induce immunopathology.     

Fusion between Leishmania amazonensis and Leishmania major parasitophorous vacuoles: live imaging of coinfected macrophages

Protozoan parasites of the genus Leishmania alternate between flagellated, elongated extracellular promastigotes found in insect vectors, and round-shaped amastigotes enclosed in phagolysosome-like Parasitophorous Vacuoles (PVs) of infected mammalian host cells. Leishmania amazonensis amastigotes occupy large PVs which may contain many parasites; in contrast, single amastigotes of Leishmania major lodge in small, tight PVs, which undergo fission as parasites divide. To determine if PVs of these Leishmania species can fuse with each other, mouse macrophages in culture were infected with non-fluorescent L. amazonensis amastigotes and, 48 h later, superinfected with fluorescent L. major amastigotes or promastigotes. Fusion was investigated by time-lapse image acquisition of living cells and inferred from the colocalization of parasites of the two species in the same PVs. Survival, multiplication and differentiation of parasites that did or did not share the same vacuoles were also investigated. Fusion of PVs containing L. amazonensis and L. major amastigotes was not found. However, PVs containing L. major promastigotes did fuse with pre-established L. amazonensis PVs. In these chimeric vacuoles, L. major promastigotes remained motile and multiplied, but did not differentiate into amastigotes. In contrast, in doubly infected cells, within their own, unfused PVs metacyclic-enriched L. major promastigotes, but not log phase promastigotes--which were destroyed--differentiated into proliferating amastigotes. The results indicate that PVs, presumably customized by L. major amastigotes or promastigotes, differ in their ability to fuse with L. amazonensis PVs. Additionally, a species-specific PV was required for L. major destruction or differentiation--a requirement for which mechanisms remain unknown. The observations reported in this paper should be useful in further studies of the interactions between PVs to different species of Leishmania parasites, and of the mechanisms involved in the recognition and fusion of PVs.     

Immunomodulatory dendritic cells inhibit Th1 responses and arthritis via different mechanisms

Dendritic cells (DCs) are professional APCs which have the unique ability to present both foreign and self-Ags to T cells and steer the outcome of immune responses. Because of these characteristics, DCs are attractive vehicles for the delivery of therapeutic vaccines. Fully matured DCs are relatively well-defined and even used in clinical trials in cancer. DCs also have the potential to influence the outcome of autoimmunity by modulating the underlying autoimmune response. To gain a better appreciation of the abilities and mechanisms by which immunomodulatory DCs influence the outcome of T cell responses, we studied several immunomodulatory DCs (TNF-, IL-10-, or dexamethasone-stimulated bone marrow-derived DCs) side by side for their ability to modulate T cell responses and autoimmune diseases. Our data show that these differentially modulated DCs display a different composition of molecules involved in T cell activation. Although, all DC subsets analyzed were able to inhibit the induction of collagen-induced arthritis, the modulation of the underlying immune response was different. Vaccination with TNF- or IL-10-modulated DCs altered the Th1/Th2 balance as evidenced by the induction of IL-5- and IL-10-secreting T cells and the concomitant reduction of the IgG2a-IgG1 ratio against the immunizing Ag. In contrast, DCs modulated with dexamethasone did not affect the ratio of IL-5-producing vs IFN-gamma-producing T cells and tended to affect the Ab response in a nonspecific manner. These data indicate that distinct mechanisms can be used by distinct DC subsets to change the outcome of autoimmunity.     

Immature human dendritic cells infected with Leishmania infantum are resistant to NK-mediated cytolysis but are efficiently recognized by NKT cells

Dendritic cells (DC) play an important role in innate and adaptive immunity, interacting with T cells, NK, and NKT cells. A critical step in the interaction of the parasitic protozoa Leishmania with their host is the evasion of both innate and adaptive immunity, producing a long-lasting chronic infection. There is growing evidence that these parasites can modify the Ag-presenting and immunoregulatory functions of DCs. The cells and mechanisms involved in innate immune response against Leishmania are still poorly understood. In this study, we investigated how Leishmania infantum infection affects DC interactions with NK and invariant NKT (iNKTs) cells in humans. We found that infected immature DCs (iDCs) do not up-regulate HLA class I molecules. Despite this, iDCs become resistant to killing mediated by autologous NK cells due to the up-regulation of HLA-E expression, which protects target cells from NK-mediated lysis through interaction with the inhibitory receptor CD94/NKG2A. Furthermore, iDCs infected with L. infantum up-regulate CD1d cell surface expression and consequently can be efficiently recognized and killed by iNKT cells that produce IFN-gamma. These data suggest that L. infantum could be able to evade NK recognition; in contrast, iNKTs may play an important role in the immune response against Leishmania.     

