Infection of Human Mononuclear Phagocytes and Macrophage-Like THP1 Cells with Leishmania donovani Results in Modulation of Expression of a Subset of Chemokines and a Chemokine Receptor
This work has not been presented anywhere in written or any other form. Guidelines set forth by the ethical committee of both the institutions involved in this work were followed in the conduct of research involving human samples.
Abstract
The expression of chemokines and chemokine receptors was studied in Leishmania donovani (LD)-infected human mononuclear phagocytes and the human monocytic cell line THP1. Our studies showed that LD infection caused the upregulation of three β chemokines (macrophage inflammatory protein-1 alpha (MIP-1α), MIP-1β and RANTES (regulated on activation normal T cell expressed and secreted)), one α chemokine (interleukin-8 (IL-8)) and the CC chemokine receptor 5 (CCR5) but not CCR1, as evident from reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The CCR5 upregulation in human mononuclear phagocytes and THP1 cells was also evident by confocal microscopy. The possible association of such upregulation in relation to Leishmania and human immunodeficiency virus (HIV) coinfection was discussed.
Introduction
Leishmania are protozoan parasites that infect human macrophages and cause a spectrum of clinical diseases including self-healing skin lesions, diffuse cutaneous or mucosal disease as well as potentially fatal visceral disease [1]. Clinical symptoms result from replication of the parasites in macrophages of the dermis, nasopharyngeal mucosa and the mononuclear phagocytic system (reviewed in [1–3]). Active disease is associated with an ineffective parasite-specific cell-mediated immune response [4, 5]. The coinfection of Leishmania and human immunodeficiency virus (HIV) is emerging as a new and frightful disease and is becoming increasingly frequent. Cases have been reported in as many as 25 countries and are currently considered an ominous threat in Spain, Italy and Portugal [6, 7].
Under normal physiological condition, the process of leucocyte recruitment is controlled by chemokines, which are chemotactic cytokines belonging to the superfamily of polypeptide mediators [8, 9]. The chemokines are broadly classified as CXC or alpha chemokines, CC or beta chemokines and C or gamma chemokines based on the location of cystine residues at the N-terminal end [9]. It was originally thought that the CXC chemokines primarily attract neutrophils drive acute inflammation while the CC chemokines mainly attract leucocytes [10–12]. Besides being promising targets for treating inflammatory, allergic, infectious and autoimmune diseases, there is a major thrust to develop anti-CCR5 (CC chemokine receptor 5)-based therapies for HIV-1 [13], because this chemokine receptor serves as a coreceptor for HIV-1 infection [13–16]. The role of some of the important alpha and beta chemokines in leishmaniasis is not very clear. There is a report that stationary phase Leishmania major promastigotes induced rapid and transient expression of transcripts of chemokines, JE (human macrophage/monocyte chemotactic & activating factor (MCAF)/macrophage chemoattractant protein-1 (MCP-1)) and KC (human growth related protein (GRO)) but not macrophage inflammatory protein-1 alpha (MIP-1α), C10 and RANTES (regulated on activation normal T cell expressed and secreted) in bone marrow-derived BALB/c macrophages [17]. There are supporting as well as conflicting reports on the role of MIP-1α in leishmaniasis. While there are reports of overexpression of MIP-1α as the host-protective chemokine [18–20], there are also reports of MIP-1α playing a deleterious role in the outcome of Leishmania donovani (LD) infection [21]. So we became interested to know the role of LD infection in the modulation of expression of CC chemokines like MIP-1α and MIP-1β, RANTES and interleukin-8 (IL-8) in human mononuclear phagocytes and human monocytic cell line (THP1) model. THP1 cells have many characteristics of human monocytes, including morphology, surface-membrane receptor, oncogene expression, production of a number of cytokines and the capacity to undergo maturational changes when induced with phorbol esters [22]. With the growing number of Leishmania and HIV-1 coinfection cases [23], we also became interested to see whether LD causes any modulation of expression of these β chemokines and their receptors CCR5/CCR1. Here we report that LD causes the upregulation of expression of all three beta chemokines – MIP-1α, MIP-1β, RANTES – one alpha chemokine IL-8 and very importantly upregulates the CCR5 expression in both human mononuclear phagocytes and THP1 cells.
