Abstract
The role of Mycobacterium avium isolates in modulating human immunodeficiency virus type 1 (HIV-1) replication was examined by use of an in vitro, resting T cell system. Two human clinical isolates (serotypes 1 and 4) but not an environmental M. avium isolate (serotype 2) enhanced HIV-1 replication. The M. avium-induced HIV-1 replication was not associated with cell activation or differential cytokine production or utilization. Addition of matrix metalloproteinase (MMP) inhibitors and their in vivo regulators, tissue inhibitors of metalloproteinases-1 and -2, abrogated M. avium-induced HIV-1 replication 80%–95%. The MMP inhibitors did not have any effect on the HIV-1 protease activity, suggesting that they may affect cellular processes. Furthermore, MMP-9 protein was differentially expressed after infection with clinical M. avium isolates and paralleled HIV-1 p24 production. Collectively, these data suggest that M. avium-induced HIV-1 replication is mediated, in part, through the induction of MMP-9.
Mycobacterium avium is a facultative intracellular pathogen that infects host monocytes and macrophages [1]. Infected macrophages become activated and subsequently produce proinflammatory cytokines, specifically tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1β [2, 3]. It has been hypothesized that this inflammatory response, which is important for M. avium bacterial containment and elimination, may be responsible for increased replication of human immunodeficiency virus type 1 (HIV-1). TNF-α has been shown to be an important cytokine for the enhancement of HIV replication in monocytes and T cells [4, 5]. Data from in vitro studies suggest that the interactions between M. avium bacilli and HIV result in increased replication of both. HIV-1 replication in a monocytoid cell line co-infected with HIV-1 and M. avium bacilli was more than 3× that in the same cells infected with HIV-1 alone [6].
Furthermore, monocytes preincubated with HIV-1 or its envelope component showed enhanced intracellular growth of M. avium bacteria, primarily because of the inability of HIV-infected monocytes to phagocytize [6, 7]. The most prevalent M. avium serotype isolated from AIDS patients is serotype 4 [8]. Several other serotypes, including serotype 1, are also isolated from AIDS patients but less frequently than serotype 4; however, environmental serotypes, such as serotype 2, are only occasionally (∼2%) recovered from AIDS patients [8]. In mice, clinical isolates were found to be virulent, while environmental M. avium strains were eliminated or did not proliferate [9, 10]. Together, these studies suggest that there may be differences between environmental and clinical M. avium isolates that enable the clinical isolates to disseminate in immunocompromised individuals and to influence the pathogenesis of these diseases in co-infected persons.
Matrix metalloproteinases (MMPs) are a family of zinc-containing enzymes that share common structural domains but have different substrate specificities, cellular sources, and inducibility. MMPs are important for cell migration and affect a variety of cell surface antigens, including adhesion molecules. Collectively, the MMPs can cleave virtually all of the extracellular matrix constituents [11]. They are regulated through the endogenously produced tissue inhibitor of metalloprotei nases (TIMPs) [12].
Macrophages produce many types of MMPs, whereas T cells produce MMP-2 and MMP-9 [13]. In addition to preparing tissue matrices for chemotaxis across basement membranes, MMPs are also important for releasing cell membrane-bound proteins, such as proinflammatory cytokines [14]. Proinflammatory cytokines, such as TNF-α and IL-1, enhance MMP production, whereas IL-10 and interferon (IFN)-γ suppress such production and modulate HIV-1 replication [4, 5, 11]. Recently, the HIV regulatory protein, Tat, has been shown to up-regulate the production of MMP-9 in monocytes and results in the recruitment of these cells, potentially leading to local tissue damage associated with progression to AIDS [15, 16]. However, little is known about the interactions between opportunistic pathogens, specifically M. avium, and MMPs on HIV replication in T cells.
Because particular M. avium isolates are recovered more frequently from HIV-infected persons, we postulated that these clinical M. avium strains might modulate HIV replication differently than environmental M. avium strains. The high bacterial tissue burden in AIDS patients implied that clinical M. avium isolates can interact with circulating and potentially infected T cells. The interactions between M. avium and HIV-1-infected T cells have not been previously examined primarily because of the lack of in vitro infection models. Typically, exogenous stimuli, such as IL-2, which would mask the potential stimulatory effects of the copathogens, are used. In this study, we used an in vitro, resting T cell model system to evaluate the interactions between HIV-1 and environmental and clinical M. avium strains. We show that all M. avium isolates tested can activate T cells, but only the clinical M. avium isolates enhance HIV-1 replication in infected T cells. The mechanism by which HIV-1 replication is enhanced may involve MMP-9, suggesting the need for bacterium-T cell interactions, and is unique to clinical M. avium strains.