Lutzomyia longipalpis salivary peptide maxadilan alters murine dendritic cell expression of CD80/86, CCR7, and cytokine secretion and reprograms dendritic cell-mediated cytokine release from cultures containing allogeneic T cells

Leishmania protozoan parasites, the etiologic agent of leishmaniasis, are transmitted exclusively by phlebotomine sand flies of the genera Phlebotomus and Lutzomyia. In addition to parasites, the infectious bite inoculum contains arthropod salivary components. One well-characterized salivary component from Lutzomyia longipalpis is maxadilan (MAX), a vasodilator acting via the type I receptor for the pituitary cyclic AMP activating peptide. MAX has been shown to elicit immunomodulatory effects potentially dictating immune responses to Leishmania parasites. When exposed to MAX, both resting and LPS-stimulated dendritic cells (DCs) show reduced CD80 and CD86 expression on most DCs in vitro. However, CD86 expression is increased significantly on a subpopulation of DCs. Furthermore, MAX treatment promoted secretion of type 2 cytokines (IL-6 and IL-10) while reducing production of type 1 cytokines (IL-12p40, TNF-alpha, and IFN-gamma) by LPS-stimulated DCs. A similar trend was observed in cultures of MAX-treated DCs containing naive allogeneic CD4(+) T cells: type 2 cytokines (IL-6 and IL-13) increased while type 1 cytokines (TNF-alpha and IFN-gamma) decreased. Additionally, the proinflammatory cytokine IL-1beta was increased in cultures containing MAX-treated mature DCs. MAX treatment of LPS-stimulated DCs also prevented optimal surface expression of CCR7 in vitro. These MAX-dependent effects were evident in DCs from both Leishmania major-susceptible (BALB/c) and -resistant (C3H/HeN) murine strains. These data suggest that modification of DC phenotype and function by MAX likely affects crucial cellular components that determine the pathological response to infection with Leishmania.     

The amastigote forms of Leishmania are experts at exploiting host cell processes to establish infection and persist

Leishmania are dimorphic protozoan parasites that live as flagellated forms in the gut of their sandfly vector and as aflagellated forms in their mammalian hosts. Although both parasite forms can infect macrophages and dendritic cells, they elicit distinct responses from mammalian cells. Amastigotes are the parasites forms that persist in the infected host; they infect cells recruited to lesions and disseminate the infection to secondary sites. In this review I discuss studies that have investigated the mechanisms that Leishmania amastigotes employ to harness the host cell's response to infection. It should be acknowledged that our understanding of the mechanisms deployed by Leishmania amastigotes to modulate the host cell's response to infection is still rudimentary. Nonetheless, the results show that amastigote interactions with mammalian cells promote the production of anti-inflammatory cytokines such as IL-10 and TGF-beta while suppressing the production of IL-12, superoxide and nitric oxide. An underlying issue that is considered is how these parasites that reside in sequestered vacuolar compartments target host cell processes in the cytosol or the nucleus; does this occur through the release of parasite molecules from parasitophorous vacuoles or by engaging and sustaining signalling pathways throughout the course of infection?     

