tuberculosis Mycobacterium Apoptotic Response to Mediated ...

The Journal of Immunology

HIV Impairs TNF-␣ Mediated Macrophage Apoptotic Response to Mycobacterium tuberculosis1 Naimish R. Patel,2* Jinping Zhu,* Souvenir D. Tachado,* Jianmin Zhang,* Zhi Wan,† Jussi Saukkonen,† and Henry Koziel* The factors that contribute to the exceptionally high incidence of Mycobacterium tuberculosis (MTb) disease in HIVⴙ persons are poorly understood. Macrophage apoptosis represents a critical innate host cell response to control MTb infection and limit disease. In the current study, virulent live or irradiated MTb (iMTbRv) induced apoptosis of differentiated human U937 macrophages in vitro, in part dependent on TNF-␣. In contrast, apoptosis of differentiated HIVⴙ human U1 macrophages (HIVⴙ U937 subclone) was markedly reduced in response to iMTbRv and associated with significantly reduced TNF-␣ release, whereas apoptosis and TNF-␣ release were intact to TLR-independent stimuli. Furthermore, reduced macrophage apoptosis and TNF-␣ release were independent of MTb phagocytosis. Whereas surface expression of macrophage TLR2 and TLR4 was preserved, IL-1 receptor associated kinase-1 phosphorylation and NF-␬B nuclear translocation were reduced in HIVⴙ U1 macrophages in response to iMTbRv. These findings were confirmed using clinically relevant human alveolar macrophages (AM) from healthy persons and asymptomatic HIVⴙ persons at clinical risk for MTb infection. Furthermore, in vitro HIV infection of AM from healthy persons reduced both TNF-␣ release and AM apoptosis in response to iMTbRv. These data identify an intrinsic specific defect in a critical macrophage cellular response to MTb that may contribute to disease pathogenesis in HIVⴙ persons. The Journal of Immunology, 2007, 179: 6973– 6980.

A

pproximately 13 million people worldwide are coinfected with HIV and Mycobacterium tuberculosis (MTb)3 (1) with mortality as high as 20% in some areas (1). In contrast to other opportunistic infections, MTb infection occurs at a high rate in HIV⫹ subjects even in early clinical stages of HIV disease and with relatively preserved CD4⫹ T lymphocytes (2– 4). However, the molecular mechanism(s) accounting for the high rates of MTb infection and disease pathogenesis in the context of HIV infection are poorly understood. Alveolar macrophages (AMs) are critical components of an effective immune response to MTb (5, 6), and AM apoptosis represents a critical host defense mechanism to promote to MTb elimination (7, 8). Apoptosis reduces MTb viability in vitro (9 –11), and increased apoptosis is observed in vivo in bronchoalveolar lavage (BAL) specimens (12) and in lung tissue specimens (9)

*Division of Pulmonary, Critical Care and Sleep Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215; and †Boston Medical Center, Boston University School of Medicine, Boston, MA 02118 Received for publication May 21, 2007. Accepted for publication September 6, 2007. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institutes of Health Grants NIH R01HL063655 (to H.K.), NIH Loan Repayment Program (to N.P.), K08-AI064014 (to N.P.), and American Lung Association Biomedical Research Grant (to M.P.). These data were presented in part at the 2004 American Thoracic Society International Meeting, Orlando, FL, and the 2005 American Thoracic Society International Meeting, San Diego, CA. 2

Address correspondence and reprint requests to Dr. Naimish Patel, Pulmonary, Critical Care and Sleep Medicine, Beth Israel Deaconess Medical Center, Kirstein Hall, Room KSB-23, 330 Brookline Avenue, Boston, MA 02215. E-mail address: [email protected]

3 Abbreviations used in this paper: MTb, Mycobacterium tuberculosis; IRAK, IL-1 receptor associated kinase; AM, alveolar macrophage; BAL, bronchoalveolar lavage; MOI, multiplicity of infection; RFU, relative fluorescence unit; sTNFR1, soluble TNF receptor 1.

Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org

from MTb disease subjects. Virulent MTb isolates produce significantly less apoptosis than attenuated strains by inhibiting TNF-␣ mediated apoptotic signaling (13, 14). Although MTb avoids elimination and maintains persistence in part by arresting macrophage phagosomal acidification and maturation (5), activation of apoptotic pathways is associated with restoration of phagosomal maturation (15), which may promote MTb elimination. Macrophage apoptosis may also promote Ag presentation to T cells via MHC class I and CD1 by bystander dendritic cells (16). Thus, through multiple mechanisms, AM apoptosis results in effective MTb elimination, and specific defects in macrophage apoptotic pathways could contribute to MTb persistence and disease pathogenesis. AM can be infected with HIV in vitro (17) and in vivo (18), but unlike CD4⫹ T lymphocytes, HIV infection of macrophages does not result in cell death, but rather persistent HIV infection (19). Persistent HIV infection of macrophages may lead to targeted dysfunction of critical components of host defense function such as receptor-mediated phagocytosis, respiratory burst, TLR (TLR4) function, and NF-␬B signaling (20 –24), which may in part contribute to disease pathogenesis of opportunistic infections such as Pneumocystis pneumonia. Although only 1–10% of AMs are infected in vivo with HIV (18, 21), macrophages from HIV⫹ individuals are associated with impaired MTb-mediated release of MIP-1␣ and RANTES, which may in turn promote MTb growth (25). Recognizing that MTb disease occurs at high rates in HIV⫹ persons even with relatively preserved CD4⫹ T cell counts, dysfunction of other immune cells, such as macrophages, may contribute to MTb disease in HIV⫹ persons. Recognizing the importance of apoptosis in the host response to MTb challenge, the purpose of this study was to investigate macrophage apoptosis in response to virulent MTb in vitro, and to examine the influence of HIV infection on macrophage apoptosis. Studies focused entirely on human macrophages, first using differentiated human macrophage U937 cells and HIV⫹ U1 cells (HIV⫹ subclone of U937), and then results were confirmed using

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HIV IMPAIRS MTb-MEDIATED HUMAN ALVEOLAR MACROPHAGE APOPTOSIS

clinically relevant human AMs, comparing macrophages from healthy individuals to asymptomatic HIV⫹ persons at high clinical risk for MTb infection.

lyzed by flow cytometry. Cells that were Annexin V-FITC positive and propidium negative were considered to be in early apoptosis.

Materials and Methods

Adherent isolated macrophages (6 well plate, 3 ⫻ 106 cells/well) were incubated with MTb for 0 –120 min, macrophage nuclear extracts were prepared by using the NE-PER kit (Pierce) according to the manufacturer’s protocol, and ELISA specific for p65 was performed using the Mercury Transfactor p65 kit according to manufacturer’s protocol (Clontech). Protein loading was standardized using Bio-Rad Bradford assay.

Human macrophages Macrophage cell lines. Initial experiments used human promonocytic U937 (American Tissue Cell Company, ATCC) and HIV-infected U1 cell lines (AIDS Research and Reference Reagent Program, Bethesda, MD). U1 cells (HIV infected subclone of U937) contain one integrated copy of HIV-1 proviral DNA, and are characterized by low levels of constitutive viral expression (26) that can be modulated with specific cytokines and PMA (27). Cells were cultured in complete RPMI 1640 medium (10% heat-inactivated FCS, 2 mM glutamine, 100 U/ml penicillin, 100 ␮g/ml streptomycin), except for experiments using live mycobacteria where ceftriaxone (1 ␮g/ml) was substituted for streptomycin. Cells were harvested during exponential growth phase, washed, and then differentiated into macrophages using PMA (100 nM) at 37°C in with 5% CO2 for 24 h, washed three times with PBS, and incubated an additional 24 h before use. Human AMs. Prospectively recruited healthy and asymptomatic HIV-seropositive (HIV⫹) individuals were without evidence for active pulmonary disease and had normal spirometry. Healthy individuals were without known risk factors for HIV infection and were confirmed to be HIV seronegative by ELISA, which was performed according to the instructions of the manufacturer (Abbott Diagnostics). Because of a high prevalence of smokers in our HIV⫹ population (approximately half are smokers), healthy control subjects were matched for smoking. Asymptomatic HIV⫹ subjects had a CD4 T cell count of ⬎200 cells/mm3, serum viral load ⬎10,000 HIV-1 RNA copies/ml, were not on HAART therapy, and had no history of opportunistic infections. Lung immune cells were obtained by BAL using standard technique (22). All procedures were performed on adults after informed consent following protocols approved by the Beth Israel Deaconess Medical Center Institutional Review Board. The cells were separated from the pooled BAL fluid and AMs were isolated by adherence for ⱖ72 h to plastic-bottom tissue culture plates as previously described (22). Isolation of AM from all healthy and HIV⫹ persons yielded cells which were ⱖ98% viable as determined by trypan blue dye exclusion and demonstrated ⬎95% positive nonspecific esterase staining.

