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Viral Gene Expression in HIV Transgenic Mice Is Activated by Mycobacterium tuberculosis and Suppressed after Antimycobacterial Chemotherapy

  1. Charles A. Scanga1,
  2. Andre Bafica1 and
  3. Alan Sher1
  1. 1Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland
  1. Reprints or correspondence: Alan Sher, Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 50 South Dr., MSC 8003, Bethesda, MD 20892 (asher{at}niaid.nih.gov)
 
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Abstract

We used human immunodeficiency virus (HIV) transgenic (Tg) mice incorporating the entire viral genome to study the effect of Mycobacterium tuberculosis infection on the induction of integrated HIV gene expression. When aerogenically infected with M. tuberculosis these mice displayed a progressive increase in pulmonary gag and env mRNA levels and of p24 antigen production by cultured splenocytes. In situ hybridization of lungs revealed increased viral transcription associated with areas of inflammation and acid-fast bacilli. By neutralizing tumor necrosis factor (TNF) in vivo after M. tuberculosis exposure, we found that, although initially TNF independent, the increased HIV expression triggered by M. tuberculosis was highly dependent on this cytokine by 4 weeks after infection. Furthermore, treatment with antimycobacterial drugs markedly reduced HIV transgene expression. These studies establish an animal model for investigating the influence of M. tuberculosis on latent HIV expression and for testing therapeutic regimens for reducing the disease burden in patients with acquired immunodeficiency syndrome-associated tuberculosis

Of the 1.7 million people who died from tuberculosis in 2004, almost 250,000 were infected with HIV, and, in parts of sub-Saharan Africa, HIV is present in >50% of adults with tuberculosis [1]. HIV infection increases susceptibility to tuberculosis proportionally with the level of immune deficiency [2]. Clinical studies have also suggested that the progression of AIDS is accelerated by Mycobacterium tuberculosis coinfection [3, 4] and that tuberculosis is associated with marked increases in HIV levels [5–12]. Nevertheless, although some human studies have suggested that successful antituberculosis chemotherapy reduces plasma viremia [5], others have not [13, 14]

HIV can integrate into the DNA of memory CD4+ T cells and macrophages and create a long-lived latent viral reservoir that is refractory to antiviral therapy. In this postintegration state, viral gene expression is controlled by the long terminal repeat (LTR) of the viral genome, which can be transactivated by several cellular transcription factors [15]. Stimulation of cells latently infected with HIV can therefore trigger abundant viral gene expression and replication and accelerate progression to AIDS. In particular, macrophages have been implicated as a major source of latent HIV expression during coinfection with opportunistic pathogens [16, 17]. M. tuberculosis targets and replicates within macrophages and may therefore provide a particularly potent stimulus for this viral reservoir. Indeed, M. tuberculosis has been shown in numerous in vitro studies to promote HIV replication within infected macrophages, although the precise mechanism involved in this process of “immune activation” has not been completely identified [18]. Infected macrophages produce proinflammatory cytokines, such as tumor necrosis factor (TNF), that may stimulate the LTR in virally infected cells [19, 20]. Additionally, Toll-like receptor (TLR) signaling [21, 22] and the transcription factor CCAAT enhancer binding protein-β (CEBP-β) [12] have been postulated to play a role in mycobacterial-induced immune activation

One impediment to delineating the complex interactions that occur in individuals coinfected with HIV and M. tuberculosis is the lack of an animal model for both infections that mimics human disease and is amenable to immunologic experimentation. Nonhuman primates are used for the study of mycobacterial and retroviral coinfections [23–26], but these models are expensive and difficult to manipulate, and they use simian immunodeficiency virus as an HIV surrogate. Although mice can be infected with M. tuberculosis they cannot support active HIV infection unless reconstituted with human cells [27] or engineered to express human cyclin T1 [28]. Nevertheless, transgenic (Tg) mice that incorporate the HIV genome [29–31] have proved to be useful for certain types of studies and have been used successfully for assessment of the effects of microbial and other stimuli on the induction of latent viral expression. We have used the HM166 HIV Tg mouse strain, which incorporates the entire NL4-3 HIV clone, including the LTR [31], to characterize the induction of viral gene and protein expression by a number of pathogens—including Toxoplasmagondii [32], Plasmodium chabaudi [33], and M. avium [21, 34]—and to study the roles played by TLRs [21] and CD40 [35] in this process. In a related study, Browning Paul et al. [30], using a different HIV Tg mouse line containing the JR-CSF viral strain, demonstrated that intravenous infection with M. tuberculosis resulted in increased plasma viral RNA levels 2 weeks after administration of the pathogen