Neutrophil-dendritic cell interaction plays an important role in live attenuated Leishmania vaccine induced immunity

BackgroundNeutrophils are involved in the initial host responses to pathogens. Neutrophils can activate T cell responses either independently or through indirect involvement of Dendritic cells (DCs). Recently we have demonstrated direct neutrophil-T cell interactions that initiate adaptive immune responses following immunization with live attenuated Leishmania donovani centrin deleted parasite vaccine (LdCen^-/-). However, neutrophil-DC interactions in T cell priming in vaccine immunity in general are not known. In this study we evaluated the interaction between neutrophils and DCs during LdCen^-/- infection and compared with wild type parasite (LdWT) both in vitro and in vivo.Methodology/findingsLdCen^-/- parasite induced increased expression of CCL3 in neutrophils caused higher recruitment of DCs capable of inducing a strong proinflammatory response and elevated co-stimulatory molecule expression compared to LdWT infection. To further illustrate neutrophil-DCs interactions in vivo, we infected LYS-eGFP mice with red fluorescent LdWT/LdCen^-/- parasites and sort selected DCs that engulfed the neutrophil containing parasites or DCs that acquired the parasites directly in the ear draining lymph nodes (dLN) 5d post infection. The DCs predominantly acquired the parasites by phagocytosing infected neutrophils. Specifically, DCs containing LdCen^-/- parasitized neutrophils exhibited a proinflammatory phenotype, increased expression of costimulatory molecules and initiated higher CD4⁺T cell priming ex-vivo. Notably, potent DC activation occurred when LdCen^-/- parasites were acquired indirectly via engulfment of parasitized neutrophils compared to direct engulfment of LdCen^-/- parasites by DCs. Neutrophil depletion in LdCen^-/- infected mice significantly abrogated expression of CCL3 resulting in decreased DC recruitment in ear dLN. This event led to poor CD4⁺Th1 cell priming ex vivo that correlated with attenuated Tbet expression in ear dLN derived CD4⁺ T cells in vivo.ConclusionsCollectively, LdCen^-/- containing neutrophils phagocytized by DC markedly influence the phenotype and antigen presenting capacity of DCs early on and thus play an immune-regulatory role in shaping vaccine induced host protective response.     

Distinctive lack of CD48 expression in subsets of human dendritic cells tunes NK cell activation

CD48 is a glycosyl phosphatidylinositol anchor protein known to be virtually expressed by all human leukocytes. Its ligand, 2B4, is a signaling lymphocyte activation molecule-related receptor involved in NK cell activation. Because dendritic cells (DCs) are strong inducers of NK cell functions, we analyzed the expression of CD48 in different human DC subsets. We observed that monocytes differentiating in DCs promptly down-regulate CD48. Similarly, DCs isolated from inflamed lymph nodes generally do not express CD48. Plasmocytoid DCs do not express CD48 either, whereas myeloid DCs harbored in blood, bone marrow, and thymus express it. In addition, we showed that CD48 expression in DCs affects NK cell functions during NK/DC cross-talk, because NK cells obtained from normal donors and from X-linked lymphoproliferative disease patients are, respectively, triggered or inhibited by DCs expressing surface CD48. Remarkably, IFN-gamma production by lymph node NK cells, in contrast to blood NK cells, can be negatively modulated by 2B4/CD48 interactions, indicating a 2B4 inhibitory pathway in lymph node NK cells. Therefore, the CD48 deficiency of DCs harbored in inflamed lymph nodes that we report in this study might be relevant to successfully activate lymph node NK cells in the early phase of the immune response. Our results show that distinct subsets of human DCs, differently from all other mononuclear hemopoietic cells, specifically do not express CD48. Moreover, the expression of CD48 depends on the anatomic location of DCs and might be related to the tissue-specific 2B4 function (activating or inhibitory) of the NK cells with which they interact.     

Leishmania amazonensis-dendritic cell interactions in vitro and the priming of parasite-specific CD4(+) T cells in vivo

The progressive disease following Leishmania amazonensis infection in mice requires functional CD4(+) T cells, which are primed to a disease-promoting phenotype during the infection. To understand how these pathogenic T cells are generated and the role of dendritic cells (DCs) in this process, we use DCs of susceptible BALB/c and resistant C3H/HeJ mice to examine parasite-DC interactions in vitro as well as the effector phenotype of T cells primed by parasite-exposed DCs in vivo. Our results demonstrate that amastigotes and metacyclics efficiently enter and activate DCs of both genetic backgrounds. Infection with amastigotes fails to induce CD40-dependent IL-12 production, but rather potentiates IL-4 production in BALB/c DCs. Upon transfer into syngeneic recipients, amastigote-exposed BALB/c DCs prime parasite-specific Th cells to produce significantly higher levels of IL-4 and IL-10 than their C3H/HeJ counterparts. Transfer studies with IL-4(-/-) DCs indicate that this enhanced Th2 priming seen in BALB/c mice is partially due to the IL-4 production by amastigote-carrying DCs. These results suggest that L. amazonensis amastigotes may condition DCs of a susceptible host to a state that favors activation of pathogenic CD4(+) T cells, and thereby provide a new perspective on the pathogenesis of cutaneous leishmaniasis and protozoan parasite-host interactions in general.     

