HIV-infected patients are at increased risk of developing active Mycobacterium tuberculosis (MTB) disease from reactivated latent infection or exogenous infection, and active MTB in HIV-infected patients has been shown to accelerate HIV disease progression.1-3 The immune background profile of individuals living in sub-Saharan Africa is characterized by chronic activation.4-7 In this respect, increased susceptibility to de novo infection with HIV and/or accelerated HIV disease progression in individuals from sub-Saharan Africa might be attributable to the chronic and persistent immune activation by ongoing immune responses to various endemic infections that prevail on the continent.7 It is worth noting that in patients with tuberculosis (TB), there is evidence of immune activation.8-11 In addition, the level of immune activation of peripheral monocytes from patients with active pulmonary TB is sufficient to enhance susceptibility to productive infection with HIV in vitro,12 and MTB (and its antigens) can upregulate HIV replication in vitro.10,13-17 In HIV-infected patients, mycobacteria also seem to augment HIV replication as measured in the peripheral circulation,10 lung,15,17-19 and lymphoid tissues,20,21 through mechanisms involving immune activation.10,21 It is interesting to note that increased expression of CCR5 and/or CXCR4, major chemokine HIV coreceptors, as well as their chemokine ligands, has been demonstrated in human monocyte-derived macrophages, alveolar macrophages, and CD4+ T cells in the course of in vivo and in vitro MTB infection,16,19,21-23 and the expression of these coreceptors seems to depend on the state of activation of the cells.24-26 Although the lung could be a preferential organ for HIV replication during active TB,15,17-19 it is worth noting that a significant increase (as high as 3- to 160-fold) in plasma HIV viremia has been observed during the acute phase of MTB disease,10,27 possibly related to mycobacteremia, which is frequently observed in HIV-coinfected patients.28 Indeed, CD4+ T cells, the principal target for HIV,29,30 contribute to more than 95% of the total virus production.31 In contrast to what has been observed in MTB disease in the Western world, where plasma HIV viremia often declines after successful TB treatment,10 few studies have shown that the HIV viral load remains unchanged during or after completion of successful anti-TB treatment in Africans.11,27,32,33 The pathogenesis of the persistently elevated plasma HIV viremia in patients coinfected with TB in Africa remains undefined, although several studies have suggested a role of the persistently activated immune system.11,33
In the present study, therefore, we studied the expression of chemokine receptors CCR5 and CXCR4 on CD4+ T cells and plasma chemokine levels of macrophage inflammatory protein (MIP)-1α, MIP-1β, regulated on activation normal T expressed and secreted (RANTES), and stromal cell-derived factor (SDF)-1α among TB patients with or without HIV coinfection during the first 2 months of anti-TB treatment. Our findings indicate that the persistently increased expression of chemokine receptors and chemokines may provide a potential mechanism of increased replication of HIV and may contribute to the persistence of HIV viremia observed in Africans despite anti-TB treatment.
PATIENTS AND METHODS
HIV-1-infected patients with TB (n = 21; CD4+ T-cell count range: 20-439 cells/μL, median = 149 cells/μL; plasma viremia range: 2200-840,000 copies/mL, median = 120,000 HIV-1 RNA copies/mL) were prospectively recruited and followed at 2 clinical centers (the All African Leprosy Research and Training Center [ALERT] and Higher 23 Health Center, both in Addis Ababa, Ethiopia). HIV-negative persons with TB only (n = 15; CD4+ T-cell count range: 215-837 cells/μL, median = 464 cells/μL) were also included as controls. Patients were enrolled after thorough clinical evaluation by primary clinicians at the respective health centers. The diagnosis of TB was made after demonstration of acid-fast bacilli in sputum samples confirmed by positive culture for MTB and radiologic or histologic evidence compatible with TB. Blood was obtained from each patient before or less than 7 days after initiation of anti-TB chemotherapy and during follow-up visits 2 months after enrollment. After TB diagnosis, all patients received directly observed therapy with isoniazid at a dose of 5 mg/kg of body weight (300 mg maximum), rifampicin at a dose of 10 mg/kg (600 mg maximum), pyrazinamide at a dose of 15 to 30 mg/kg (2 g maximum), and ethambutol at a dose of 15 to 25 mg/kg. All HIV-infected patients were antiretroviral naive, because the drugs were not available in Ethiopia at the time of the study. Informed consent was obtained from all study participants, and the study protocol has been reviewed and approved by institutional and national ethical clearance committees.