Materials and Methods
HIV-1 isolates
HIV-1 LAI stocks were prepared from infected A3.01 cells, as described elsewhere [17]. The LAI stocks were aliquoted and stored at −70°C until use. Primary HIV-1 isolates were obtained through the NIH AIDS Research and Reference Program. Stocks were prepared from primary isolates of subtype A isolates from Uganda (UG031 and UG029) and subtype B isolates from Brazil and Haiti (BR003 and HT596) by growing them once in phytohemagglutmin-P (Difco, Detroit), IL-2-activated peripheral blood mononuclear cells (PBMC). Supernatants were collected, filtered, aliquoted, and stored at −70°C until use. Co-receptor utilization of primary isolates has been described elsewhere [18].
Mycobacterium avium stock
Stocks of M. avium bacteria were prepared by growing 1 veterinary isolate (serotype 2; ATCC no. 35712) and 2 human clinical isolates (serotype 1 [19] and serotype 4 [20]) in cultures of human macrophages for 7 days. The macrophages were lysed in 0.1% triton ×-100, and the bacteria were plated from the cultures on Middlebrook 7H11 agar containing 0.1% glycerol, oleic acid, albumin, dextrose, and catalase (Difco). Colonies subsequently were transferred and grown in Middlebrook 7H9 broth made with endotoxin-free water and containing 0.1% glycerol, albumin, dextrose, and catalase (Difco) for 10–14 days. The broth-grown strains were harvested, pelleted, and washed twice in fresh 7H9 broth. The mycobacteria were resuspended in fresh 7H9 broth, aliquoted, and frozen at −70°C. After 1 week, one aliquot was thawed and plated on supplemented Middlebrook 7H11 media to determine the number of colony-forming units per milliliter.
Acute in vitro infection
CD8 and CD8/14 depletion was done, as described elsewhere [21]. In brief, PBMC were obtained from healthy, HIV-negative persons by leukophoresis and were immediately isolated through a ficoll gradient and cryopreserved. Individual tubes were suspended in RPMI 1640 (Gibco, Grand Island, NY) and incubated with anti-CD8 immunomagnetic beads (Dynal, Oslo) according to the manufacturer's instructions. For CD8/CD14-depleted PBMC, cells were allowed to adhere to a plastic flask for 2 h at 37°C in 7% CO2. The nonadherent cells were removed, and anti-CD8 and -CD 14 immunomagnetic beads were added according to the manufacturer's instructions. The depleted PBMC were cultured in RPMI 1640 supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM glutamine, and 10% fetal bovine serum (C-RPMI) without the addition of exogenous mitogens or cytokines. The experiments were done in medium without streptomycin, and the results were identical to those obtained with medium containing the antibiotic. To deter contamination and ensure there was no growth of the M. avium bacilli, all experiments reported here were done with streptomycin. One million CD8- or CD8/CD14-depleted PBMC per milliliter were incubated with HIV-1 (MOI of 0.001) and with or without the M. avium isolates (1 mycobacterial cfu to 1 cell). After being incubated for 12 h at 37°C in 7% CO2, the cells were washed 3× with C-RPMI and resuspended, and 2 mL were placed in a 24-well plate. One-milliliter aliquots were taken every 2 days for 10–14 days; fresh medium was added to replenish the cultures. All aliquots were stored at −70°C until needed. Viral replication was measured by use of a p24gag antigen-capture ELISA (Coulter Immunology, Miami).
Compounds
Pentoxifylline was obtained from Sigma (St. Louis). BB3103 was a gift from Alan Drummond (British Biotech, Oxford, UK). MMP inhibitor I was purchased from Calbiochem-Novabiochem (San Diego). TIMP-1 and TIMP-2 were purchased from Chemicon (Temicula, CA). To evaluate the effectiveness of the compounds, we initially titrated each, using 10-fold dilutions, and evaluated them for toxicity by inhibition of M. avium-induced proliferation uptake, by the ability to inhibit M. avium-induced HIV-1 replication, and by cell viability as determined by trypan blue exclusion. The effective dose of each compound was added at the indicated concentration to the cultures on day 0 and every other day thereafter. The ability of the compounds to inhibit p24gag antigen production was calculated, and the data are presented as percent inhibition: [1-(treated wells/control wells)] × 100 ± SE.