In the present article, we have extended these earlier observations and demonstrated a major effect of M. tuberculosis on viral gene and protein expression in HM166 HIV Tg mice during the course of infection initiated by the natural aerosol route. Furthermore, we show that this response can be markedly suppressed by either antibiotic therapy or TNF neutralizing antibodies. Our findings thus establish a murine model for investigating the influence of M. tuberculosis on latent HIV expression and for evaluating potential interventions that might be used to suppress the progression of disease in patients with AIDS-associated tuberculosis

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Materials and Methods

MiceThe HM166 HIV Tg mouse line was derived [31, 32] by pronuclear injection of FVB/N mouse embryos with a full-length clone of the NL4-3 HIV-1 strain. The mice were bred under specific pathogen-free conditions in an American Association of Laboratory Animal Care-accredited facility at the National Institutes of Health. Both male and female mice were used, and they were between 6 and 12 weeks old. Wild-type FVB/N mice were obtained from Taconic Farms

Mycobacterial infectionsFor in vivo infection, mice were placed in a nose-only aerosolization system (CH Technologies) and infected with 50–100 cfu of the virulent H37Rv M. tuberculosis strain. To assess mycobacterial load, lungs and spleens were harvested, homogenized in PBS that contained 0.05% Tween 20 (Sigma), plated onto 7H11 agar, and colonies enumerated 21 days later. For in vitro infections, M. tuberculosis was added at an MOI ∼2 to wells that contained macrophages. Intracellular mycobacteria were quantitated after 3 days by washing the monolayers with PBS, lysing the cells with 1% saponin (Sigma), and plating lysate dilutions onto 7H11 agar. Mycobacterial colonies were counted after 21 days

In vivo treatmentsTo neutralize TNF in vivo, mice received twice weekly intraperitoneal (ip) injections of 0.5 mg of MP6-XT22 monoclonal antibody. Control mice were injected with normal rat IgG. For antituberculosis chemotherapy, mice were treated with isoniazid and pyrazinamide (0.1 and 15 mg/mL, respectively; both from Sigma) dissolved in drinking water supplied ad libitem

Macrophage and spleen-cell cultureMice were injected ip with thioglycolate, and peritoneal exudate cells were harvested 3 days later. Cells were plated at 2.5×105 cells/well in a 96-well plate in Dulbecco’s modified Eagle medium (Invitrogen) that contained 10% fetal bovine serum, 2 mmol/L l-glutamine, 25 mmol/L HEPES, and 0.1 mmol/L nonessential amino acids. The next day, nonadherent cells were removed, and the adherent peritoneal macrophages were refed with fresh medium. Cells were primed overnight with 100 U/mL recombinant interferon (IFN)-γ (provided by Genentech) and then infected with M. tuberculosis as described above. For ex vivo splenocyte cultures, spleen cells from HIV Tg mice were prepared as described elsewhere [36] and seeded at 8×105 cells/well in round-bottom 96-well plates. TNF and HIV p24 in supernatants were measured by ELISA using commercial kits (TNF, R&D Systems; p24, Coulter). Reactive nitrogen intermediates were assayed using Greiss reagent

Measurement of HIV gene expression by real-time reversetranscription-polymerase chain reaction (RT-PCR)Total RNA was isolated from lung and spleen and reverse-transcribed as described elsewhere [36]. Real-time RT-PCR was performed on an ABI Prism 7900 device (Applied Biosystems) using SYBR Green PCR Master Mix (Applied Biosystems). Relative expression was determined by the comparative cycle-threshold method, in which each sample was normalized to hprt and expressed as a fold increase compared with uninfected controls. The following primer pairs were used: for hprt 5′-GTTGGTTACAGGCCAGACTTTGTTG-3′ (forward) and 5′-GAGGGTAGGCTGGCCTATAGGCT-3′ (reverse); for env 5′-GGGGACCAGGGAGAGCATT-3′ (forward) and 5′-TGGGTCCCCTCCTGAGGA-3′ (reverse); for gag 5′-CCAGATGAGAGAACCAAGGG-3′ (forward) and 5′-TTGTGAAGCTTGCTCGGCTCT-3′ (reverse); and for tnf 5′-AAAATTCGAGTGACAAGCCTGTAG-3′ (forward) and 5′-CCCTTGAAGAGAACCTGGGAGTAG-3′ (reverse)