In vitro HIV infection To assess the direct effect of HIV-1 infection on macrophage apoptotic response, AM from healthy individuals were infected in vitro as previously described (22), using a monocytotropic (R5) isolate (HIV Bal). Productive HIV-1 infection was verified by the measurement of HIV p24 Ag in the culture supernatants by ELISA (Dupont).

Microbial organisms and reagents Virulent (H37Rv) mycobacteria inactivated via irradiation and mycobacterial lipoarabinomannan (Aralam) were obtained from J. Belisle (Colorado State University, Fort Collins, CO) and the National Institute of Allergy and Infectious Diseases Tuberculosis Research materials contract N01-AI75320. Stocks of live virulent (H37Rv) and attenuated (H37Ra) isolates were created using published protocols (25). Stocks were thawed, vortexed, and sonicated using a bath sonicator for 15 s at 500 W and allowed to stand for 10 min, and the upper 200 ␮l of solution were used for experiments (25). Pneumocystis organisms were obtained from Pneumocystis infected rat model (21).

Macrophage apoptosis measurements Macrophage apoptosis was determined by two independent assays: 1) ELISA apoptosis assay. Adherent macrophages in 96-well microtiter trays (4 –10 ⫻ 104 cells/well) were incubated with H37Ra, H37Rv, or iH37Rv MTb isolates (multiplicity of infection, MOI 10:1) for 1–5 days, and apoptosis was measured on cell lysates using an Ag-capture ELISA for histone and fragmented DNA (Cell Death Detection ELISA Plus, Roche Applied Science) according to the manufacturer’s protocol. Staurosporine (protein kinase C inhibitor; 5 ␮M) was used as a positive control for apoptosis, and for select experiments, TNF-␣ neutralizing Abs or isotype controls (mAb 210, R&D Systems) were added to culture supernatants to neutralize TNF-␣ bioactivity before addition of MTb. 2) Annexin V Apoptosis assay. Differentiated macrophages in 24-well low-binding plate (5 ⫻ 105 cell/well, Costar) were washed and incubated an additional 24 h. Cells were then incubated with MTb (MOI 10:1) or staurosporine (5 ␮M) for 24 h. Cell suspensions were labeled with Annexin V-FITC and propidium iodide according to manufacturer’s protocol (BD Pharmingen), and ana-

NF-␬B ELISA

Cytokine detection in cultured supernatants by ELISA Isolated adherent macrophages (24-well plate, 5 ⫻ 105 cells/well) are incubated with MTb isolates (MOI 10:1) LPS, or okadaic acid (0.05 ␮g/ml) for 24 h at 37°C in humidified 5% CO2. Culture supernatants were harvested and centrifuged to remove cellular debris, and aliquots are stored at – 80°C until assayed. Specific immunoreactivity to TNF-␣ (R&D Systems) was measured by ELISA, as described previously (24).