Cytokine measurement and neutralization
Cytokme mRNA expression for IL-1β, IL-6, IL-10, TNF-α, and IFN-γ was measured, as described elsewhere [22]. Cytokme production for IL-1β, IL-6, IL-10, IL-12, TNF-α, and IFN-γ was measured by ELISA according to the manufacturer's instructions (BioSource International, Camarillo, CA). To determine whether cytokines were involved in HIV-1 enhancement, neutralizing cytokine monoclonal antibodies (MAbs) (R&D Systems, Minneapolis) were added to the wells. The MAbs used were anti-IL-1β; (1 μg/mL), anti-IL-2 (10 μg/mL), anti-IL-4 (1 μg/mL), anti-IL-5 (1 μg/mL), anti-IL-6 (1 μg/mL), anti-IL-10 (1 μg/mL), anti-TNF-α (1 μg/mL), anti-transforming growth factor β 31 (10 μg/mL), and antigranulocyte macrophage colony-stimulating factor (10 μg/mL). The concentrations indicated are at saturating conditions (as determined on the basis of the manufacturer's neutralization curve) and were similar to concentrations used elsewhere [21]. The ability of the neutralizing cytokine MAb to inhibit p24gag antigen production was calculated, and the data are presented as percent inhibition (see above).
Cellular proliferation and activation
Twenty thousand CD8-or CD8/CD14-depleted PBMC were added to a 96-well, U-bottom plate in triplicate. Environmental and human clinical M. avium isolates were added to the PBMC with or without the treatments described above. Each treatment was added to the appropriate wells at the same concentration as that used in the acute infections. The cells were cultured for 6 days at 37°C in 7% CO2 and pulsed with 0.5 μ Ci 3H-thymidine per well for 18 h prior to harvest; the cpm incorporated were counted on a Matrix 96 direct Beta Counter (Packard Instruments, Downers Grove, IL). The expression of cellular activation markers was assessed by use of flow cytometry on CD8- or CD8/CD14-depleted PBMC cultured with the M. avium isolates for 6 days. Cells were stained with fluorescein isothio-cyanate-anti-CD3 (Becton Dickinson, Mountain View, CA) and either phycoerythrin (PE)-anti-HLA-DR (Becton Dickinson), PE-anti-CD69 (Becton-Dickinson), or PE-anti-CD98 (Immunotech, Westbrook, ME). Samples were analyzed by use of Consort 30 software (Becton-Dickinson).
Detection of MMPs
CD8-depleted PBMC were cultured for 6 days with or without M. avium bacilli in serum-free medium (AIM V; Gibco). Supernatants were collected every 2 days, passed through a 0.45-μm filter, and stored at −70°C until use. MMP-2 and MMP-9 were quantitated by use of ELISA (Amersham, Arlington Heights, IL) according to the manufacturer's instructions. MMP production was confirmed by use of zymography, as described elsewhere [15].
Effect of MMP inhibitors on HIV-1 protease
The chronically infected, TNF-α-inducible cell line OM10.1 [23] was grown in C-RPMI. MMP inhibitors were cultured at the indicated concentrations with the OM10.1 cells 2 h before TNF-α induction (20 U/mL). A protease inhibitor (saquinavir, 0.25 μg/mL; gift from Roche Pharmaceuticals, Nutley, NJ), was used as a positive control for p55gag precursor accumulation. After 16 h, the cells were washed twice with PBS and lysed in lysing buffer (0.1 M Tris-buffered saline, pH 7.5, 0.5% Triton ×-100, 0.1% deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride). The equivalent of 50,000 cells were separated through a 10% SDS-polyacrylamide gel. The proteins were blotted onto a polyvinylidene difluoride membrane and incubated with anti-HIV-1 sera. The HIV-1 proteins were developed by use of a high-titered anti-HIV serum recognized by a goat anti-human IgG alkaline phosphatase conjugate (Sigma) and a 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium kit (Kirkegaard & Perry Laboratories, Gaithersburg, MD). To ensure the activity of saquinavir, a reverse transcriptase (RT) assay was used [23].
Results
M. avium clinical isolates enhance HIV-1 replication
To evaluate the influence of pathogens on HIV replication, we utilized a resting CD8-depleted PBMC in vitro model, which could be reproducibly infected with HIV-1 without the addition of exogenous stimulants [21]. Addition of a clinical M. avium serotype 4 strain to CD8-depleted PBMC increased HIV replication 50 × over the p24 levels of the environmental serotype 2 strain by day 6 (figure 1A). Kinetic analysis demonstrated that the p24 levels remained high through day 10 after infection (figure 1A). Similar results were obtained with a second clinical M. avium strain (serotype 1; figure 1A). Another nontuberculous mycobacterium (Mycobacterium smegmatis) did not enhance HIV-1 replication (data not shown).
Clinical Mycobacterium avium isolates, serotypes 1 and 4, enhance human immunodeficiency virus type 1 (HIV-1) replication independently of mycobacterial processing or growth. A, CD8-depleted peripheral blood mononuclear cells (PBMC) were infected with LAI (MOI 0.001) and cultured with environmental (serotype 2) and clinical (serotypes 1 and 4) M. avium isolates (1 cfu/1 cell). Supernatant samples were collected every 2 days, and HIV-1 replication was measured by use of p24gag ELISA. B, CD8/CD14-depleted PBMC were infected with LAI (MOI 0.001) and cultured with environmental (serotype 2) and clinical (serotype 4) M. avium strains (1 cfu/1 cell). C, Heat-killed M. avium serotype 4 isolate (serotype 4 HK) and viable serotype 4 isolate were cultured with LAI-infected CD8-depleted PBMC (1 cfu/1 cell). HIV-1 replication was measured by use of p24gag ELISA. Data presented are representative of at least 4 independent experiments.
