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Leishmania infantum Promotes Replication of HIV Type 1 in Human Lymphoid Tissue Cultured Ex Vivo by Inducing Secretion of the Proinflammatory Cytokines TNF- and IL-1¹

Research Center in Infectious Diseases, Centre Hospitalier de l’Université Laval Research Center, and Laval University, Québec, Canada

	Abstract

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Parasitic infections such as leishmaniasis can modulate the life cycle of HIV-1 and disease progression. Coinfection with HIV-1 and Leishmania has emerged as a serious threat in countries where both pathogenic agents are widespread. Although there are numerous clinical reports illustrating the cofactor role played by Leishmania in HIV-1-infected patients, there is still no information on the contribution of Leishmania to the biology of HIV-1 in human lymphoid tissue that is considered a major in vivo site of virus production. In this study we explored the modulatory effect of Leishmania on the process of HIV-1 infection using ex vivo cultured human tonsillar tissue. We found that the protozoan parasite Leishmania enhances both HIV-1 transcription and virus production after infection of human tonsillar tissue infected ex vivo with viral strains bearing various coreceptor usage profiles. Studies conducted with pentoxifylline and neutralizing Abs revealed that the Leishmania-mediated increase in HIV-1 production was linked to a higher production of TNF- and IL-1. Our findings help to unravel the molecular mechanism(s) through which the two microorganisms interact and provide information that may be useful for the design of more effective therapeutic strategies aimed at controlling disease progression in persons dually infected with HIV-1 and Leishmania. This work also indicates that histocultures of human lymphoid tissue infected by both pathogens represent an ideal experimental cell system to dissect interactions occurring between HIV-1 and an opportunist pathogen in a human microenvironment that approximates conditions prevailing under physiological situations.

	Introduction

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Human immunodeficiency virus type 1 has been recognized as the causative agent of AIDS (1). One of the most striking clinical features of this retroviral infection remains the long latency period between the initial viral invasion, the onset of AIDS, and, eventually, the establishment of an immunodeficiency state (1, 2). Progression to AIDS among HIV-1-infected individuals has been shown to be associated with the active replication of HIV-1 (3). Critical events that occur during the virus life cycle, such as reverse transcription, integration of the viral genome into the host DNA, and subsequent viral replication, are all tightly associated with the host cell machinery (4). For example, virus gene expression relies heavily on a number of host cell transcription factors (e.g., NF-B, NFAT, and AP-1) due to a similarity between the architecture of the regulatory elements of HIV-1 and that of some cellular genes, such as the IL-2 promoter. Consequently, although activation of the normal immune response mediated by invading pathogens is crucial to mount a protective host response, the same response may be detrimental by favoring virus replication and HIV-1-associated disease progression. It has been postulated that several microorganisms can act as cofactors in HIV-1 disease progression by increasing the level of systemic immune activation (5).

Protozoan parasites of the genus Leishmania are transmitted by sand flies during a blood meal and cause a wide spectrum of diseases affecting the skin, mucous membranes, and viscera. Leishmania species cause a chronic infection, called leishmaniasis, in mammals and are obligate intracellular parasites (6). Since the mid-1980s, there have been numerous reports of leishmaniasis patients concurrently infected with HIV-1. In addition, visceral leishmaniasis (VL),³ which is mainly caused by Leishmania infantum, has emerged as an opportunistic disease among HIV-1-seropositive patients (7). Leishmania is now considered to be a cofactor in HIV-1 infection, because it fulfills the following three criteria. First, Leishmania and HIV-1 infection overlap in many areas of the world, such as southern Asia, sub-Saharan Africa, South America, and particularly southern Europe (8). Therefore, Leishmania is highly prevalent in the same human population as HIV-1. Secondly, both pathogens can reside within the same target cells, i.e., macrophages and dendritic cells (9). Thus, infection of the same cell type by two different pathogens may have important effects on the immune response and influence the biology of both microorganisms. Thirdly, the Leishmania parasite and its major surface molecule, lipophosphoglycan (LPG), have been shown to increase HIV-1 replication (10, 11, 12). Data from clinical studies have indicated that the virus load in HIV-1/Leishmania-coinfected patients is higher than that in HIV-1-infected controls without coinfection (13). Recently, L. donovani and LPG were shown to induce HIV-1 production in CD8-depleted PBMC from asymptomatic HIV-1-infected donors (14). However, experimental data from these in vitro studies were obtained with suspensions of PBMCs that lack the complex intercellular interactions and the rich cellular repertoire that are typical of lymphoid tissues where both HIV-1 and Leishmania are concentrated at a given time period after infection.