Cell-free BAL soluble receptor protein measurements Archived samples of cell-free BAL fluid from a mixed population of HIV⫹ subjects (all asymptomatic with CD4⫹ T cell count ⬎200 cell/ml, and a majority on HAART therapy) and healthy subjects (matched for smoking) were used to measure levels of soluble TNFR1 and soluble TNFR2 by ELISA. Soluble receptor measurements were normalized for BALassociated dilution using urea nitrogen measurements as previously described (18, 28).

RNA isolation and RT-PCR Total RNA was isolated from macrophages after 6 h incubation and RTPCR was performed according to the manufacturer’s protocol Thermoscript PCR system (Invitrogen Life Technologies) as previously described (24). The following primer was used for TNF-␣ RT-PCR: 5-AGC CCA TGT TGT AGC AAA CC-3 and 5 -GGA AGA CCC CTC CCA GAT AG-3.

Western blotting Cell cytoplasmic protein extracts were prepared using standard ice-cold RIPA buffer with protease and phosphatase inhibitors. Western blotting was performed utilizing a standard protocol (21) with Abs specific to phospho-IL-1 receptor associated kinase (IRAK) (Thr209) and total IRAK-1 protein (Cell Signaling Technology).

Flow cytometry surface receptor analysis Human TLR2 and TLR4 expression was measured via surface Ab labeling (mAb TL2.1 and HTA125, Santa Cruz Biotechnology) in macrophage cell suspensions with an Epics XL flow cytometer (Beckman Coulter) as previously published (24). Results were recorded as mean relative fluorescence units (RFU) and the percentage of the population staining positive.

Phagocytosis analysis Irradiated MTb and Pneumocystis were labeled with FITC (Sigma-Aldrich) according to the previously published protocol (29). Fluorescent microscopy was performed on isolated adherent macrophages on glass coverslips as previously published (22). Flow cytometric phagocytic analysis was performed by culturing isolated differentiated macrophages (5 ⫻ 105 cells/ well) in low binding 24-well plates (Costar), and then incubating with FITC-MTb or FITC-Pneumocystis for 1 h. Flow cytometry was performed before and after quenching of extracellular FITC signal with trypan blue.

Statistical methods All data was analyzed using nonparametric methodology (Mann-Whitney U test), and a p ⬍ 0.05 was considered to be significant. For ELISA data, cytokine protein levels or absolute OD was compared. For flow cytometry analysis RFU were compared. Experiments were repeated a minimum of three times.

Results Reduced MTb-mediated apoptosis in HIV-infected human macrophages MTb, including live virulent H37Rv (MTbRv), irradiated H37Rv (iMTbRv), and live avirulent H37Ra MTb (MTbRa), induced apoptosis of U937 macrophages by 24 h as determined by ELISA


6975 (Fig. 1A). In contrast, apoptosis was markedly reduced in HIV⫹ U1 macrophages in response to each MTb isolate. Importantly, apoptosis in response to staurosporine (nonspecific proapoptotic agent (30)) was similar comparing U937 and HIV⫹ U1 macrophages. As an independent measure of apoptosis, Annexin V staining measurement using flow cytometry (Fig. 1B) confirmed reduced MTb-mediated apoptosis in HIV⫹ U1 macrophages. Reduced TNF-␣ release by HIV-infected human macrophages in response to MTb

FIGURE 1. Reduced macrophage apoptosis of HIV-infected human macrophages in response to virulent MTb. A, Adherent differentiated human macrophage U937 and HIV⫹ U1 cells were incubated with irradiated virulent H37Rv MTb (iMTbRv), live virulent H37Rv MTb (MTbRv), or live attenuated H37Ra MTb (MTbRa) at MOI 10:1, or staurosporine (SS; 5 ␮M) for 24 h, and macrophage apoptosis was measured by ELISA. Data are shown as percentage change in OD compared with unstimulated conditions (mean ⫾ SEM; n ⫽ 3). B, Flow cytometry determination of apoptosis by Annexin V staining. Differentiated human macrophage U937 and HIV⫹ U1 cells were incubated with iMTbRv for 24 h. Following Annexin V-FITC and propidium iodide labeling, cells were analyzed by flow cytometry. For each histogram panel of U937 and U1 cells, data demonstrate Annexin V positive staining of PI-negative cell population (right tracing) compared with unstained cells (left tracing). Quantitative analysis of flow cytometry data compare unstimulated (Unstim) to iMTbRv stimulated U937 and U1 cells. Data are presented as relative fluorescence units (RFU; mean ⫾ SEM) (n ⫽ 3). ⴱ, p ⬍ 0.05.