In situ hybridizationTo simultaneously detect mycobacteria and HIV-expressing cells, in situ hybridization using an HIV-specific riboprobe was performed, followed by acid-fast staining, as described elsewhere [34], by Molecular Histology. Briefly, paraffin sections of lung were dewaxed, treated with protease, and hybridized with a 33P-labeled antisense probe corresponding to 9 kb of the HIV-1 genome. A sense probe was used as a control. After hybridization, slides were dipped in NTB 2 emulsion, exposed for 4 days, and developed in D-19 (Eastman Kodak). Autoradiograms were stained with Kinyoun's carbol fuchsin and carefully decolorized to preserve the silver grains

Statistical analysisNonparametric statistical analysis was done by 2-tailed, Mann-Whitney U test at 95% confidence intervals

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Results

Normal control of M. tuberculosis infection by HIV Tg miceGenetic background can play an important role in determining the outcome of murine infection with M. tuberculosis [37]. Because the susceptibility of the FVB/N strain used to make the HM166 HIV Tg mice had not been previously described and because it was possible that the transgene itself might influence host resistance to M. tuberculosis we first tested whether our HIV Tg mice could control aerosol infection with the virulent H37Rv bacterial strain. When mycobacterial loads were assessed in lung tissue (figure 1A), infected HIV Tg mice displayed bacterial growth kinetics typical of those seen in genetically resistant mouse strains [37] and showed 100% survival over the course of 12 months (data not shown). Moreover, after priming with IFN-γ, thioglycolate-elicited peritoneal macrophages from both HIV Tg and from wild-type FVB/N mouse strains were able to restrict the replication of M. tuberculosis (figure 1B) and produced comparable levels of TNF and of nitric oxide (figure 1C). These data confirmed that the FVB/N mouse displays normal control of bacterial growth and is suitable for studies of long-term infection and demonstrated that incorporation of the HIV transgene does not detectably impair macrophage responses to the pathogen

Figure 1
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Figure 1

Resistance of HIV transgenic (Tg) mice to Mycobacterium tuberculosis infection. A HIV Tg mice aerogenically infected with ∼50 cfu M. tuberculosis Lungs were removed from 3 mice per time point, homogenized in PBS/Tween, and plated onto 7H11 agar. Mycobacterial colonies were enumerated 3 weeks later. B Peritoneal macrophages from wild-type FVB/N or HIV Tg mice, left unprimed (black bars) or primed with interferon-γ (white bars) overnight and infected with M. tuberculosis (MOI, ∼2) for 3 days. Cells were then lysed and plated onto 7H11 agar for the enumeration of intracellular mycobacteria. C Supernatants from the same cell cultures, analyzed 3 days after infection by ELISA for concentrations of tumor necrosis factor (TNF) (black bars) and by Greiss assay for reactive nitrogen intermediates (NOx) (hatched bars) In panel A, each point is the mean ± SE of 3 mice and is representative of 2 experiments. Bars in panels B and C are means ± SDs and are representative of 3 experiments

Enhancement of HIV gene expression by infection with M. tuberculosis. We next examined the influence of aerosol infection with M. tuberculosis on viral gene expression in HIV Tg mice. To do so, we first measured HIV p24 production by cultured splenocytes at different time points after infection and compared this response with bacterial colony-forming units in the same organ. Infection with M. tuberculosis induced marked increases in ex vivo p24 production that were first detected at day 42 and appeared to progressively increase over the course of the next 100 days, even after bacterial loads in the same tissue reached a plateau (figure 2A). Increased HIV gene expression was also evident in vivo in the lungs of aerogenically infected mice. At both 21 and 50 days after infection, when substantial numbers of mycobacteria are present in lungs (figure 1A), the levels of both gag and env RNA as measured by real time RT-PCR were 15–30 times higher than the background expression of these genes observed in uninfected HIV Tg mice (figure 2B)