Materials and methods
Culture of parasites, THP1 cells and human mononuclear phagocytes LD strain AG83 (MHOM/IN/1983/AG83) originally obtained from an Indian Kala-azar patient was maintained in golden hamster, as described previously [24]. Promastigotes were cultured at 24 °C in M199 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 20 mm HEPES, pH 7.4, 4 mm NaHCO3, 100 U/ml of penicillin and 100 µg/ml of streptomycin (all from Gibco BRL, Grand Island, NY, USA).
THP1 cells (CD14+, CD15+, derived from a patient with acute monocytic leukaemia) [25] were obtained from the National Center for Cell Science (Pune, Maharashtra, India). Cells were cultured in RPMI-1640 medium containing 4.5 g of glucose per litre, 10 mm HEPES, 1 mm sodium pyruvate and 10% (v/v) fetal bovine serum. The cells were maintained in tissue culture flasks (Nunc A/S Roskilde, Denmark) in 5% CO2 incubator at 37 °C.
Human mononuclear phagocytes were isolated and cultured, as described previously [26–29] with slight modifications. Briefly, peripheral blood was obtained by venipuncture from adult healthy donors using heparin as anticoagulant. Peripheral blood mononuclear cells (PBMCs) were separated on a Ficoll–Hypaque density gradient (Sigma Chemical Company, St. Louis, MO, USA), washed twice, counted and resuspended in RPMI without serum [29]. Mononuclear phagocytes were purified from PBMCs by adherence to glass Petri plates or glass coverslips at 37 °C with 5% CO2 for 2 h. Nonadherent cells were removed by washing three times with warm (37 °C) RPMI. Mononuclear phagocytes were covered with complete RPMI containing 10% autologous serum and incubated for 3 days to allow the cells to reach a resting state before infection.
Infection of THP1 cells THP1 cells have been successfully utilized as an in vitro model for Leishmania infantum and Leishmania aethiopica infection [30, 31]. Prior to infection, the cells were induced to become adherent, matured macrophage-like phenotype by the addition of 20 nm phorbol 12-myristate 7-acetate (PMA) to the culture for 12 h [32]. There are reports in the literature that mature, and not immature macrophages, are the host to intracellular parasites [33, 34]. PMA-treated adherent THP1 (P-THP1) cells were washed three times and cultured in fresh medium before infection. Cell viability was determined to be >97% by the Trypan blue dye exclusion method. About 1 × 106 cells were infected for 6, 12 and 24 h at parasite/cell multiplicities of 10 : 1 [30, 31, 35]. Free parasites were removed by washing the cells two times with culture medium without fetal bovine serum. During infection, the cells were cultured in tissue culture Petri dishes (Tarsons, Calcutta, West Bengal, India).
Infection of human mononuclear phagocytes Infection of mononuclear phagocytes with Leishmania promastigotes was performed, as previously described [26, 27] with minor modifications. Briefly, the Petri plates or coverslips containing adhered phagocytes were washed with serum-free RPMI three times and covered with RPMI containing 10% autologous serum. Parasites were added to the cells (10 : 1) and incubated for 2 h at 37 °C with 5% CO2. Based on our prior experiments to determine the kinetics of infection of phagocytes, 2 h incubation was found to allow optimum infection of cells. Following infection, monolayers of phagocytes were washed with serum-free RPMI three times to remove extracellular parasites.
Oligonucleotide primers Oligonucleotide primers specific for humans were used to PCR amplify cDNA. β-Actin primers were used as a control to evaluate the expression of a housekeeping gene. The primers for all the chemokines and chemokine receptors were custom made from Gibco BRL and were kindly gifted by Professor P. Mohanakumar, University of Washington, St. Louis, MO, USA. The primers for β-actin were custom made from Gibco BRL. The sequences of the primers are given in Table 1.