Proinflammatory cytokines have been recognized to be important mediators in HIV-1 pathogenesis (15). Of these cytokines, TNF-, which is primarily produced by activated monocytes and macrophages, has been reported to play a major role in the up-regulation of HIV-1 gene expression via a positive action on the NF-B signaling pathway (16). Another proinflammatory cytokine, IL-1, has been demonstrated to be capable of stimulating virus replication in infected monocyte-derived macrophages (17). An elevated production of TNF- and IL-1 has been detected in Leishmania-infected macrophages (18, 19). Moreover, the plasma level of TNF- has been shown to be raised in disseminated forms of leishmaniasis (20, 21). Therefore, it is likely that in HIV-1/Leishmania-coinfected subjects, HIV-1 uses a portion of the Leishmania-mediated cytokine network to its own advantage.

To better understand the complex interactions between HIV-1 and Leishmania in human lymphoid tissue, experiments were conducted in tonsil histocultures that preserve their general cytoarchitecture, including a network of follicular dendritic cells (FDC) (22, 23). Histocultures of tonsillar tissues infected ex vivo allowed us to assess whether the reported capacity of Leishmania to induce proinflammatory cytokine production can affect HIV-1 gene expression in lymphoid tissue.

	Materials and Methods

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Production of virus stocks

Viruses were prepared by transient calcium phosphate transfection of human 293T cells with an infectious molecular clone of HIV-1, i.e., NL4-3 (T-tropic/X4), JR-CSF (macrophage-tropic/R5), or 89.6 (dual-tropic/R5X4). The primary clinical HIV-1 isolate 029 (R5X4) was produced in mitogen-stimulated PBMCs obtained from healthy donors. Reporter viruses were pseudotyped by cotransfection of 293T cells with pNL4.3.LUC.R⁺E^- and an expression vector coding for the vesicular stomatitis virus envelope glycoprotein G (VSV-G). All virus preparations underwent a single freeze-thaw cycle before infection. Virus stocks were normalized for virion content using a p24 Ab capture assay developed in our laboratory (see below).

Leishmania parasites

L. infantum promastigotes were maintained in semidefined medium containing FBS and antibiotics. The promastigotes used for the infection were prepared from day -7 or -8 cultures (stationary phase). Before infection, L. infantum promastigotes were washed once in RPMI 1640 medium supplemented with FBS. Promastigotes were then resuspended in the same medium. L. infantum was selected because it constitutes the main etiologic agent of VL, which is the disease type mostly involved in HIV-1/Leishmania coinfection.

Infection of human lymphoid tissue cultured ex vivo with HIV-1 particles and Leishmania parasites

Human tonsils were obtained from patients who underwent routine tonsillectomy. Tonsils were received within a few hours of the operation and washed thoroughly with PBS containing antibiotics. The tissues were then dissected into small pieces of 2–3 mm³ in diameter and were cultured in RPMI 1640 supplemented with FBS and antibiotics on collagen sponge gels at the air-liquid interface for up to 15 days. The culture medium was changed every 2–3 days. The tonsillar tissue blocks were left untreated or were exposed to HIV-1 (i.e., fully competent or pseudotyped reporter viruses; 5 ng of p24 in 5–10 µl of volume/tissue block) and/or L. infantum parasites (1,000–20,000 parasites in 5 µl of volume/tissue block). Coinfection with Leishmania and HIV-1 was performed by exposing tonsil tissues to the pathogens one after the other, with either L. infantum or HIV-1 first. This experimental strategy was adopted to parallel in vivo conditions prevailing in dually infected persons. Infection was achieved by slowly applying HIV-1 and/or L. infantum on top of the tissue blocks. It should be noted that several blocks of human tonsillar tissue from an individual donor were tested (i.e., a total of eight tissue blocks in four different wells) to normalize for variation in cell number and composition. Each tissue block received equal amounts of HIV-1 and/or L. infantum parasites.