Clinical Mycobacterium avium isolates, serotypes 1 and 4, enhance human immunodeficiency virus type 1 (HIV-1) replication independently of mycobacterial processing or growth. A, CD8-depleted peripheral blood mononuclear cells (PBMC) were infected with LAI (MOI 0.001) and cultured with environmental (serotype 2) and clinical (serotypes 1 and 4) M. avium isolates (1 cfu/1 cell). Supernatant samples were collected every 2 days, and HIV-1 replication was measured by use of p24gag ELISA. B, CD8/CD14-depleted PBMC were infected with LAI (MOI 0.001) and cultured with environmental (serotype 2) and clinical (serotype 4) M. avium strains (1 cfu/1 cell). C, Heat-killed M. avium serotype 4 isolate (serotype 4 HK) and viable serotype 4 isolate were cultured with LAI-infected CD8-depleted PBMC (1 cfu/1 cell). HIV-1 replication was measured by use of p24gag ELISA. Data presented are representative of at least 4 independent experiments.
Because clinical M. avium strains are isolated from HIV-infected persons late in the course of their disease, typically when the R5X4 viral isolates begin to emerge [24], we examined the ability of clinical M. avium isolates to enhance primary R5 and R5X4 HIV-1 isolate replication. HIV-1 isolates representing subtype A were obtained from Uganda (UG031 and UG029), and those of subtype B were from Brazil and Haiti (BR003 and HT596). Analysis of co-receptor usage of these isolates has shown that UG031 and BR003 exclusively use CCR5 (R5), whereas UG029 and HT596 use multiple co-receptors (R5X4) [18]. M. avium serotype 1 and 4 strains enhanced HIV-1 replication from all primary isolates (figure 2) regardless of their genotype or co-receptor usage. The variability in the amount of p24 induced reflects the different rates of M. avium-induced primary HIV-1 replication. As observed with LAI, M. avium serotype 2 strain had no effect on primary HIV-1 isolate replication (figure 2).
Clinical Mycobacterium avium isolates enhance R5 and R5X4 primary human immunodeficiency virus type 1 (HIV-1) isolate replication. Primary HIV-1 isolates representing R5 (UG031, BR003) and R5X4 (UG029, HT596) isolates were cultured with environmental (serotype 2) and clinical (serotypes 1 and 4) M. avium isolates in CD8-depleted peripheral blood mononuclear cells. HIV-1 replication was measured by use of p24gag ELISA. Reference line at 7 pg/mL indicates values below cutoff. Data presented are representative of 3 independent experiments. NSI, non-syncytium-inducing; sero 1, 2, 4, serotype 1, 2, 4, respectively; SI, syncytium-inducing.
Clinical Mycobacterium avium isolates enhance R5 and R5X4 primary human immunodeficiency virus type 1 (HIV-1) isolate replication. Primary HIV-1 isolates representing R5 (UG031, BR003) and R5X4 (UG029, HT596) isolates were cultured with environmental (serotype 2) and clinical (serotypes 1 and 4) M. avium isolates in CD8-depleted peripheral blood mononuclear cells. HIV-1 replication was measured by use of p24gag ELISA. Reference line at 7 pg/mL indicates values below cutoff. Data presented are representative of 3 independent experiments. NSI, non-syncytium-inducing; sero 1, 2, 4, serotype 1, 2, 4, respectively; SI, syncytium-inducing.
Clinical M. avium isolates enhance HIV-1 replication independently of mycobacterial antigen processing and growth
In a normal immune response, M. avium bacteria are phagocytized and killed by macrophages that subsequently present the antigens to T cells [25, 26]. To test the possibility that M. avium-induced HIV-1 replication was dependent on mycobacterial antigen processing, we removed monocytes/macrophages from the in vitro, resting T cell model. Addition of the serotype 4 M. avium strain to the CD8/14-depleted cell population resulted in a 5- to 10-fold increase in HIV-1 replication, whereas the addition of a serotype 2 M. avium strain did not (figure 1B). These data are similar to the clinical M. avium-induced HIV-1 replication in the CD8-depleted model (figure 1A) and suggest that antigen processing by macrophage was not necessary for enhancement of HIV-1 replication. Furthermore, the HIV-1 enhancement was not dependent upon active mycobacterial growth or newly synthesized mycobacterial components, because a heat-killed serotype 4 strain enhanced HIV-1 replication to the same magnitude as the live serotype 4 M. avium strain (figure 1C). The addition of heat-killed serotype 2 M. avium bacilli did not enhance HIV-1 replication, a finding that was similar to results obtained from the addition of live serotype 2 M. avium (data not shown).