| Primers | Sequence |
|---|---|
| β-Actin | (F) 5′ATGGATGATGATATCGCCGCC-3′ |
| (R) 5′CTAGAAGCATTTGCGGTGGAC-3′ | |
| MIP-1α | (F) 5′CGCCTGCTGCTTCAGCTACCTCCCGGCA-3′ |
| (R) 5′TGGACCCCTCAGGCACTAAGCTCCAGGTCG-3′ | |
| MIP-1β | (F) 5′ACCCTCCCACCGCCTGCTGCTTTTCTTCAC-3′ |
| (R) 5′GTTGCAGGTCATACACGTACTCCTGGACCC-3′ | |
| RANTES | (F) 5′GGCACGCCTCGCTGTCATCCTCA-3′ |
| (R) 5′CTTGATGTGGGCACGGGGCAGTG-3′ | |
| IL-8 | (F) 5′ATGACTTCCAAGCTGGCCGTGGCT-3′ |
| (R) 5′TCTCAGCCCTCTTCAAAAACTTCTC-3′ | |
| CCR5 | (F) 5′TCACTTGGGTGGTGGCTGTG-3′ |
| (R) 5′CCTGCATAGCTTGGTCCAAC-3′ | |
| CCR1 | (F) 5′ACAAAGAGCTCTGCTGCCTC-3′ |
| (R) 5′CCCACTTCTTATTGGGGTCA-3′ |
- MIP, macrophage inflammatory protein; RANTES, regulated on activation normal T cell expressed and secreted; IL-8, interleukin-8; CCR, CC chemokine receptor.
RNA isolation and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) Total cellular RNA was extracted from infected or control cells directly in culture plates by adding TRIZOL reagent (Life Technologies, Invitrogen Corporation, Carlsbald, CA, USA), according to manufacturer's instruction. To eliminate contaminating genomic DNA, total RNA was subjected to DnaseI (amplification grade, Gibco BRL, Life Technology, USA) treatment, as specified by the manufacturer. RNA concentration and purity was determined by measuring at A260 and A280; samples were immediately stored at −70 °C. RT-PCR was performed, as mentioned before [36] with slight modifications. Briefly, reverse transcription was performed with SuperScript II RNase H– (Gibco BRL). Thirty cycles of PCR amplification was carried out with PCR SuperMix High Fidelity (Gibco BRL) using the forward and reverse primers, on a Perkin Elmer Model 9700 DNA thermal cycler with initial denaturation at 94 °C for 2 min followed by denaturation at 94 °C for 30 s, primer annealing at 55 °C for 30 s and extension at 72 °C for 1 min in a final volume of 50 µl. Identical aliquots were processed in parallel without addition of reverse transcriptase (RT), in order to ensure that any possible residual genomic DNA was not serving as a template in the PCR amplification.
To assess the integrity of the starting RNA as well as to control the variability in the RNA or cDNA handling during the RT-PCR method and as a standard for semiquantitative comparisons, the levels of β-actin transcripts were determined simultaneously for every PCR sample by coamplification with primers specific for β-actin [34]. PCR amplification products were electrophoresed on 1.5% agarose gels and stained with ethidium bromide.
Confocal microscopy Human mononuclear phagocytes and P-THP1 cells (1 × 106), both infected and uninfected, were washed three times with wash buffer (phosphate-buffered saline (PBS), pH 7.4, containing 2% FCS) and incubated with PBS containing 1% bovine serum albumin at 4 °C for 30 min. Cells were washed twice and suspended in wash buffer. About 25 µl of cells (1 × 105) were transferred to a 5 ml tube for staining. About 10 µl of fluorescein-conjugated anti-CCR5 immunoglobulin G2b (IgG2b) antibody (R&D Systems, Minneapolis, MN, USA) was added to the cells and incubated in dark for 45 min, according to manufacturer's instruction. Following incubation, cells were washed twice in 4 ml of wash buffer to remove unreacted anti-CCR5 reagent and finally resuspended in 300 µl of wash buffer for analysis in laser scanning confocal microscopy (Zeiss LSM 510 model). Similarly, cells were stained with unrelated fluorescein-conjugated antibody of the same isotype to find out the specificity of binding of anti-CCR5 antibody.
Densitometric and statistical analysis The band intensity of the amplified chemokine, chemokine receptor and β-actin products was analysed using a model GS 700 Densitometer and molecular analyst™, version 1.5 software (Biorad, Hercules, CA, USA). The results are expressed as a ratio of expression of chemokine/chemokine receptor to β-actin expression to control the equivalent loading in agarose gel. Results are expressed as mean ± standard deviation (SD) for individual sets of experiments. Each experiment was performed thrice, and the representative data from one of these experiments is presented in the manuscript. The variation among experiments was less than 3%.