HIV-1 infection assay

The kinetics of HIV-1 replication were analyzed by serial measurements of extracellular p24 using an in-house double Ab enzymatic test, as described previously (24). Briefly, at the indicated number of days postinfection, culture supernatants bathing the tissue blocks were collected, lysed, and added to an anti-p24 Ab-precoated plate. After exposing the samples to a second anti-p24 Ab, which recognizes a p24 epitope different from that recognized by the first Ab, the unknown p24 value was calculated on the basis of regression analysis of p24 standards prepared from known p24 concentration samples. HIV-1 transcriptional activity upon coinfection with recombinant HIV-1-based reported viruses and L. infantum parasites was measured by assessing the luciferase assay. Briefly, tissue blocks were exposed to L. infantum parasites for up to 6 days to establish a latent infection state. Next, VSV-G pseudotypes were added, and the tonsil tissues were incubated for another 2 days. At the end of the experiment, tonsillar tissue cells were mechanically lysed, and luciferase units representing HIV-1 transcriptional activity were measured with a microplate luminometer (MLX; Dynex Technologies, Chantilly, VA).

Cytokine production and blocking studies

Levels of TNF- and IL-1 in the culture supernatants bathing the infected tissue blocks were quantitated with commercially available ELISA kits according to the manufacturer’s instructions (Cedarlane Laboratories, Hornby, Canada). The TNF- inhibitor pentoxifylline (PTX; 1-(5'-oxohexyl)-3,7-dimethylxanthine; Sigma-Aldrich, St. Louis, MO; 100 µM final concentration), the neutralizing anti-TNF- Ab (R&D Systems, Minneapolis, MN; 500 ng/ml final concentration), or the neutralizing anti-IL-1 Ab (R&D Systems; 1 µg/ml final concentration) was added to the culture medium at the time of infection and then every 3 days with each medium change.

Flow cytometry

Flow cytometric analysis was performed on tonsillar cells mechanically isolated from noninfected control and infected tonsil tissues. A mechanical cell isolation method was chosen over an enzymatic one to avoid the enzymatic digestion of cell surface markers, which might affect the flow cytometry results. Briefly, cells were isolated from tissue blocks on day 6 postinfection and stained with the appropriate mAbs, i.e., anti-HLA-DR (clone L-243), anti-ICAM-1 (clone RR1.1.1), and anti-CD25 (clone 7G7B6) Abs, as well as the Tritest kit (BD Biosciences, Mountain View, CA), which comprises a mixture of anti-CD3-PerCP, anti-CD4-FITC, and anti-CD8-PE Abs.

Statistical analysis

Results presented are expressed as the mean ± SD of four separate wells that each contained two blocks of tissue from the same donor. Statistical significance between groups for different donors was determined by ANOVA. Calculations were made with Microsoft Excel (Redmond, WA). A value of p < 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Virus transcription is augmented upon concurrent infection of human lymphoid tissue with L. infantum promastigotes and HIV-1

A previous study (12) has shown that Leishmania, through one of its major surface molecule, i.e., LPG, acts as a potent activator of HIV-1 transcription in an established human CD4-expressing T cell line. We tested whether Leishmania parasites can also accentuate virus transcription in intact lymphoid tissue, an organ known as a major reservoir of HIV-1 in vivo. This goal was achieved by first inoculating human tonsillar tissue blocks with increasing concentrations of L. infantum promastigotes ranging from 1,000–20,000 parasites/tissue block. The parasitic infection was allowed to establish itself for 6 days before infection with VSV-G-pseudotyped HIV-1-based reporter viruses. Forty-eight hours later, tonsillar cells were mechanically isolated from the tissue, and the virus-encoded luciferase activity was monitored in cell lysates. Given that the reporter gene in the tested recombinant virus is placed under the control of the regulatory elements of HIV-1, i.e., the long terminal repeat (LTR) domain, the measurement of luciferase activity represents a sensitive method for monitoring virus transcriptional activity. Our data show that L. infantum promastigotes increased HIV-1 LTR-directed transcriptional activity 2- to 6-fold compared with the group infected solely with HIV-1 (Fig. 1).

