Parasites of the genus Leishmania
generate severe clinical syndromes, ranging from self-healing cutaneous to often fatal visceral leishmaniasis, which afflict millions of people worldwide (2
). All Leishmania
species express a surface glycocalyx, composed of related glycolipids, including lipophosphoglycan and glycoinositol phospholipids, and this glycocalyx is involved in many aspects of parasite pathogenicity (15
). Components of this surface coat are likely to be involved in the activation of innate immunity and may thus also have an important influence on the induction of the adaptive immune response against Leishmania
The CD1 family consists of nonpolymorphic antigen-presenting molecules that are expressed on antigen-presenting cells and induced on peripheral blood monocytes (PBMs) by exposure to granulocyte-macrophage colony-stimulating factor (GM-CSF) and other cytokines (21
). Group I CD1 molecules, which include human CD1a, CD1b, and CD1c, have been shown to present mycobacterial lipids and glycolipids to specific T cells. These can potentially contribute to antimicrobial resistance by production of gamma interferon (IFN-γ) (24
) and the antimicrobial protein granulysin (27
). The group I CD1 proteins are thus likely to be involved in the protective immune response against complex intracellular pathogens such as Leishmania
species, which are know to harbor abundant glycolipid antigens.
Intracellular pathogens, however, have evolved various survival strategies to avoid immune recognition of infected host cells. These include down-regulation of antigen-presenting molecules by Mycobacterium
), capture and degradation of antigen-presenting molecules by Leishmania mexicana
and Leishmania amazonensis
), and interference with intracellular antigen-loading by Leishmania major
). Here we demonstrate that Leishmania donovani
promastigotes block lipid antigen presentation in infected human DCs and down-regulate group I CD1 expression. Both processes may potentially be associated with the establishment of a disease-promoting immune response and chronic infection.
We tested in vitro the ability of Leishmania
-infected dendritic cells (DCs) to present antigen to a well-characterized CD1b-restricted T-cell line (DN1) (17
), which is specific for the mycobacterial lipid mycolic acid (4
). Human blood monocytes were isolated from random donor leukocyte concentrates and differentiated to immature DCs by a 3-day incubation with GM-CSF (300 U/ml; Immunex Corporation, Seattle, Wash.) and interleukin 4 (IL-4; 200 U/ml; gift from Schering Corporation, Kenilworth, N.J.). Irradiated (5,000 rads) immature DCs were infected with stationary-phase L. donovani
promastigotes (MHOM/SD/1S-2D, referred to as LD1S) at a multiplicity of 10 parasites per host cell, and infected cells as well as uninfected controls were incubated at a 1:1 ratio with DN1 T cells in the presence of 5 μg of Mycobacterium tuberculosis
sonicate per ml. Uninfected DCs efficiently presented mycobacterial mycolic acids, as judged by incorporation of radiolabeled thymidine into proliferating activated T cells (Fig. , top) and their robust secretion of IFN-γ (middle). In contrast, infected DCs were unable to stimulate T-cell proliferation, and a substantial decrease in T-cell IFN-γ production was observed. T cells were not activated by incubation with infected DCs alone or Leishmania
sonicate (not shown), a result expected from the known specificity of the DN1 T-cell line to mycobacterial mycolic acid (4
FIG. 1. Leishmania-infected DCs inhibit CD1b-restricted T-cell activation. (A) T-cell proliferation and activation assay. L. donovani LD1S-infected DCs were incubated 2 days postinfection with CD1b-restricted T cells (DN1 cells) in the presence or absence of (more ...)
Leishmania infection had also a profound effect on DC cytokine production induced by exposure to the M. tuberculosis sonicate. Antigen-treated, uninfected DCs produced increased levels of IL-12 as a result of their maturation following CD1b-T-cell-receptor ligation. In contrast, IL-12 production was completely abolished in the presence of intracellular Leishmania (Fig. , bottom). Together, these data indicated that Leishmania disrupts the primary immune recognition of lipid antigens in infected human DCs and subverts the protective immune response by inhibition of T-cell activation and host cell IL-12 production.
Since the ability of DCs to phagocytose Leishmania
promastigotes in culture has been questioned (31
), we confirmed that infection of DCs under the conditions used in our experiments led to intracellular parasitism. As determined by nuclear staining, 85 to 90% of DCs were generally infected in our experiments (Fig. , top). Only a minor fraction of the parasites were associated with the host cell surface, as shown by immunofluorescence staining of nonpermeabilized DCs with antiphosphoglycan antibody CA7AE (29
). More than 90% of the parasites were labeled only following host cell permeabilization, indicating predominant intracellular parasite localization.