Results
Parasite burden
We did a time kinetics of THP1 cell infection by LD and considered three time points – early (6 h), middle (12 h) and late (24 h). The per cent infected cells at 6, 12 and 24 h were 68, 96 and 93, respectively (Fig. 1A), and the values of intracellular parasites per 100 THP1 cells at the corresponding time points were 266 ± 5, 679 ± 11 and 626 ± 11, respectively (Fig. 1B). At 12 and 24 h time points, the expression of β-actin was checked and found to be unaltered, indicating that the cells remained intact during the course of our investigation.
Time kinetics of phorbol 12-myristate 7-acetate (PMA)-treated adherent THP1 (P-THP1) cell infection by Leishmania donovani (LD). Panels A and B show per cent infected cells and number of intracellular parasites/100 cells, respectively, at indicated time points.
In case of human mononuclear phagocytes, 96% cells were found to be infected at 2 h post infection, and the number of intracellular LD per 100 cells was 319 (data not shown).
Modulation of expression of chemokines and chemokine receptors in human mononuclear phagocytes
We analysed the expression of one α chemokine (IL-8) and three β chemokines (MIP-1α, MIP-1β and RANTES) in human mononuclear phagocytes infected with LD. At 2 h post infection, 96% of the cells were found to be infected, and based on our prior experiments to determine the kinetics of infection of phagocytes, 2 h incubation was found to allow optimum infection of cells. We noticed that at 2 h post infection, there was enhanced expression of the chemokines MIP-1α, MIP-1β and RANTES but not IL-8 (Fig. 2). Interestingly, among the four chemokines studied, RNA expression of RANTES was significantly higher in infected mononuclear phagocytes than in uninfected control cells. Identical experiments with mononuclear phagocytes with either latex beads or dead parasites failed to show any enhancement of expression, as seen in the case of live parasites, indicating that infection by live LD promastigotes was a prerequisite for activation of chemokines in P-THP1 cells (data not shown).
Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of chemokine gene expression in human mononuclear phagocytes, as indicated. In each case, expression of chemokines and its densitometric data as the means ± standard errors of the mean of the ratio of the chemokine to β-actin band from the same sample are presented at 2 h post infection. □, uninfected mononuclear phagocytes; 
, Leishmania donovani (LD)-infected mononuclear phagocytes. Upper and lower panels show the expression of β-actin and chemokines, respectively. MIP, macrophage inflammatory protein; RANTES, regulated on activation normal T cell expressed and secreted; IL-8, interleukin-8.
We extended our studies on the expression of chemokine receptors under identical conditions. Both CCR5 and CCR1 are common receptors to the beta chemokines tested in this study, and CCR5 has been implicated in HIV-1 infection [15, 16]. In our study, we found that LD infection caused significant enhancement in the expression of transcripts of CCR5 but not CCR1 (Fig. 3). Staining of uninfected (Fig. 4A) and infected (Fig. 4B) mononuclear phagocytes with monoclonal antihuman CCR5-FITC (fluorescein isothiocyanate) antibody and subsequent analysis by confocal microscopy revealed significantly higher expression of CCR5 on the surface of infected cells than that on uninfected cells. Unrelated isotype FITC-IgG2b failed to stain the cells (data not shown).
Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of chemokine receptor gene expression in human mononuclear phagocytes. In each case, expression of chemokine receptors and its densitometric data as the means ± standard errors of the mean of the ratio of the chemokine receptor to β-actin band from the same sample are presented at 2 h post infection. □, uninfected mononuclear phagocytes; , Leishmania donovani (LD)-infected mononuclear phagocytes. Upper and lower panels show the expression of β-actin and chemokine receptors, respectively. CCR, CC chemokine receptor.
Confocal laser scanning immunofluorescence of human mononuclear phagocytes. Uninfected (A) and infected (B) human mononuclear phagocytes were stained with monoclonal antihuman CC chemokine receptor 5 (CCR5)-FITC (fluorescein isothiocyanate) antibody.