View larger version (16K): [in this window] [in a new window]   FIGURE 1. Leishmania up-regulates HIV-1 LTR-directed transcriptional activity in human tonsillar tissues cultured ex vivo. Human tonsillar tissue blocks were incubated in the presence or the absence of L. infantum promastigotes (1,000–20,000 parasites/tissue block). Six days postinfection, tissue blocks were inoculated with VSV-G-pseudotyped reporter HIV-1 particles (5 ng of p24/tissue block). After an additional incubation period of 48 h, cells were mechanically isolated from the tissue, and HIV-1 LTR-driven transcriptional activity was monitored by measuring luciferase activity in the cell lysates. Presented in this study for each treatment is the mean ± SD of four separate wells that contained two blocks of tissue each from the same donor (i.e., a total of eight tissue blocks for each condition). The data shown are representative of independent experiments performed with three different donors. A statistically significant difference was obtained when comparing samples inoculated with HIV-1 only (mean ± SD, 4.5 ± 0.7) and samples infected with HIV-1 and 5,000 parasites (mean ± SD, 28.9 ± 3.8) for the three donors tested (p = 0.0053).

To more closely parallel physiological conditions and to take into account the variety of cellular tropisms exerted by distinct HIV-1 isolates, we next tested the possible modulatory effect of Leishmania on the replicative capacity of fully infectious laboratory and clinical strains of HIV-1 exhibiting different coreceptor tropisms (i.e., X4, R5, and R5X4). Experiments were conducted by infecting tonsillar tissue first with L. infantum and next with complete HIV-1 particles. We also performed experiments in which infection was initiated with HIV-1 before the addition of increasing concentrations of parasites. This experimental strategy was adopted to reproduce the in vivo conditions prevailing in dually infected persons. Kinetic studies demonstrated that coinfection with L. infantum leads to a marked increase in HIV-1 production for all viral strains tested and under all experimental conditions used. Indeed, the addition of L. infantum before inoculation with HIV-1 was found to augment progeny virus production in human lymphoid tissue ex vivo infected with prototypic X4 (i.e., NL4–3), R5 (i.e., JR-CSF), and R5X4 (i.e., 89.6) laboratory variants as well as with an R5X4 field isolate of HIV-1 (i.e., 029; Fig. 2). Similar observations were made when HIV-1 was applied before Leishmania, as shown in Table I, which shows peaks of p24 levels.

View larger version (14K): [in this window] [in a new window]   FIGURE 2. Leishmania enhances replication of different laboratory and clinical isolates of HIV-1 in human lymphoid tissue. Human tonsillar tissues were first inoculated with various doses of Leishmania (1,000–20,000 parasites/tissue block) before the addition 5 h later of fully infectious laboratory (A, NL4–3; B, JR-CSF; C, 89.6) and clinical variants of HIV-1 (D, 029; 5 ng of p24/tissue block). Half the supernatant was harvested from each well at the indicated time points. Virus production was assessed by measuring p24 released into the culture medium. Presented in this study for each treatment are the mean ± SD of four separate wells that each contained two blocks of tissue from the same donor (i.e., a total of eight tissue blocks for each condition). Data shown are representative of independent experiments performed with five different donors. Statistically significant differences were obtained when comparing samples inoculated with HIV-1 only (mean ± SD, 9.0 ± 4.2, 5.7 ± 1.4, 10.3 ± 2.8, and 13.5 ± 4.2 for panels A, B, C, and D, respectively) and samples infected with HIV-1 and 5,000 parasites (mean ± SD, 18.5 ± 7.4, 12.7 ± 2.7, 16.6 ± 4.1, and 25.6 ± 8.3 for A, B, C, and D, respectively) for the five donors tested (p = 0.0029, 0.0004, 0.0014, and 0.0037 for A, B, C, and D, respectively).

A unique feature of HIV-1 infection is the establishment of a state of chronic T cell activation. For example, a significant number of cells from this cellular subset express activation markers, such as HLA-DR and IL-2R (CD25) (25). Interestingly, it has been reported that the Leishmania-mediated induction of HIV-1 replication in CD4-expressing T lymphocytes was linked to cellular proliferation and increased expression of immune activation markers (14). As the virus life cycle is closely associated with the activation state of the host cell, and a progressive loss of CD4⁺ T cells is also observed in HIV-1-infected individuals, we thus evaluated the number of both CD8⁺ and CD4⁺ T lymphocytes and measured the expression of immune activation markers upon infection with Leishmania and HIV-1 used either alone or in combination. Flow cytometric analyses revealed that HIV-1-directed depletion of CD4⁺ T cells is not amplified by addition of L. infantum (Fig. 3A). Moreover, surface expression levels of immune activation markers, such as HLA-DR, ICAM-1, and IL-2R, are not increased by the presence of Leishmania in samples also inoculated with HIV-1 (Fig. 3B).