Figure 2
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Figure 2

Increased viral gene expression after aerosol infection of HIV transgenic (Tg) mice with Mycobacterium tuberculosis. A HIV Tg mice infected by aerosol with ∼50 cfu M. tuberculosis Splenocytes were obtained at the indicated times, and p24 levels in culture supernatants were assayed 3 days later by ELISA (white squares) Portions of spleens were also homogenized and plated on 7H11 agar for the determination of mycobacterial load (black circles) Each point is the mean of at least 3 mice, and bars are SDs (p24) or SEs (colony-forming units [cfu]). B RNA extracted from lungs of HIV Tg mice at the indicated times after aerosol M. tuberculosis infection. RNA was subjected to real-time reverse transcription-polymerase chain reaction using primers specific for the HIV gag (black bars) and env (hatched bars) genes. Data were corrected for hprt expression and are expressed relative to gene expression in lungs from uninfected HIV Tg mice. Bars represent the mean ± SD of at least 3 mice

To more precisely localize the sites of HIV activation in the lungs of HIV Tg mice after M. tuberculosis infection, lung tissue was subjected to in situ hybridization using a radiolabled antisense riboprobe derived from the HIV genome. In lungs of uninfected HIV Tg mice, widely scattered, diffuse hybridization foci were observed (figure 3A). In corresponding sections from M. tuberculosis-infected mice, these foci were more numerous and conspicuously more intense (figure 3B). When the latter tissues were reacted with a control sense riboprobe, only a few scattered silver grains were evident (not shown). A more detailed examination revealed that most of the HIV gene expression detected in lungs from M. tuberculosis-infected mice was associated with areas of granulomatous inflammation (figure 3B), and colocalization of silver grains with acid-fast bacilli was commonly observed in the sections (figure 3C). These HIV-specific in situ hybridization reactions were first detected in increased numbers at day 57 and, as expected, remained high 104 days after infection (figure 3D). These data clearly demonstrated that infection of HIV Tg mice with M. tuberculosis results in increased viral expression at sites of bacterial replication and established a model for studying the interaction of tuberculosis with latent HIV expression

Figure 3
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Figure 3

Increased pulmonary HIV RNA expression colocalizing with mycobacteria after Mycobacterium tuberculosis infection of HIV transgenic (Tg) mice. Representative lung sections from uninfected HIV Tg mice (A) and from HIV Tg mice 104 days after M. tuberculosis infection (B and C) were subjected to in situ hybridization using a HIV-specific antisense riboprobe (black dots) followed by acid-fast staining for the detection of mycobacteria (red rods) Original magnifications, ×40 (A and B) and ×100 (C). D Foci of silver grain accumulation enumerated in lung sections from HIV Tg mice at the indicated times after M. tuberculosis infection. Bars are means ± SDs of at least 3 mice per time point

Role played by TNF in M. tuberculosis-induced HIV expressionA major mechanism of immune activation of HIV involves the induction of viral LTR function by signaling pathways stimulated by cytokines such as TNF that are produced in response to immunization or coinfection. TNF is strongly induced by M. tuberculosis infection and, although it plays an important role in host resistance to that pathogen, it has also been implicated as a mediator of HIV induction in patients with tuberculosis [38, 39]. It was therefore of interest to determine whether the increase in HIV gene expression seen in M. tuberculosis-infected HIV Tg mice was dependent on bacterial-induced TNF. As predicted, aerogenic infection of HIV Tg mice resulted in increased and sustained tnf gene expression in lungs that closely resembled in its kinetics the induction of gag in the same tissues (figure 4). When TNF was depleted in vivo by the administration of neutralizing anti-TNF, pulmonic mycobacterial burdens were found to be significantly higher at both 2 and 4 weeks after infection (figure 5A), consistent with observations published elsewhere [40] and confirming the efficacy of the neutralization regimen. When HIV proviral expression in lungs was measured by real-time RT-PCR, the transcription of both gag and env viral genes was enhanced at 2 weeks after infection in the anti-TNF-treated mice at levels similar to those of mice that received control antibody (figure 5B). By contrast, by 4 weeks after infection, a major reduction in the expression of both viral genes was observed in mice that received anti-TNF (figure 5B). Significantly, this ablation occurred in the face of increased bacterial loads in the same mice (figure 5A). These findings suggested that M. tuberculosis induces HIV transgene expression by both TNF-dependent and -independent mechanisms, with the former playing a more substantial role later during infection