The role of IL-10 in promoting disease progression in leishmaniasis

To determine the role of IL-10 in cutaneous leishmaniasis, we examined lesion development following Leishmania major infection of genetically susceptible BALB/c mice lacking IL-10. Whereas normal BALB/c mice developed progressive nonhealing lesions with numerous parasites within them, IL-10(-/-) BALB/c mice controlled disease progression, and had relatively small lesions with 1000-fold fewer parasites within them by the fifth week of infection. We also examined a mechanism whereby Leishmania induced the production of IL-10 from macrophages. We show that surface IgG on Leishmania amastigotes allows them to ligate Fc gamma receptors on inflammatory macrophages to preferentially induce the production of high amounts of IL-10. The IL-10 produced by infected macrophages prevented macrophage activation and diminished their production of IL-12 and TNF-alpha. In vitro survival assays confirmed the importance of IL-10 in preventing parasite killing by activated macrophages. Pretreatment of monolayers with either rIL-10 or supernatants from amastigote-infected macrophages resulted in a dramatic enhancement in parasite intracellular survival. These studies indicate that amastigotes of Leishmania use an unusual and unexpected virulence factor, host IgG. This IgG allows amastigotes to exploit the antiinflammatory effects of Fc gamma R ligation to induce the production of IL-10, which renders macrophages refractory to the activating effects of IFN-gamma.     

Immunomodulatory Effects of Four Leishmania infantum Potentially Excreted/Secreted Proteins on Human Dendritic Cells Differentiation and Maturation

Leishmania parasites and some molecules they secrete are known to modulate innate immune responses through effects on dendritic cells (DCs) and macrophages. Here, we characterized four Leishmania infantum potentially excreted/secreted recombinant proteins (LipESP) identified in our laboratory: Elongation Factor 1 alpha (LiEF-1α), a proteasome regulatory ATPase (LiAAA-ATPase) and two novel proteins with unknown functions, which we termed LiP15 and LiP23, by investigating their effect on in vitro differentiation and maturation of human DCs and on cytokine production by DCs and monocytes. During DCs differentiation, LipESP led to a significant decrease in CD1a. LiP23 and LiEF-1α, induced a decrease of HLA-DR and an increase of CD86 surface expression, respectively. During maturation, an up-regulation of HLA-DR and CD80 was found in response to LiP15, LiP23 and LiAAA-ATPase, while an increase of CD40 expression was only observed in response to LiP15. All LipESP induced an over-expression of CD86 with significant differences between proteins. These proteins also induced significant IL-12p70 levels in immature DCs but not in monocytes. The LipESP-induced IL-12p70 production was significantly enhanced by a co-treatment with IFN-γ in both cell populations. TNF-α and IL-10 were induced in DCs and monocytes with higher levels observed for LiP15 and LiAAA-ATPase. However, LPS-induced cytokine production during DC maturation or in monocyte cultures was significantly down regulated by LipESP co-treatment. Our findings suggest that LipESP strongly interfere with DCs differentiation suggesting a possible involvement in mechanisms established by the parasite for its survival. These proteins also induce DCs maturation by up-regulating several costimulatory molecules and by inducing the production of proinflammatory cytokines, which is a prerequisite for T cell activation. However, the reduced ability of LipESP-stimulated DCs and monocytes to respond to lipopolysaccharide (LPS) that can be observed during human leishmaniasis, suggests that under certain circumstances LipESP may play a role in disease progression.     