View larger version (27K): [in this window] [in a new window]   FIGURE 3. Leishmania-directed increase in HIV-1 production is linked with neither depletion of CD4+ T cells nor cell activation. Human tonsillar tissues were first inoculated with Leishmania (20,000 parasites/tissue block) before the addition 5 h later of fully infectious NL4–3 (5 ng of p24/tissue block). Cells were mechanically dispersed after 15 days in culture, and the percentage of CD8+ and CD4+ T cells was measured using the Tritest kit (A). Dispersed cells were also used to measure surface expression levels of HLA-DR, ICAM-1, and IL-2R by flow cytometry (B). Controls consisted of tonsil fragments cultured in the absence of L. infantum and HIV-1. Data shown are representative of independent experiments performed with three different donors. Statistically significant differences were obtained when comparing samples inoculated with HIV-1 only (mean ± SD, 33.3 ± 4.2 and 16.0 ± 3.2 for A and B, respectively) and samples infected with HIV-1 and L. infantum (mean ± SD, 36.4 ± 9.8 and 15.0 ± 4.3 for A and B, respectively) for the two donors tested (p = 0.05 and 0.04 for A and B, respectively).

Enhancement of HIV-1 production is linked with Leishmania-mediated induction of the proinflammatory cytokines TNF- and IL-1

Leishmania and its major surface molecule LPG have been shown to mediate the secretion of several cytokines, including TNF- and IL-1 (11, 20, 26, 27). Treatment of HIV-1-infected cells with these two proinflammatory cytokines results in a significant activation of virus production in a variety of cell systems, either directly through nuclear translocation of NF-B (28, 29) or indirectly via costimulation of Th2 cells (30, 31). Therefore, in an attempt to define the relative contributions of TNF- and IL-1 to the Leishmania-mediated stimulatory effect on virus production, histocultures of human lymphoid tissue were exposed to L. infantum before monitoring cytokine production. As illustrated in Fig. 4, Leishmania infection of tonsillar tissue resulted in an augmentation of both TNF- and IL-1. Interestingly, the secretion of both cytokines was also promoted by HIV-1 in this experimental cell system. More importantly, cytokine production was further increased by the presence of the two pathogens (Fig. 5). Interestingly, HIV-1 production was similarly enhanced after addition of exogenous TNF- to virus-infected tonsillar tissue (Fig. 6). Experiments performed with PTX, a phosphodiesterase inhibitor that abolishes TNF- production (32, 33), and a blocking anti-TNF- Ab provided clear evidence of the direct implication of this proinflammatory cytokine in the Leishmania-dependent augmentation of HIV-1 production in human lymphoid tissue dually infected ex vivo (Figs. 7, A and B, respectively). The increase in HIV-1 replication associated with Leishmania was also diminished by the presence of a blocking anti-IL-1 Ab (Fig. 7C). The ability of PTX and the neutralizing anti-IL-1 to inhibit the Leishmania-mediated enhancement of HIV-1 production was also demonstrated in time-course experiments (Figs. 8, A and B, respectively).

View larger version (18K): [in this window] [in a new window]   FIGURE 4. Exposure of human lymphoid tissue to Leishmania leads to secretion of TNF- and IL-1. Human tonsillar tissues were either left untouched or were inoculated with increasing doses of L. infantum promastigotes (1,000–20,000 parasites/tissue block). Six days postinfection, supernatants bathing the infected tissues were collected, and levels of TNF- (A) and IL-1 (B) were measured using commercial enzymatic tests. Presented in this study for each treatment are the mean ± SD of four separate wells that contained two blocks of tissue each from the same donor (a total of eight tissue blocks for each condition). Results shown are representative of independent experiments performed with three different donors. Statistically significant differences were obtained when comparing samples inoculated with HIV-1 only (mean ± SD, 45.9 ± 8.0 and 23.8 ± 11.8 for A and B, respectively) and samples infected with HIV-1 and 5,000 parasites (mean ± SD, 239.8 ± 60.0 and 59.3 ± 16.3 for A and B, respectively) for the three donors tested (p = 0.0343 and 0.0288 for A and B, respectively).