Modulation of expression of chemokines and chemokine receptors in THP1 cells
In order to evaluate whether the results obtained from infection of human mononuclear phagocytes with LD could be corroborated by similar studies in cell line, we analysed the expression of the above chemokines in human macrophage-like THP1 cells infected with LD at 12 and 24 h post infection. As about 32% of the cells remained uninfected at 6 h time point and as most of the cells were infected at 12 and 24 h time point, we analysed the expression of the indicated genes at the later two time points. We noticed that at 12 h time point with PMA alone, there was upregulation of all the three β chemokines (MIP-1α, MIP-1β and RANTES) and the α chemokine (IL-8). Enhanced expression of chemokines (MIP-1β and IL-8) and chemokine receptors by PMA in blood monocytes and also in THP1 cells has been reported previously [22, 37–39]. There was further enhancement of expression of all the above chemokines in LD-infected P-THP1 cells (Fig. 5A–D). Interestingly, similar to the results obtained in human mononuclear phagocytes, there was considerable enhancement in the expression of RANTES in P-THP1 cells infected with LD than in uninfected P-THP1 cells (Fig. 5C). However, at 24 h time point, the expression of all the four chemokines decreased considerably. There was no difference in MIP-1α, MIP-1β, RANTES and IL-8 expression between THP1 cells and LD-infected THP1 cells at 24 h time point. It may be recalled that the decrease in expression of the above chemokines was not because of cell death, as β-actin expression remained unaltered. This was again confirmed by the Trypan blue dye exclusion method, and cells were found to be 95% viable. Identical experiments with P-THP1 cells and subsequent stimulation with either latex beads or dead parasites failed to show any enhancement of expression, as seen in the case of live parasites (data not shown), indicating that intracellular parasite replication was a prerequisite for activation of chemokines in P-THP1 cells.
Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of chemokine gene expression in phorbol 12-myristate 7-acetate (PMA)-treated adherent THP1 (P-THP1) cells. In each case, expression of chemokines and its densitometric data as the means ± standard errors of the mean of the ratio of the chemokine to β-actin band from the same sample are presented at 12 and 24 h time points. □, P-THP1 cells; 
, Leishmania donovani (LD)-infected P-THP1 cells. Panels A, B, C and D show the expression of MIP-1α, MIP-1β, RANTES and IL-8, respectively. β-Actin expression at the two time points is shown only in panel A. MIP, macrophage inflammatory protein; RANTES, regulated on activation normal T cell expressed and secreted; IL-8, interleukin-8.
We analysed the RNA expression for the chemokine receptors CCR5 and CCR1 in uninfected and LD-infected P-THP1 cells. In our study, we found that PMA stimulation caused enhanced expression of both CCR5 and CCR1 in THP1 cells (data not shown). Interestingly, when P-THP1 cells were infected with LD, there was further enhancement in the expression of CCR5 but not CCR1 (Fig. 6A,B). We stained uninfected (Fig. 7A) and infected (Fig. 7B) P-THP1 cells with monoclonal antihuman CCR5-FITC antibody and analysed by confocal microscopy. Our results indicated that infection and intracellular replication of LD specifically caused significant augmentation in surface expression of CCR5 protein in 12 h-infected P-THP1 cells, as there was no difference in the expression of CCR5 between control P-THP1 cells and cells treated with dead parasites or latex beads. At 24 h time point, although CCR5 expression decreased considerably, slightly higher expression of CCR5 was still observed in infected P-THP1 cells than in uninfected cells. Unrelated isotype FITC-IgG2b failed to stain the cells (data not shown).
Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of chemokine receptor gene expression in phorbol 12-myristate 7-acetate (PMA)-treated adherent THP1 (P-THP1) cells. In each case, expression of chemokine receptors and its densitometric data as the means ± standard errors of the mean of the ratio of the chemokine receptor to β-actin band from the same sample are presented at 12 and 24 h time points. □, P-THP1 cells; 
, Leishmania donovani (LD)-infected P-THP1 cells. Panels A and B show the expression of CCR5 and CCR1, respectively. β-Actin expression at the two time points are shown only in panel A. CCR, CC chemokine receptor.
Confocal laser scanning immunofluorescence of P-THP1 cells. Uninfected (A) and infected (B) phorbol 12-myristate 7-acetate (PMA)-treated adherent THP1 (P-THP1) cells were stained with monoclonal antihuman CC chemokine receptor 5 (CCR5)-FITC (fluorescein isothiocyanate) antibody.
Discussion
A recent study [40] suggests that selective induction and general suppression of gene expression in LD infection play an important role in allowing the parasite to survive and proliferate within its host macrophage cells. It has been shown that there is a 2.9- and 6.3-fold upregulation of MIP-1α and MIP-1β gene expression, respectively, in LD-infected bone marrow-derived macrophages of BALB/c origin mice [40]. There are reports that human monocytes infected with L. major produce significant level of IL-8 in the supernatant [41, 42] and MIP-1α is expressed in diffuse cutaneous lesions, as evident from the immunohistochemical localization [43].