Fluorescence-Activated Cell Sorter Analysis
Cells were stained with the following antibody (Ab) combinations: CD3 (fluorescein isothiocyanate [FITC]) or CD8 (FITC) and CD4 (phycoerythrin [PE]) and CD45 (perdinin-chlorophyll-A protein [PerCP]), human leukocyte antigen D-related (HLA-DR; FITC) and 2D7 or 12G5 (PE) and CD4 (PerCP). PE-conjugated CCR5 (2D7) and CXCR4 (12G5) monoclonal antibodies (mAbs) were obtained from Pharmingen (La Jolla, CA). mAbs (FITC-, PE-, PerCP-conjugated) to CD3, CD4, CD8, CD45, and HLA-DR were obtained from Becton Dickinson (San Jose, CA). For each stain, thawed peripheral blood cells were incubated with appropriate mAbs for 30 minutes, after which stained cells were analyzed using a 3-color fluorescence-activated cell sorter scan (FACScan) with Cellquest software (Becton Dickinson). Expression of HLA-DR, CCR5, and CXCR4 on CD4+ T-cell subsets was analyzed by a combination of side scatter and setting of a gate around the CD4-PerCP-stained cells. At least 2500 live cells were acquired before the analysis was performed.
Plasma HIV-1 Viremia
Plasma HIV viremia was quantitated by using a nucleic acid-based amplification assay (NASBA; Organon Teknika, The Netherlands). HIV RNA concentrations below the detection limit of the NASBA assay (<80 copies/mL of plasma) were considered at 80 copies/mL.
Enzyme-Linked Immunosorbent Assay for Chemokines
The chemokines MIP-1α, MIP-1β, RANTES, and SDF-1α were measured by commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems) with limits of detection 10, 11, 8, and 18 pg/mL, respectively.
Means were compared using the paired Student t test. Median CD4 counts and plasma HIV levels were compared using the nonparametric Mann-Whitney U test. The individual changes in CD4 counts and plasma HIV load from baseline were compared using the nonparametric Wilcoxon signed rank test. Correlation was assessed by using the Spearman rank test. A P value of less than 0.05 was considered indicative of statistical significance. The statistical analysis was performed using the SPSS statistical package (SPSS, Chicago, IL).
Levels of Immune Activation, Expression of CCR5 and CXCR4 on CD4+ T-Cell Populations, and Plasma Chemokines
Because there is evidence for immune activation in HIV-infected patients with TB,8-11 we evaluated the state of activation of the CD4+ T cells as reflected by the expression of the cellular marker HLA-DR. As shown in Figure 1, the proportion of circulating CD4+ T cells expressing HLA-DR was significantly higher in TB/HIV-coinfected patients compared with patients with TB only (median [interquartile range (IQR)]: 47.1% [32.6-57.9] vs. 13.6% [11.7-19.3], respectively; P < 0.001).
In addition, CCR5 expression within a CD4+ T-cell subpopulation from TB/HIV-coinfected patients was increased significantly compared with patients with TB only (35.0% [22.4-45.8] vs. 3.3% [2.5-7.5], respectively; P < 0.001). Overall, a 10-fold increase in CCR5 expression was observed on CD4+ T-cell subsets from individuals coinfected with TB and HIV compared with individuals infected with TB only. Similarly, CXCR4-expressing CD4+ T cells were significantly increased in TB/HIV patients compared with patients with TB only (96.9% [94.4-98.7] vs. 71.7% [60.8-76.4], respectively; P < 0.001). The level of CD4+ T cells also expressing CXCR4 was significantly greater than that of CD4+ T cells expressing CCR5, regardless of the HIV status of the patients (see Fig. 1; P < 0.001).