Clinical and environmental M. avium isolates induce similar levels of cellular proliferation and activation
To better understand the mechanism(s) involved in the induction of HIV-1 replication during M. avium co-infection, we examined cellular proliferation and activation. Proliferation for CD8-depleted PBMC infected with M. avium serotype 2 (stimulation index [SI] = 9.6) was similar to the proliferation induced by the clinical M. avium isolates (serotypes 4 and 1) (SI =16 and 11, respectively). Moreover, CD8/14-depleted PBMC showed similar proliferation values when infected with all of the M. avium isolates (data not shown). Expression of activation markers was examined on day 6 after infection of cultured CD8-depleted PBMC with or without the addition of M. avium bacilli. Clinical and environmental isolates of M. avium induced similar, albeit low, expression of HLA-DR, CD69, and CD98 (figure 3), indicating that all tested M. avium isolates induce similar cellular activation.
Mycobacterium avium-induced human immunodeficiency virus type 1 (HIV-1) enhancement is independent of cellular activation. CD8-depleted peripheral blood mononuclear cells (PBMC) were cultured for 6 days with M. avium serotype 2 and serotype 4 strains. Cells were stained for CD3, HLA-DR, CD69, and CD98. Analysis was done on CD3+ cells. Shown are 2 representative experiments. Gray, medium only; white, M. avium serotype 2 strain; black, M. avium serotype 4 strain. Similar results were found with CD8/CD14-depleted PBMC.
Mycobacterium avium-induced human immunodeficiency virus type 1 (HIV-1) enhancement is independent of cellular activation. CD8-depleted peripheral blood mononuclear cells (PBMC) were cultured for 6 days with M. avium serotype 2 and serotype 4 strains. Cells were stained for CD3, HLA-DR, CD69, and CD98. Analysis was done on CD3+ cells. Shown are 2 representative experiments. Gray, medium only; white, M. avium serotype 2 strain; black, M. avium serotype 4 strain. Similar results were found with CD8/CD14-depleted PBMC.
Clinical and environmental strains of M. avium induce similar cytokine profiles
Because cytokines, particularly TNF-α, are involved in the pathogenesis of M. avium, Mycobacterium tuberculosis, and HIV-1 [5], we measured the mRNA expression and the protein production of several cytokines, including TNF-α, IFN-7, IL-1β, IL-6, IL-10, and IL-12, after HIV-1 and M. avium bacterial infection. No differences in TNF-α, IL-6, IL-1β, and IL-10 mRNA expression were detected 16 h after infection with both HIV-1 and either the clinical or environmental M. avium isolates (data not shown). These data were confirmed by measuring the soluble cytokine levels in day-6 supernatants. IL-6 and TNF-α protein production were similar in both serotype 2 and serotype 4 M. avium-infected cultures (figure 4A). Likewise, IL-10 (20 ± 6 and 27 ± 6 pg/mL) and IFN-γ (1190 ± 208 and 1182 ± 224 pg/mL) levels were similar after culture with M. avium serotype 2 or serotype 4, respectively. IL-12 protein expression was not detected.
A, Clinical and nonclinical Mycobacterium avium isolates induce similar cytokine expression. Cytokine production was measured by ELISA for interleukin (IL)-6 and tumor necrosis factor (TNF)-α on supernatants from day 6 of culture. B, Induction of human immunodeficiency virus type 1 (HIV-1) replication by 2 clinical isolates of M. avium is not mediated through TNF-α. Neutralizing monoclonal antibodies against TNF-α or IL-6 were added to cultures at concentrations indicated in Materials and Methods. Supernatants were tested for p24 production. % inhibition was determined from day-8 culture. Data presented are representative of 4 independent experiments
A, Clinical and nonclinical Mycobacterium avium isolates induce similar cytokine expression. Cytokine production was measured by ELISA for interleukin (IL)-6 and tumor necrosis factor (TNF)-α on supernatants from day 6 of culture. B, Induction of human immunodeficiency virus type 1 (HIV-1) replication by 2 clinical isolates of M. avium is not mediated through TNF-α. Neutralizing monoclonal antibodies against TNF-α or IL-6 were added to cultures at concentrations indicated in Materials and Methods. Supernatants were tested for p24 production. % inhibition was determined from day-8 culture. Data presented are representative of 4 independent experiments
Because TNF-α and IL-6 have been shown to be important modulators for HIV-1 and M. avium, we next evaluated their effect on M. avium-induced HIV-1 replication. Neither anti-IL-6 nor anti-TNF-α MAb had any significant effect on reducing the M. avium-induced HIV-1 enhancement (figure 4B); however, anti-TNF-α MAb did block Plasmodium falciparum-induced HIV-1 replication [21]. Furthermore, neutralizing MAb against IL-2, IL-4, IL-5, and IL-10 had no effect on the M. avium-induced HIV enhancement (data not shown).