Figure 4
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Figure 4

Coordinate expression of tnf and HIV gag genes in lungs after Mycobacterium tuberculosis infection of HIV transgenic (Tg) mice. Lungs were removed from HIV Tg mice at the indicated times after aerosol M. tuberculosis infection, RNA was isolated and subjected to real-time reverse transcription-polymerase chain reaction using tnf- and HIV gag-specific primers. Data were corrected for hprt expression and expressed relative to gene expression in lungs from uninfected HIV Tg mice. Each point represents the mean ± SD of at least 3 mice

Figure 5
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Figure 5

Partial dependence of Mycobacterium tuberculosis-induced HIV gene expression on tumor necrosis factor (TNF). A HIV transgenic mice infected with M. tuberculosis and then given twice-weekly control (gray bars) or anti-TNF (black bars) monoclonal antibody (MAb) by intraperitoneal injection. At the indicated times, lungs were removed, homogenized, and plated onto 7H11 agar for the determination of mycobacterial burden. B RNA purified from lung samples and analysis of the expression of the HIV genes gag (top) and env (bottom) by real-time reverse transcription-polymerase chain reaction. Bars are the mean from at least 3 mice, and error bars are SEs (A) or SDs (B) Asterisks are statistically significant (P<.05) differences in message levels between anti-TNF- and control antibody-treated mice

Reduction in HIV gene expression after antimycobacterialchemotherapy As noted above, an important issue in the treatment of AIDS-associated tuberculosis concerns the effect of antituberculosis chemotherapy on the progression of HIV disease in dually infected individuals. To address this question experimentally, M. tuberculosis-infected HIV Tg mice were treated with isoniazid and pyrazinamide beginning at day 56 after aerosol exposure, and mycobacterial loads and HIV gene expression were monitored over time. As expected from previous studies [41], antibiotic treatment resulted in dramatically lower mycobacterial loads in both spleen (figure 6A) and lungs (figure 6B) of the infected mice and was followed by a rebound in colony-forming units in both organs after the cessation of chemotherapy on day 100. When ex vivo HIV p24 expression by cultured splenocytes was analyzed in the same drug-treated Tg mice, a marked reduction was observed that lagged behind the decrease in bacterial counts and persisted after the increase in mycobacterial load that occurred after antibiotic withdrawal (figure 6C). Similarly, a decrease in gag mRNA expression was observed in the lungs of mice treated with isoniazid and pyrazinamide, again without evidence of a significant rebound within the first 55 days after the cessation of chemotherapy (figure 6D). Given the important role played by TNF in stimulating HIV gene expression in M. tuberculosis-infected HIV Tg mice established in figure 5, we investigated whether a decrease in TNF levels paralleled the decrease in HIV p24 after antibiotic treatment. Indeed, splenocytes cultured from HIV Tg mice after mycobacterial infection produced increasing amounts of TNF that were significantly reduced when mice were treated with antimycobacterial drugs (figure 7). Together, these data confirmed the link among viral gene activity, mycobacterial load, and TNF in the Tg mouse model and suggested that antibiotic treatment of M. tuberculosis can have long-lasting effects on latent HIV expression