Because the expression of chemokine receptors seems to depend on the state of activation of the cells,23-25 we further analyzed whether CCR5 and CXCR4 expression on CD4+ T cells correlated with cellular activation. As shown in Figure 2, the proportion of CCR5+ CD4+ T cells also expressing HLA-DR was significantly higher in cells from TB/HIV patients than in those from TB patients without HIV (20.6% [11.4-35.3] vs. 2.0% [1.3-3.8], respectively; P < 0.001). Similarly, the proportion of CXCR4+ CD4+ T cells also expressing HLA-DR was significantly higher in TB/HIV patients than in patients with TB only (48.4% [29.6-54.9] vs. 9.1% [6.7-10.8], respectively; P < 0.001). The proportion of activated CXCR4+ CD4+ T cells was higher than that of activated CCR5+ CD4+ T cells in both patient groups. In addition, the proportion of CD4+ T cells expressing CCR5 or CXCR5 positively correlated with the activation state of the cells, with Spearman correlation coefficients of 0.73 (P < 0.0001) for CD4+ T cells expressing CCR5 and 0.53 (P = 0.001) for CD4+ T cells expressing CXCR4 (Fig. 3). Moreover, the plasma levels of chemokines in TB/HIV-coinfected patients were not significantly different from those of patients with TB only (Fig. 4).
Levels of Cellular Activation, Chemokine Expression, and Plasma Chemokines During Antituberculosis Treatment in HIV-Coinfected Patients
Because efficient induction of HIV replication is initiated through immune activation,34 we evaluated changes in the level of expression of the activation marker HLA-DR, chemokine receptor expression on CD4+ T cells, and plasma chemokine levels. Patients with MTB disease were treated with anti-TB chemotherapy, and we evaluated the effect of anti-TB treatment during the initial 2 months on the virologic and immunologic parameters. Treatment of TB was associated with a significant increase in the absolute CD4 count only in those patients who were not coinfected with HIV (Table 1). There was no significant change in the CD4+ T-cell percentage or absolute cell count in the TB/HIV group, however. In addition, the plasma HIV load remained persistently elevated during anti-TB treatment. Although 24% of patients had a significant decrease in plasma HIV-1 viremia, 48% exhibited a significant increase (>0.5 log10) and 20% had no significant change during anti-TB treatment.
Lack of significant change in CD4 cell count and plasma HIV viremia also paralleled lack of significant change in the proportion of activated cells, chemokine receptor expression on CD4+ T cells, and plasma chemokine levels during anti-TB treatment. Thus, compared with the baseline levels, the percentage of CD4+ T cells also expressing HLA-DR remained sustained after 2 months of successful treatment of TB (see Fig. 1, 47.1% [32.6-57.9] vs. 49.9% [39.5-56.8], respectively; P = 0.281). Also, no significant change was noted in the levels of the CCR5- and CXCR4-expressing CD4+ T-cell population (see Fig. 1). The percentage of CCR5+ CD4+ T cells expressing the activation marker HLA-DR remained persistently elevated after 2 months of anti-TB treatment (see Fig. 2). Likewise, we observed no significant change with regard to plasma chemokine levels measured at baseline compared with 2 months after anti-TB treatment (see Fig. 4).
In this study, we noted that during anti-TB treatment, there was no significant increase in CD4 cell count among the TB/HIV-coinfected patients compared with patients who were not coinfected with HIV. These findings are similar to those reported by Morris et al.33 Poor CD4 T-cell restoration during TB treatment may be the consequence of uncontrolled HIV-1 replication.