MMP inhibitors block clinical M. avium-induced HIV-1 replication
To better understand the mechanism of M. avium-induced HIV-1 replication, we evaluated broad-acting inhibitors of cytokine production, including pentoxifylline, a non-specific phosphodiesterase inhibitor, and BB3103, an MMP inhibitor that blocks the processing of cytokine precursors [27]. Both compounds were chosen because they have been shown to be effective at reducing HIV replication [28, 29]. Pentoxifylline had no effect on clinical M. avium-induced HIV replication (table 1). However, BB3103 was able to reduce the induced HIV replication by 92% (table 1). To determine whether the effect was due to MMP inhibitors in general and not just to BB3103, we also used the MMP inhibitor I and found that it blocked M. avium-induced HIV-1 replication by 82% (table 1). Because the MMP inhibitors are broad acting and may influence other proteinases, we added TIMP-1 and TIMP-2, specific in vivo inhibitors of MMPs, to the cultures. TIMP-1 blocked clinical M. avium-induced HIV-1 replication by 81%, whereas TIMP-2 blocked replication by 84% (table 1). These data show that both broad and specific MMP inhibitors block clinical M. avium-induced HIV-1 replication.
Matrix metalloproteinase (MMP) inhibitors block clinical Mycobacterium avium-induced human immunodeficiency virus type 1 replication.
Matrix metalloproteinase (MMP) inhibitors block clinical Mycobacterium avium-induced human immunodeficiency virus type 1 replication.
MMP inhibitors do not modulate the HIV-1 protease function
The HIV-1 protease is an aspartyl proteinase, which is closely related to the MMP family. It is involved in virus maturation by processing the precursor group-specific antigen (gag) proteins. To determine whether the MMP inhibitors modulate the function of the HIV-1 protease, as evidenced by the accumulation of the p55gag precursor, we added them to a latent, inducible HIV-1 cell line, OM10.1 [23]. The OM10.1 cell line was used because it is a well-characterized model and to ensure high quantities of HIV proteins to analyze by Western blot. Addition of TNF-α resulted in an increase in the p55 and p24gag proteins, compared with the medium control (figure 5, lower panel), and a concomitant increase in RT activity (figure 5, upper panel). The therapeutic protease inhibitor, saquinavir, prevented gag processing, as indicated by the accumulation of the p55gag precursor protein, and completely blocked RT activity (figure 5). Neither BB3103 nor MMP inhibitor I caused the p55gag precursor to accumulate at the 10-μM concentration that was used to suppress clinical M. avium-induced HIV-1 replication, suggesting that they do not directly affect the HIV protease function. While the p55gag and p24 bands were fainter for the BB3103-treated lanes, the proteins were present, and both inhibited RT activity by the TNF-α-induced OM10.1 cells by ∼50% (figure 5, upper panel).
Matrix metalloproteinase (MMP) inhibitors do not affect human immunodeficiency virus type 1 (HIV-1) protease activity. OM10.1 cells were cultured with BB3103 (MMP inhibitor that blocks processing of cytokine precursors; 100, 10, and 1 μM) and MMP inhibitor I (100, 10, and 1 μM) for 2 h prior to addition of 20 U/mL tumor necrosis factor (TNF)-α. As positive control, therapeutic protease inhibitor (saquinavir; 0.25 μg/mL) was used. Arrows indicate p55gag precursor protein and p24gag core protein (lower panel). RT activity was measured to correlate virus inhibition and compound activity (upper panel). cpm, counts per minute.
Matrix metalloproteinase (MMP) inhibitors do not affect human immunodeficiency virus type 1 (HIV-1) protease activity. OM10.1 cells were cultured with BB3103 (MMP inhibitor that blocks processing of cytokine precursors; 100, 10, and 1 μM) and MMP inhibitor I (100, 10, and 1 μM) for 2 h prior to addition of 20 U/mL tumor necrosis factor (TNF)-α. As positive control, therapeutic protease inhibitor (saquinavir; 0.25 μg/mL) was used. Arrows indicate p55gag precursor protein and p24gag core protein (lower panel). RT activity was measured to correlate virus inhibition and compound activity (upper panel). cpm, counts per minute.