We next tested if inhibition of the CD1-mediated responses was associated with transcriptional down-regulation of CD1 expression. Human DCs obtained from seven healthy random donors were infected with stationary-phase promastigotes as described above, and total RNA was isolated with Trizol reagent (Gibco BRL, Carlsbad, Calif.) 2 days after the infection. Abundance of CD1a, -b, and -c mRNA was determined by quantitative reverse transcription (RT)-PCR. Three distinct patterns of regulation were observed (Fig. ): two donors (donors 1 and 2) showed no or only moderate down-regulation of the three CD1 isoforms, two donors (donors 3 and 4) showed substantial inhibition of CD1 expression ranging from two- to fivefold, and three donors (donors 5, 6, and 7) showed strong down-regulation above 20-fold compared to the uninfected control. With the exception of donor 5, the level of inhibition was comparable between the three isoforms, suggesting a process of coordinate down-regulation of the genes. However, the overall heterogeneity of this phenomenon between the various donors suggests the presence of unknown host factors that determine susceptibility to CD1 down-regulation.
FIG. 2. Leishmania-infected DCs down-regulate CD1 expression. (A) RT-PCR analysis. Infected DCs and uninfected controls from seven donors were cultured for 48 h, and total RNA was extracted. TaqMan one-step RT-PCR (Applied Biosystems) was carried out with the (more ...)
Consistent with reduced abundance of CD1 mRNA, we observed a reduction in CD1 surface expression in Leishmania
-infected DCs. Surface expression of CD1a (using monoclonal antibody OKT6 [20
]), CD1b (using monoclonal antibody BCD1b3 [5
]), and CD1c (using monoclonal antibody F10/21A3 [22
]) was determined by flow cytometry in DCs 2 days after infection with stationary-phase promastigotes and compared to that in uninfected controls. As seen at the transcriptional level, the response of the various donors was heterogenous (Fig. , left), including no down-regulation (donor 8), moderate (2-fold) down-regulation (donors 9 and 10), or strong (5- to 10-fold) down-regulation (donors 11 to 13). Inhibition of CD1 expression was not observed following incubation of DCs with 1 nm latex beads (Sigma, St. Louis, Mo.) or heat-inactivated Leishmania
promastigotes (56°C, 30 min) (Fig. , right). Thus, CD1 down-regulation appeared to be an active process driven by live Leishmania
and not a consequence of phagocytosis of inert particles or nonviable parasite-derived material.
heavily colonizes the bone marrow of the infected host, where it may interfere with the differentiation of immune effector cells. We therefore tested the effect of intracellular Leishmania
on induction of CD1 expression during differentiation of PBMs into immature DCs. PBMs of three donors were infected with stationary-phase promastigotes and incubated for 3 days with GM-CSF-IL-4. Surface marker expression of CD1a, -b, and -c, as well as major histocompatibility complex (MHC) class I (using monoclonal antibody PA2.6 [(8
]) and class II (using monoclonal antibody L243 [13
]), was quantified thereafter by flow cytometry and compared to results for cytokine-treated uninfected controls. As expected, uninfected PBMs differentiated into immature DCs and induced expression of all three group I CD1 isoforms (Fig. , left). In contrast, PBMs showed markedly reduced induction of CD1a, -b, and -c expression if previously infected with Leishmania
(Fig. , right). No effect of either cytokine-treatment or Leishmania
infection on MHC class I and class II expression was observed.
FIG. 3. Leishmania inhibits cytokine-induced CD1 expression in PBMs. (Left) Infected (open histograms) and uninfected (closed histograms) PBMs were incubated with GM-CSF and IL-4 and analyzed for group I CD1, MHC class I, and MHC class II expression by flow cytometry. (more ...)
Expression of CD1 is a hallmark of DCs (7
), and our results on the inhibition of CD1 expression by Leishmania
-infected DCs suggest a pathway for immune evasion by these parasites. Indeed, visceral Leishmania
infection has been shown to impair DC functions and has been associated with depletion of follicular DCs from germinal centers in infected spleens (3
). DCs have a high turnover rate with an average life span of approximately 2 days (12
), and thus, a constant supply of de novo differentiated cells is required to maintain immune functions. Leishmania
bone marrow infection may potentially disrupt this supply or modify the functional properties of DCs, including their antigen presentation and T-cell-activation capacities. Our study supports this hypothesis and suggests that L. donovani
actively modulates the expression pattern of infected immature DCs and leukocyte precursors toward an immune-suppressive phenotype, processes that may have important implications for innate and acquired antileishmanial immunity and may be relevant for the pathophysiology of chronic leishmaniasis.