Figure 6
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Figure 6

Reduction in HIV gene expression after antituberculosis chemotherapy. HIV transgenic (Tg) mice were aerogenically infected with Mycobacterium tuberculosis and some mice were treated with isoniazid and pyrazinamide for 44 days beginning on day 56 of infection (antibiotic treatment period; gray bar). Mycobacterial burdens were measured in the spleens (A) and lungs (B) of untreated mice (white squares) and antibiotic-treated mice (black circles) at the indicated time points. C Splenocytes prepared from M. tuberculosis-infected HIV Tg mice that had been treated with antibiotics (black circles) or left untreated (white squares) HIV p24 was measured in supernatants 3 days later. D RNA extracted from lung tissue at the indicated days after M. tuberculosis infection of HIV Tg mice that had been treated with antibiotics (black bars) or left untreated (white bars) HIV gag expression was analyzed by real-time reverse transcription-polymerase chain reaction. Each point represents the mean ± SE of at least 3 mice (A and B) or SDs (C) and each bar is the mean ± SD of at least 3 mice (D) Asterisks are statistically significant (P<.05) differences in message levels between antibiotic-treated and untreated mice

Figure 7
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Figure 7

Induction of tumor necrosis factor (TNF) production by Mycobacterium tuberculosis infection and reduction after antituberculosis chemotherapy. Splenocytes were prepared from the same M. tuberculosis-infected HIV transgenic mice as those described in figure 6, and the concentration of TNF in the supernatant was measured 3 days later. Mice represented by white bars did not receive antibiotics; those represented by the black bar received a 4-week course of antimycobacterial drugs. Each bar depicts the mean of at least 3 mice ± SDs

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Discussion

An association between M. tuberculosis infection and the progression of AIDS has been noted in a number of clinical studies [3, 4], but the direct investigation of the in vivo mechanisms underlying this disease interaction has been hampered by the lack of a suitable experimental system amenable to immunologic dissection. Here, we have confirmed and extended the initial observation [30] that M. tuberculosis inoculation increases viral RNA levels in HIV Tg mice by developing an aerogenic M. tuberculosis infection model in HM166 HIV Tg mice. This work has allowed us to more thoroughly analyze the effects of this pathogen on proviral gene expression and to examine the mechanisms underlying that response

In accord with previous findings on HIV Tg mice intravenously infected with M. tuberculosis [30] or M. avium [34], HM166 Tg mice exposed to aerosol M. tuberculosis showed a progressive increase in HIV p24 production by splenocytes and enhanced viral mRNA expression in lungs that appeared to correlate, at least at earlier time points, with increased bacterial load. These findings mirror those of a clinical study in which the development of tuberculosis resulted in a significant increase in plasma viremia in a subset of prospectively monitored HIV patients [7]. Similarly, in agreement with in situ HIV localization experiments performed on tissue from patients with mycobacterial infections [12, 16, 42], the enhanced pulmonary HIV mRNA expression seen in M. tuberculosis-infected HIV Tg mice appeared to associate with granulomatous lesions containing numerous bacilli. Although it was not directly assessable with the in situ technology used here, it is likely that macrophages are the major cell type responsible for enhanced viral gene expression in response to M. tuberculosis given that this population was shown to be the primary source of HIV RNA after M. avium infection of the same HIV Tg mouse strain [34]. Human studies have also revealed macrophages to be a major viral reservoir from which opportunistic infections stimulate enhanced HIV replication [16, 42], although, to our knowledge, this issue has yet to be addressed in patients with tuberculosis

An important issue raised in previous research on AIDS-associated tuberculosis concerns the role played by TNF in driving HIV replication in individuals coinfected with M. tuberculosis Several studies have correlated M. tuberculosis-induced TNF with increased HIV expression in vitro [19, 20, 43], as well as in vivo [38, 39, 44–46]. Conversely, treatment of patients coinfected with HIV and M. tuberculosis with the TNF antagonist thalidomide [6] or the TNF receptor-Fc chimera etanercept [47] did not reduce viral loads. In the present article, we addressed the role played by M. tuberculosis-induced TNF in the induction of HIV by neutralizing the cytokine in HIV Tg mice throughout the course of aerogenic M. tuberculosis infection. Anti-TNF treatment did not reduce viral expression 2 weeks after infection, even though it resulted in an increased bacterial burden, which suggests that early viral transcription is independent of TNF. However, by 4 weeks after mycobacterial exposure, TNF depletion resulted in a major reduction in pulmonary HIV expression, despite the occurrence of a concomitant increase in bacterial load. Anti-TNF treatment did not totally abolish HIV transactivation, which suggests the continued contribution of TNF-independent signaling or incomplete TNF neutralization. Thus, the present results firmly support that TNF plays a major role in the regulation of HIV transcription during M. tuberculosis coinfection and provide an experimental model for investigating anti-TNF therapy as an adjunct in the treatment of AIDS-associated tuberculosis