Differential induction of MMP-9 by M. avium serotype 4 clinical isolate
Because T cells produce only MMP-2 and MMP-9 in response to immune activation, we analyzed culture supernatants for their production. M. avium isolates were cultured with CD8-depleted PBMC for 6 days, and supernatants were tested for the presence of MMP-2 and MMP-9 by use of ELISA and for their activity by use of zymography. Minimal amounts of MMP-2 were detected in day-4 and day-6 supernatants from PBMC infected with the clinical and environmental strains of M. avium (data not shown). However, the M. avium serotype 4 clinical isolate induced more MMP-9 (58.7 ± 10.5 ng/mL) than did the M. avium serotype 2 environmental isolate (9 ± 7.6 ng/mL) by day 6 of culture (figure 6A). Gelatin zymography, which measures the ability of MMPs to degrade gelatin in an acrylamide matrix, confirmed the differential induction of MMP-9 by the clinical M. avium isolate and showed that it was functional (figure 7). Furthermore, the disparate induction of MMP-9 correlated with the induction of HIV-1 replication by the clinical but not the environmental M. avium isolates (figure 6B).
Clinical Mycobacterium avium isolate induces matrix metalloproteinase (MMP)-9, which correlates with human immunodeficiency virus type 1 enhancement. CD8-depleted peripheral blood mononuclear cells were cultured with environmental (serotype 2) and clinical (serotype 4) M. avium isolates. A, MMP-9 production was measured in culture supernatants by ELISA according to manufacturer's suggestions. B, Virus expression was measured by ELISA for p24gag. Data presented are representative of 4 independent experiments.
Clinical Mycobacterium avium isolate induces matrix metalloproteinase (MMP)-9, which correlates with human immunodeficiency virus type 1 enhancement. CD8-depleted peripheral blood mononuclear cells were cultured with environmental (serotype 2) and clinical (serotype 4) M. avium isolates. A, MMP-9 production was measured in culture supernatants by ELISA according to manufacturer's suggestions. B, Virus expression was measured by ELISA for p24gag. Data presented are representative of 4 independent experiments.
Clinical Mycobacterium avium isolate differentially induces matrix metalloproteinase (MMP)-9. CD8-depleted peripheral blood mononuclear cells were cultured in serum-free medium with clinical or environmental M. avium isolates. Supernatants were collected every 2 days and passed through 0.45-μm filter. MMP activity was measured in supernatants by digesting gelatin in acrylamide gel matrix. Clear zones are indicative of 92-kDa MMP-9 enzyme. Sero 2 and sero 4, serotypes 2 and 4, respectively.
Clinical Mycobacterium avium isolate differentially induces matrix metalloproteinase (MMP)-9. CD8-depleted peripheral blood mononuclear cells were cultured in serum-free medium with clinical or environmental M. avium isolates. Supernatants were collected every 2 days and passed through 0.45-μm filter. MMP activity was measured in supernatants by digesting gelatin in acrylamide gel matrix. Clear zones are indicative of 92-kDa MMP-9 enzyme. Sero 2 and sero 4, serotypes 2 and 4, respectively.
Discussion
In this study, we analyzed the potentiating effects of mycobacteria on HIV-1 replication. We applied a model that does not use external stimulants, such as IL-2 or phytohemagglutinin-P, which would conceal the effects produced by the mycobacteria by driving HIV-1 replication through autocrine/paracrine cytokine production [4]. Instead, fetal bovine serum was used to prime the cells, which subsequently became susceptible to the suboptimal stimulation by the bacterial pathogen. CD8+ PBMC have been shown to affect HIV-1 infection in vitro, possibly through the production of suppressor factors [30]; therefore, elimination of CD8+ cells results in a reproducible HIV-1 infection. We demonstrate that 2 clinical M. avium isolates (serotypes 1 and 4) enhanced HIV-1 replication to a greater extent than did an environmental M. avium isolate (serotype 2), which paralleled the HIV-1 replication of the virus-only cultures. The enhancement of HIV-1 replication was shown to be independent of M. avium-induced cell proliferation, activation, and antigen processing.
Immune activation has a major impact on HIV replication, thus affecting the morbidity and mortality of HIV-1-infected persons. In vitro experiments have shown that the production of HIV-1 is dependent on the activation state of the target cell [31–34]. Activation of the immune system through vaccination has shown transient increases in HIV-1 production in vivo [35, 36]. A recent study showed that the onset of M. avium complex bacteremia induced a significant rise in HIV RNA levels [37]. However, a second report suggested that M. avium complex did not influence the HIV RNA levels but that increasing TNF-α levels for both cases and controls were associated with a generalized increase in HIV RNA levels in persons with advanced AIDS [38]. Furthermore, M. tuberculosis has been shown to enhance HIV-1 replication in blood monocytes [39, 40]. The ability of HIV-1 to enter the cell and the frequency of infected cells were similar in monocytes from M. tuberculosis patients and normal controls [40]. Collectively, these studies suggest that the enhanced replication may be a result of immune activation through cytokine modulation. Although these data imply that immune activation is important for enhanced HIV-1 replication, our data suggest that it may be required but is not sufficient since all examined M. avium strains induced similar cellular proliferation and activation, as evidenced through comparable stimulation indices and activation marker expression.