The role of dendritic cells in tumor microenvironments and their uses as therapeutic targets

Dendritic cells (DC), which consist of several different subsets, specialize in antigen presentation and are critical for mediating the innate and adaptive immune responses. DC subsets can be classified into conventional, plasmacytoid, and monocyte-derived DC in the tumor microenvironment, and each subset plays a different role. Because of the role of intratumoral DCs in initiating antitumor immune responses with tumor-derived antigen presentation to T cells, DCs have been targeted in the treatment of cancer. By regulating the functionality of DCs, several DC-based immunotherapies have been developed, including administration of tumor-derived antigens and DC vaccines. In addition, DCs participate in the mechanisms of classical cancer therapies, such as radiation therapy and chemotherapy. Thus, regulating DCs is also important in improving current cancer therapies. Here, we will discuss the role of each DC subset in antitumor immune responses, and the current status of DC-related cancer therapies.     

Dendritic cells in the recognition of intestinal microbiota

Mucosal dendritic cells (DCs) constantly survey the luminal microenvironment which contains commensal microbiota and potentially harmful organisms regulating pathogen recognition and adaptive as well as innate defense activation. Distinct mechanisms are beginning to emerge by which intestinal antigen sampling and handling is achieved ensuring specificity and contributing to redundancy in pathogen detection. Distinct DC subsets are associated with these mechanisms and regulate specific innate or adaptive immune responses to help distinguish between commensal microbiota, pathogens and self antigens. Understanding DC biology in the mucosal immune system may contribute to the unraveling of infection routes of intestinal pathogens and may aid in developing novel vaccines and therapeutic strategies for the treatment of infectious and inflammatory diseases.     

Consequences of the natural propensity of Leishmania and HIV-1 to target dendritic cells

Recent studies have shown that both Leishmania and HIV type-1 (HIV-1) hijack dendritic cell (DC) functions to escape immune surveillance using an array of elaborate strategies. Leishmania has developed a variety of adaptations to disrupt cellular defense mechanisms, whereas HIV-1 targets DCs to achieve a more efficient dissemination. The capacity of Leishmania and HIV-1 to target DCs through a common cell-surface molecule, namely DC-SIGN (dendritic cell specific ICAM-3-grabbing non-integrin), points to a possible dangerous liaison between these two pathogens. This review explores our knowledge of how Leishmania and HIV-1 interact dynamically with DCs, and how they exploit this cell type for their reciprocal benefit.     

Inhibition of myeloid dendritic cell accessory cell function and induction of T cell anergy by alcohol correlates with decreased IL-12 production

Alcohol consumption inhibits accessory cell function and Ag-specific T cell responses. Myeloid dendritic cells (DCs) coordinate innate immune responses and T cell activation. In this report, we found that in vivo moderate alcohol intake (0.8 g/kg of body weight) in normal volunteers inhibited DC allostimulatory capacity. Furthermore, in vitro alcohol treatment during DC differentiation significantly reduced allostimulatory activity in a MLR using naive CD4(+) T cells, and inhibited tetanus toxoid Ag presentation by DCs. Alcohol-treated DCs showed reduced IL-12, increased IL-10 production, and a decrease in expression of the costimulatory molecules CD80 and CD86. Addition of exogenous IL-12 and IL-2, but not neutralization of IL-10, during MLR ameliorated the reduced allostimulatory capacity of alcohol-treated DCs. Naive CD4(+) T cells primed with alcohol-treated DCs showed decreased IFN-gamma production that was restored by exogenous IL-12, indicating inhibition of Th1 responses. Furthermore, CD4(+) T cells primed with alcohol-treated DCs were hyporesponsive to subsequent stimulation with the same donor-derived normal DCs, suggesting the ability of alcohol-treated DCs to induce T cell anergy. LPS-induced maturation of alcohol-treated immature DCs partially restored the reduced allostimulatory activity, whereas alcohol given only during DC maturation failed to inhibit DC functions, suggesting that alcohol primarily impairs DC differentiation rather than maturation. NFkappaB activation, a marker of DC maturation was not affected by alcohol. Taken together, alcohol both in vitro and in vivo can impair generation of Th1 immune responses via inhibition of DC differentiation and accessory cell function through mechanisms that involve decreased IL-12 induction.