View larger version (14K): [in this window] [in a new window]   FIGURE 5. TNF- and IL-1 production is increased by Leishmania or HIV-1 infection, but cytokine release is further augmented upon coinfection. Human tonsillar tissues were inoculated with L. infantum (20,000 parasites/tissue block), HIV-1 (JR-CSF when measuring TNF- and NL4–3 when measuring IL-1; 5 ng of p24/tissue block), or both pathogens. Controls consisted of uninfected tonsil histocultures. Six days postinfection, supernatants bathing the infected tissues were collected, and levels of TNF- (A) and IL-1 (B) were measured using commercial enzymatic tests. Presented in this study for each treatment are the mean ± SD of four separate wells that contained two blocks of tissue each from the same donor (a total of eight tissue blocks for each condition). Data shown are representative of independent experiments performed with three different donors. Statistically significant differences were obtained when comparing samples inoculated with HIV-1 only (mean ± SD, 160.3 ± 21.4 and 56.3 ± 19.2 for A and B, respectively) and samples infected with HIV-1 and L. infantum (mean ± SD, 266.3 ± 42.7 and 95.7 ± 20.1 for A and B, respectively) for the three donors tested (p = 0.0165 and 0.0032 for A and B, respectively).

View larger version (14K): [in this window] [in a new window]   FIGURE 6. Production of progeny virus is increased by addition of exogenous TNF- to the tonsillar tissue. Human tonsillar tissues were inoculated with NL4–3 (5 ng of p24/tissue block) in the absence or the presence of TNF- (0.1 and 1 ng/ml). Half the supernatant was harvested from each well at the indicated time points. Virus production was assessed by measuring p24 released into the culture medium. Presented in this study for each treatment are the mean ± SD of four separate wells that contained two blocks of tissue each from the same donor (a total of eight tissue blocks for each condition). Results shown are representative of independent experiments performed with four different donors. Statistically significant differences were obtained when comparing samples inoculated with HIV-1 (mean ± SD, 65.0 ± 7.4) and samples infected with HIV-1 and treated with 1 ng/ml TNF- (mean ± SD, 162.6 ± 10.1) for the four donors tested (p = 0.0004).

View larger version (11K): [in this window] [in a new window]   FIGURE 7. Leishmania-mediated augmentation of HIV-1 production is diminished by treatment with PTX or neutralizing Abs. Human lymphoid tissue histocultures were inoculated with HIV-1 (JR-CSF when measuring TNF- and NL4–3 when measuring IL-1; 5 ng of p24/tissue block) or with both pathogens (L. infantum, 20,000 parasites/tissue block). In some instances samples were also treated either with PTX (100 µM; A), neutralizing anti-TNF- Ab (500 ng/ml; B), or neutralizing anti-IL-1 Ab (1 µg/ml; C). Six days postinfection, supernatants bathing the infected tissues were collected, and virus production was assessed by measuring p24 released into the culture medium. Presented in this study for each treatment are the mean ± SD of four separate wells that contained two blocks of tissue each from the same donor (a total of eight tissue blocks for each condition). Data shown are representative of independent experiments performed with three different donors. Statistically significant differences were obtained when comparing samples inoculated with HIV-1 only (mean ± SD, 19.1 ± 4.2, 27.6 ± 9.7, and 15.5 ± 1.6 for A, B, and C, respectively) and samples infected with HIV-1 and L. infantum (mean ± SD, 37.3 ± 3.9, 46.0 ± 15.6, and 25.4 ± 4.0 for A, B, and C, respectively) for the three donors tested (p = 0.0105, 0.0398, and 0.0211 for A, B, and C, respectively).