The observation of TNF-independent HIV gene expression at early times after infection is consistent with previous studies that indicated that, in addition to TNF, other mycobacteria-induced signals can promote the induction of HIV. For example, mycobacteria possess potent ligands for several TLRs and trigger TLR-dependent immune responses in vivo [48, 49]. Indeed, M. tuberculosis products can transactivate the HIV LTR in a TLR2-dependent manner [22], and we have shown that TLR2 plays a major role in driving viral expression in HIV Tg mice infected with M. avium even though this pathway is not essential for TNF induction in these mice [21, 50]. Other HIV stimulatory pathways triggered by mycobacteria that might explain the early TNF-independent induction of HIV in our M. tuberculosis-infected HIV Tg mice include those involving the transcription factor CEBP-β [12]. The existence of these TNF-independent signaling pathways that trigger HIV gene expression may explain the failure of TNF antagonist therapies to significantly reduce HIV burdens in coinfected patients [6, 47]

A major finding of the present study is that antibiotic treatment of M. tuberculosis-infected HIV Tg mice results in significantly reduced viral mRNA and protein expression with an attendant decrease in TNF production. This issue has previously been addressed in several epidemiologic studies that had differing results. In one clinical investigation of 7 individuals coinfected with HIV and M. tuberculosis plasma HIV RNA levels decreased when antituberculosis treatment was successful but not when antibiotic therapy failed [5]. This is contrary to the results of 3 larger studies performed in distinct patient groups in areas of Africa where HIV is endemic in which tuberculosis treatment was not consistently associated with reduced HIV plasma loads [13, 14, 51]. These more recent reports documented a reduction in inflammation markers after antituberculosis treatment, including decreases in TNF receptor [51], C-reactive protein [51], interleukin-6 [14, 51], and neopterin [13] levels. However, each study also reported persistent immune activation after therapy, as evidenced by sustained TNF levels [51] and CD38 and HLA-DR expression by CD8+ lymphocytes [14]. This continued immune activation despite antimycobacterial treatment could reflect continued exposure to other pathogens in the African subjects studied—a situation that may contrast with that of the patients examined in the report by Goletti et al. [5]. The results presented here, in M. tuberculosis-infected HIV Tg mice bred under specific pathogen-free conditions, are consistent with the above hypothesis and establish a model for studying the effects of reducing the mycobacterial burden on HIV expression in a setting in which tuberculosis itself is the major proviral inducer

The global impact of AIDS-associated tuberculosis is enormous, yet our understanding of how M. tuberculosis affects HIV expression is restricted by the limitations of clinical studies and in vitro systems. We report here that M. tuberculosis infection of HIV Tg mice resembles human disease in several aspects and, thus, provides a manipulable in vivo model for the study of viral dynamics during tuberculosis. It is of interest that both modulation of TNF levels and antibiotic treatment were effective in reducing viral expression in this in vivo system, given that both approaches have previously been advocated on their own or in combination as clinical therapeutic modalities [47]. The tuberculosis/HIV Tg mouse model used here could therefore be used as a platform to establish conditions for these and for other intervention strategies and, at a more basic level, to better understand the immunologic mechanisms underlying the exacerbating influence of M. tuberculosis on HIV infection in vivo

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Acknowledgments

We thank Dr. Cecil Fox and his colleagues, for performing the in situ hybridization; and Drs. Carl G. Feng and Daniel L. Barber, for helpful discussions and critical review of the manuscript

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Footnotes

  • Presented in part: Keystone Symposia, Taos, New Mexico, 25–30 March 2004 (abstract 315)

    Potential conflicts of interest: none reported

    Financial support: National Institutes of Health Intramural AIDS Targeted Antiretroviral Program

  • Present affiliation: Aeras Global TB Vaccine Foundation, Rockville, Maryland

  • Received April 27, 2006.
  • Accepted August 26, 2006.
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  1. J Infect Dis. (2007) 195 (2): 246-254. doi: 10.1086/510244
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