M. avium is characterized as a facultative intracellular pathogen and can survive and multiply within macrophages. Macrophages process the mycobacterial antigens and present them to T cells, which provide protective immune responses in healthy individuals [25, 26, 41, 42]. The precise mechanism of T cell activation is thought to occur through antigen presentation and monokine production; however, we show that M. avium can stimulate T cell proliferation independently of the presence of monocytes/macrophages. The T cell stimulation was not dependent upon factors actively produced by the myco-bacteria, because bacilli from the heat-killed and the viable serotype 4 strain induced similar levels of cell proliferation and HIV-1 replication. Moreover, while heat-killed M. avium serotype 2 bacilli did induce similar levels of cell proliferation, it did not enhance HIV-1 replication, which is a finding similar to that for the viable serotype 2 strain. These data imply that the factor(s) that enhance HIV-1 replication are distinctive for the serotype 4 M. avium strain and are heat stable.
Although the identity of the factor(s) responsible for the T cell activation is currently being investigated for M. avium, recent studies have shown that bacteria-associated protein components of M. tuberculosis and Mycobacterium fortuitum can induce proliferative responses from T cells [43, 44]. However, because both environmental and clinical M. avium strains induce proliferation, any mycobacterial components that induce T cell activation also will need to be evaluated in conjunction with HIV-1 enhancement.
Not only is HIV-1 replication up-regulated by cellular activation, it is also modulated by cytokines. Both acutely and chronically HIV-1-infected cell lines regulate HIV-1 replication through autocrine/paracrine cytokine production [45, 46]. Proinflammatory cytokines, such as IL-6, IL-1/3, and TNF-α, are potent inducers of HIV-1 replication in macrophages [47]. More recently, those proinflammatory cytokines, in addition to IFN-γ, were shown to be crucial for HIV-1 replication in primary PBMC [4]. Organisms responsible for several opportunistic infections in AIDS patients also have been shown to modulate HIV-1 replication through cytokine manipulation. Recently, P. falciparum has been shown to regulate HIV-1 replication through the induction of TNF-α in primary PBMC [21].
Salmonella typhimurium and Leishmania donovani also were shown to activate HIV-1 replication in a chronically infected cell line through the induction of TNF-α [48, 49]. However, our investigation into the modulation of cytokines has yielded no substantial differences in the cytokine profiles induced by any of the M. avium isolates. Furthermore, we were unable to attribute the HIV-1 enhancement to the production of TNF-α, as shown by the addition of anti-TNF-α MAb and pentoxifylline. Unlike the other in vitro models in which proinflammatory cytokines modulated HIV-1 replication, they did not play an important role in M. avium-induced HIV-1 replication. These differences in cytokine involvement may reflect the initial priming of PBMC in other model systems. Kinter et al. [4] used IL-2-primed PBMC and were able to recover HIV-1 by day 5 after infection. Our in vitro model required the addition of a second agent (e.g., clinical M. avium strains) to efficiently induce HIV-1 by day 5. These data indicate that cytokines do not have a central role in M. avium-induced HIV-1 enhancement, and, thus, other factors may contribute to M. avium-induced HIV-1 replication.
MMPs comprise a family of zinc-containing proteinases that are intimately involved in inflammatory processes (reviewed in [13]). MMPs are typically expressed as latent pro-MMPs that must be activated through limited proteolysis [50]. The same cells that secrete the MMPs also secrete TIMP-1 and TIMP-2 [51, 52]. The controlled regulation of the degradation of the extracellular matrix is dependent on the proper interplay between proteinase and inhibitor. Previously, MMP-9 has been shown to be modulated by the HIV-1 transactivating protein, Tat. Expression of MMP-9 promoted chemotactic and invasive behavior of the monocytes, possibly playing a role in tissue damage [16]. Recently, M. tuberculosis also has been shown to induce MMP-9 release from macrophages [53].
We show for the first time that the M. avium serotype 4 clinical isolate induces MMP-9 in CD8-depleted PBMC. Because broad-acting and specific in vivo inhibitors of MMPs blocked M. avium-induced HIV-1 replication and because M. avium clinical isolates differentially induce MMP expression, these data suggest that MMPs appear to have an active role in HIV-1 replication. Although we cannot conclusively confirm that the HIV replication is exclusively due to MMP-9, MMPs appear to be involved in the clinical M. avium-induced HIV replication, implying that physical interactions between bacterium and cell need to occur. Although we have examined a limited number of M. avium isolates, these data suggest that other pathogens that can alter MMP production may also induce HIV-replication through a similar pathway.
Acknowledgments
We gratefully acknowledge C. Robert Horsburgh and Salvator T. Butera for their critical review of this manuscript and Robin Moseley for editorial assistance.