View larger version (16K): [in this window] [in a new window]   FIGURE 8. A decrease in Leishmania-mediated up-regulation of HIV-1 replication by PTX and the blocker of IL-1 is seen in time-course experiments. Human lymphoid tissue histocultures were inoculated with HIV-1 (JR-CSF when measuring TNF- and NL4–3 when measuring IL-1; 5 ng of p24/tissue block) or with both pathogens (L. infantum, 20,000 parasites/tissue block). Some samples were treated with PTX (100 µM; A) or anti-IL-1 Ab (1 µg/ml; B). At the indicated time points postinfection, half the supernatants were collected, and virus p24 production in the presence or the absence of the cytokine inhibitors was analyzed. Presented in this study for each treatment are the mean ± SD of four separate wells that contained two blocks of tissue each from the same donor (a total of eight tissue blocks for each condition). Data shown are representative of independent experiments performed with three different donors. Statistically significant differences were obtained when comparing samples inoculated with HIV-1 and L. infantum (mean ± SD, 29.7 ± 8.5) and samples infected with HIV-1 and L. infantum and also treated with PTX (mean ± SD, 9.2 ± 3.4) for the three donors tested (p = 0.0285).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Previous reports have indicated that secondary lymphoid organs constitute preferred anatomical sites for HIV-1 replication and propagation (34, 35). It has been noted that HIV-1 induces a degeneration of the FDC network and a wholesale disruption of lymphoid architecture. Interestingly, destruction of FDCs and concomitant loss of germinal centers (GCs) were seen in animals with chronic VL (36). Human lymphoid tissues contain GCs that are vital for the production of memory B and T cells. The GC microenvironment is composed of centroblasts, centrocytes, FDCs, macrophages, CD4⁺ T cells, CD8⁺ T lymphocytes, and dendritic cells. Up to now, T lymphoid/monocytic cell lines and suspensions of PBMCs cultured in vitro have been used to obtain all the present experimental data on possible Leishmania/HIV-1 interactions (11, 12, 14, 37). However, these experimental cell systems lack the complex intercellular interactions and the rich cellular repertoire typical of lymphoid tissues. Therefore, to more closely parallel the critical events that take place in human lymphoid tissue, we used histocultures of human tonsils that have been reported to preserve the general cytoarchitecture of lymphoid tissue, including a network of FDCs (22). This last observation is of high importance, considering that dendritic cells play a crucial role in the pathogenesis of HIV-1 infection as they not only handle the crucial Ags, but also interact with infectious virions. The tonsil experimental cell system supports productive infection with various HIV-1 isolates bearing different tropisms without any requirement for exogenous activation or stimulation (22).

Using human lymphoid histocultures, we first investigated the possible modulatory effect of L. infantum on HIV-1 transcriptional activity. Our results showed that L. infantum enhances HIV-1 LTR-directed gene expression when using VSV-G-pseudotyped reporter virus. Using the pseudotyping strategy with VSV-G allows bypassing the natural mode of HIV-1 entry. This integrated virus not only widely broadens the natural virus tropism, but also significantly enhances virus infectivity (38, 39). The observed Leishmania-mediated up-regulation of HIV-1 LTR-driven transcriptional activity is consistent with experiments performed in T cells (12), a major cellular reservoir of HIV-1, and in primary monocyte-derived macrophages (our unpublished observations). The ability of Leishmania to influence replication of fully infectious viruses was also tested because recombinant HIV-1-based reporter viruses pseudotyped with the VSV-G envelope are deficient in HIV-1-encoded Env and Nef, two proteins known to be essential for the virus life cycle. Leishmania was found to positively influence the replication of R5, X4, and R5X4 HIV-1 variants in ex vivo-infected human lymphoid tissue.