Induction of expression of chemokines (MIP-1β and IL-8) and chemokine receptors by PMA in blood monocytes and THP1 cells has been reported previously [22, 37, 39, 44]. Our studies indicated that LD infection upregulates the beta chemokines MIP-1α, MIP-1β and RANTES and CCR5 in human mononuclear phagocytes. In order to see whether the results observed in human mononuclear phagocytes could be mimicked in cell line, we infected THP1 cells with LD, and the results obtained were very similar to that observed in human mononuclear phagocytes. We also found that PMA induces the expression of not only MIP-1β and IL-8 but also of MIP-1α and RANTES in THP1 cells. There is a report that in the absence of any stimulus, IL-8 mRNA is undetectable in monocytes [40], and this has also been confirmed by our study in THP1 cells. In a recent study, Leishmania-induced CXC and CC chemokine mRNA expression was studied by RNase protection assay [45]. It has been shown that chemokine gene expression of RANTES, MIP-1α, MIP-1β, MIP-2, IP-10, MCP-1 and T-cell activation factor-3 (TCA-3) in the air pouch exudate cells was increased by L. major or LD after injection within air pouches. Furthermore, CCR gene expression was studied in the exudate cells. It was observed that CCR3, CCR5 and CCR2 were induced in greater magnitude by L. major than LD, whereas CCR1 expression was upregulated to a similar extent both in L. major and LD. In our system, we did not see significant enhancement of CCR1. This difference in CCR1 expression may be attributed to the different types of cells used for the investigation. Badolato et al. showed that L major infection of human peripheral blood monocytes resulted in a sustained induction of both MCAF (the human homologue of JE) and IL-8, which continued to increase throughout the 12 h period post infection [45].
Expression of the chemokines MIP-1α, MIP-1β and RANTES in THP1 cells has been previously demonstrated and their role in chemotaxis of THP1 cells is also known [46]. It has been previously demonstrated that PMA induces upregulation of MIP-1α, MIP-1β and RANTES and their receptors (CCR5, CCR1) in THP1 cells [46–48]. We found identical results in our study except for CCR1, which was less expressed. In both BALB/c and severe combined immunodeficiency mice, LD infection induced rapid hepatic accumulation of MIP-1α, MCP-1 and IP-10 RNA [21, 49]. Elevated expression of a subset of chemokines is the earliest known transcriptional response of macrophages to Leishmania infection and potentially provides a signal for the initiation of downstream immunological response that occur in vivo, such as cytokine induction and chemotaxis of mononuclear cells [21]. Predominant expression of MIP-1α has been reported earlier in the case of nonhealing diffuse cutaneous leishmaniasis in human [43]. Although we did not notice differential expression of CCR1 with respect to control versus infected cells, we did observe an increase in the expression of CCR5 at 2 h-infected human mononuclear phagocytes and 12 h-infected THP1 cells. This result can have serious implications. In human studies, CCR5 was shown to be overexpressed on macrophages following in vitro Mycobacterium tuberculosis infection and on alveolar macrophages from tuberculosis patients [50]. In a recent report, it has been shown that in CCR5–/– and MIP-1α–/– mice, when infected with LD, there is an enhanced control of parasite replication, indicating a possible deleterious role played by CCR5 and MIP-1α in LD infection [18]. Reduction in virus entry and replication has been observed in macrophages with downmodulated CCR5 and/or CD4+ expression [51].
Epidemiological data demonstrated that infections with opportunistic pathogens like Leishmania and Mycobacterium facilitates HIV disease progression in HIV-infected patients through enhanced production of interferon-γ and IL-6 [51, 52], which in turn upregulates the expression of beta chemokines. Considering the ever escalating number of cases of coinfection of HIV with Leishmania[17, 18], our study, demonstrating the role of Leishmania in inducing key chemokines and chemokine receptors in human mononuclear phagocytes and THP1 cells, is an important observation linking two serious diseases through the drama played by chemokines and chemokine receptors. As we obtained identical results in human mononuclear phagocytes and THP1 cells, current studies are underway to elucidate the mechanisms of chemokine and chemokine receptor upregulation by LD in THP1 cells.
Acknowledgments
This work was supported by Council of Scientific and Industrial Research and The Department of Science and Technology (DST), Government of India (Grant # SP/SO/D-71/98).