In addition to the CD4 molecule,29,30 the chemokine receptors CCR5 and CXCR4 have been identified as major requisites for HIV-1 entry into CD4+ T cells and macrophages.35-38 The β-chemokine receptor CCR5 is the most important coreceptor used by macrophage (M)-tropic viruses, whereas the α-chemokine receptor CXCR4 is the dominant coreceptor for T-cell line (T)-tropic viruses.35-39 The expression pattern of the chemokine receptors on various cells is believed to have an influence on susceptibility, viral tropism, and HIV disease progression.39,40 It is interesting to note that there is ample evidence for immune activation in the course of active MTB disease.8-11 In addition, the expression of these receptors has been shown to be dependent on the state of activation of the target cells.24-26
Previous studies have demonstrated that there was an increase in CCR5 expression on mononuclear cells in the lung16,19 and lymphoid tissue21 from HIV patients coinfected with mycobacteria. CD4+ T cells latently infected with HIV are a major source of circulating viremia.31 Because mycobacteremia is also frequent in HIV-infected patients28 and MTB-derived antigens have been shown to induce chemokine receptor expression on immune cells in vitro16,22,23 and chemokines,16 we undertook the present study to evaluate the impact of anti-TB treatment in TB/HIV-coinfected persons. This study demonstrates that CCR5- and CXCR4-expressing CD4+ T cells were elevated in TB patients also coinfected with HIV. The findings from the present study concur with those reported previously.16,23 In the present study, in addition, we have shown that activated CD4+ T cells express high levels of CCR5 and CXCR4 and that the expression of the coreceptors and cellular activation paralleled each other. Susceptibility to infection with HIV is associated with levels of CCR5 expression,25,41 and MTB can activate CD4+ T cells to induce HIV replication.10 Thus, increased expression of chemokine receptors within the CD4+ T-cell subpopulation may, at least in part, explain the mechanistic pathway of increased HIV replication observed during coinfection with TB. In addition, dysregulation in the chemokines might play an important role in this process.
We have previously demonstrated that acute MTB infection is associated with significant increases in plasma HIV viremia.32 The plasma HIV load remained persistently elevated during anti-TB treatment, however. The data are consistent with those from prior studies in Africa of plasma HIV viremia during MTB disease,11,27,33 but differ from those of an earlier report10 that successful treatment of MTB in Western subjects is associated with a significant reduction in HIV-1 RNA levels. Lawn et al11 demonstrated that sustained plasma HIV viremia is associated with persistently elevated concentrations of tumor necrosis factor-α (TNFα) during anti-TB treatment. Morris et al33 also recently showed that the sustained plasma HIV-1 load during anti-TB treatment was associated with persistently elevated HLA-DR and CD38 in a CD8+ T-cell population. In the present study, we observed that the levels of CCR5 and CXCR4 expressed on CD4+ T cells remained persistently elevated and paralleled the level of cellular activation as well as chemokines during anti-TB treatment. The fact that plasma viremia declines after successful treatment of infections other than TB42-44 but remains persistently elevated during coinfection with TB indicates that TB-induced immune activation remains protracted and suggests that TB infection might modulate HIV-1-specific immune responses, which are not restored once TB is successfully treated. A similar observation has been noted in treated schistosomiasis patients coinfected with HIV in Uganda.45 One could argue that the absence of change in CD4 cell count, HIV viral load, and chemokine receptors and chemokines could be the result of ineffective anti-TB treatment. Only those patients infected with a sensitive M. tuberculosis isolate were included in this specific study, however.46
In Ethiopia, the predominant circulating HIV virus is subtype C.47-49 Although CXCR4 use by HIV-1 isolates often evolves during disease progression associated with the emergence of syncytium-inducing variants,50 this is not the case from our studies in Ethiopia.49 Indeed, in advanced HIV disease, CCR5 use by non-syncytium-inducing isolates was the predominant phenomenon observed, and the emergence of syncytium-inducing viruses that use CXCR4 was found to be rare for subtype C viruses.49,51 Moreover, Morris et al52 showed that R5 HIV-1 subtype C variants are preferentially recovered from patients with active TB. Taken together, the data suggest that increased expression of CCR5 on an activated CD4+ T-cell population may provide the availability of target cells for increased replication of HIV associated with TB coinfection.
These studies suggest that expression of chemokine receptors in the course of TB along with other components of the immune activation process may provide a background for more efficient availability of target cells for enhanced replication of HIV, and hence accelerated HIV disease progression, in coinfected individuals as well as increased susceptibility of TB positive/HIV negative patients after exposure to HIV-1. Although the introduction of highly active antiretroviral therapy (HAART) may alter the sequela of increased HIV replication in the course of MTB disease, in much of the developing world, where both HIV and TB prevail, the findings underscore the need for concomitant antiretroviral treatment with anti-TB treatment or an intensive first 2 months of anti-TB treatment followed by antiretroviral therapy. Moreover, more effective strategies for treatment of latent TB infection should be provided for HIV-coinfected persons.
The authors thank the study participants for their kind collaboration and the laboratory staff of the ENARP and TB laboratory of the EHNRI for their technical assistance.
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