The precise mechanism(s) by which Leishmania may affect HIV-1 replication under in vivo conditions is poorly defined. Chronic immune activation mediated by coinfecting pathogens has been proposed to lead to increased HIV-1 production, especially in the setting of developing countries (40). We show in this study that L. infantum can augment HIV-1 production without affecting the overall cellular activation state prevailing in lymphoid tissues. The protozoan parasite Leishmania may modulate HIV-1 replication by virtue of its capacity to induce the secretion of cytokines that are known to up-regulate virus gene expression. This is exemplified by the demonstration that Leishmania infection results in the production of soluble factors such as TNF- and IL-1 (41, 42, 43), which are proinflammatory cytokines known to amplify HIV-1 gene expression (28). In the present study human tonsillar tissues cultured in vitro were found to constitutively secrete low levels of TNF- (an average of 50 pg/ml) and IL-1 (an average of 15 pg/ml) into the medium throughout the culture period. More importantly, the secretion of TNF- and IL-1 was markedly increased by exposure to whole L. infantum promastigotes. It is worth noting that TNF- and IL-1 production in tonsillar tissue cultures was also increased upon infection with HIV-1 alone, although the increase with HIV-1 alone was weaker for IL-1 than with Leishmania alone. Pretreatment of cultured blocks of human lymphoid tissue with PTX, a methylxanthine derivative that inhibits TNF- synthesis, or neutralizing Abs confirmed that these two cytokines play a major role in the observed Leishmania-induced growth of HIV-1 production. The idea that Leishmania may promote HIV-1 replication by modulating the production of TNF- in histocultures of human lymphoid tissue is supported by our previous work, which showed that TNF- plays a pivotal role in LPG-mediated virus production in a T lymphoid cell line latently infected with HIV-1 (12). This observation is also in line with previous studies illustrating that TNF- is directly responsible for Ag-dependent HIV-1 production in malaria (44) and tuberculosis (45). It should be emphasized that the peak of cytokine production appeared on day 6 postinfection, i.e., before maximal amounts of most HIV-1 variants tested are released into the surrounding medium by infected tissues. This finding coupled with the reported high concentrations of TNF- in disseminated leishmaniasis patients (20, 21) lead us to propose that this proinflammatory cytokine should be considered a key non-Ag-specific factor responsible for the Leishmania-mediated increase in HIV-1 production. Moreover, our experiments indicate that the effect of Leishmania on the virus replicative cycle in a complex cellular microenvironment such as lymphoid tissues is a multifactorial phenomenon, as IL-1 is also involved in this process.

It is now well established that TNF- achieves activation of HIV-1 gene expression via nuclear translocation of NF-B (46). Thus, it can be hypothesized that the Leishmania-dependent release of TNF- may function in an autocrine/paracrine manner to induce virus gene expression in cells harboring HIV-1, i.e., CD4⁺ T lymphocytes and macrophages. PTX, as an effective inhibitor of protein kinase C, protein kinase A, and NF-B (47, 48, 49, 50), has been shown to selectively inhibit TNF- production as well as HIV-1 transcription and virus production (51). We demonstrate in this study that PTX severely diminishes HIV-1 replication in lymphoid tissue also exposed to L. infantum. The previous demonstration that PTX diminishes plasma viral load and improves cell-mediated immunity in HIV-1-infected individuals is thus of high interest (52).

In summary, the current study offers an in-depth analysis of the multiple interactions between HIV-1 and Leishmania, a protozoan parasite now considered an important cofactor in this retroviral disease. Using a human tonsil histoculture system that allows the study of key pathogenic events taking place in lymphoid tissues, we demonstrated that Leishmania positively modulates the process of HIV-1 infection by favoring a higher production of the proinflammatory cytokines TNF- and IL-1. This work confirms the relevance of using human lymphoid tissue infected ex vivo to scrutinize the multifaceted interactions between HIV-1 and a specific opportunist human pathogen.

	Acknowledgments

 
We are grateful to Sylvie Méthot for editorial assistance. We thank M. Dufour for his technical assistance with the flow cytometry studies.

	Footnotes

 
¹ This work was supported by a grant (to M.J.T.) from the Canadian Institutes of Health Research HIV/AIDS Research Program (HOP-37781) and a Canadian Institutes of Health Research Group Grant (to M.J.T. and B.P.; MGC-14500). The work was performed by C.Z. in partial fulfillment of her Ph.D. degree at the Faculty of Medicine, Laval University. C.Z. holds a Doctoral Award from the Fonds de la Recherche en Santé du Québec. B.P. is a Burroughs Wellcome Fund New Investigator in Molecular Parasitology and a Fonds de la Recherche en Santé du Québec Scholar (senior level). M.J.T. is the recipient of a Tier 1 Canada Research Chair in Human Immuno-Retrovirology.

² Address correspondence and reprint requests to Dr. Michel J. Tremblay, Laboratory of Human Immuno-Retrovirology, Research Center in Infectious Diseases, RC709, Centre Hospitalier de l’Université Laval Research Center, 2705 Laurier Boulevard, Québec, Québec, Canada G1V 4G2. E-mail address: michel.j.tremblay{at}crchul.ulaval.ca

³ Abbreviations used in this paper: VL, visceral leishmaniasis; FDC, follicular dendritic cell; GC, germinal center; LPG, lipophosphoglycan; LTR, long terminal repeat; PTX, pentoxifylline; VSV-G, vesicular stomatitis virus envelope glycoprotein G.

Received for publication July 7, 2003. Accepted for publication December 17, 2003.

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