Recognizing the important role for TNF-␣ in the induction of cellular apoptosis (13, 14), the addition of neutralizing anti-TNF Abs to U937 human macrophages reduced apoptosis in response to each of the MTb isolates whereas isotype control Abs did not influence MTb-mediated apoptosis (Fig. 2A). Each of the MTb isolates increased TNF-␣ release by U937 human macrophages significantly by 24 h compared with unstimulated macrophages (Fig. 2B). In contrast, release of TNF-␣ by HIV⫹ U1 macrophages was significantly lower in response to each of the MTb isolates (Fig. 2B). Importantly, in response to okadaic acid, a general cellular phosphatase inhibitor (31), TNF-␣ release was similar. Furthermore, induction of TNF-␣ mRNA transcripts was significantly lower in HIV⫹ U1 macrophages in response to iMTbRv compared with U937 macrophages (Fig. 2C). Next, to further establish an important role for TNF-␣ in apoptosis of HIV-infected macrophages, HIV⫹ U1 macrophages were incubated with iMTbRv in the presence and absence of modulators of TNF-␣ activity. As above, iMTbRv failed to induce significant apoptosis of HIV⫹ U1 macrophages. However, addition of exogenous recombinant human TNF-␣, or pharmacological stimulation of endogenous macrophage TNF-␣ release with okadaic acid (24)

FIGURE 2. TNF-␣ release and the role of TNF-␣ in MTb-mediated macrophage apoptosis. A, MTb-mediated macrophage apoptosis is dependent on TNF-␣. Differentiated U937 macrophages were incubated with MTb isolates in the presence and absence of specific neutralizing Ab to TNF-␣ (anti-TNF; mAb 210, 5 ␮g/ml) or isotype control (isotype), and after 24 h, apoptosis was determined by ELISA. Data are expressed as percent change in apoptosis compared with unstimulated macrophages (n ⫽ 3). B, Reduced MTb-mediated TNF-␣ release by HIV⫹ human macrophages. Differentiated human U937 and HIV⫹ U1 macrophages were incubated with individual MTb isolates at a multiplicity of 10:1 for 24 h, in the presence and absence of a general cellular phosphatase inhibitor, okadaic acid (OA; 50 ng/ml). Cell-free culture supernatants were assayed for TNF-␣ by ELISA. C, Reduced TNF-␣ mRNA transcripts in HIV⫹ macrophages. Differentiated U937 and HIV⫹ U1 macrophages were incubated for 6 h in the presence or absence of irradiated virulent MTb (iMTbRv). Specific TNF-␣ mRNA was detected by RT-PCR, with ␤-actin as an internal control for mRNA loading. Representative gel of three experiments providing identical results. D, Recovery of apoptosis in HIV⫹ macrophages by exogenous TNF-␣ or endogenous macrophage TNF-␣ release. Differentiated HIV⫹ U1 macrophages were incubated with irradiated virulent H37Rv MTb (iMTbRv) in the presence or absence of exogenous recombinant human TNF-␣ (100 ng/ml), okadaic acid (OA; 50 ng/ml), or neutralizing anti-TNF Ab (mAb 210) for 24 h at 37°C. Apoptosis was determined by Cell Death ELISA. Data is shown as percentage change in OD from unstimulated condition. All quantitative data values represent mean ⫾ SEM. ⴱ, p ⬍ 0.05 compared with iMTbRv alone; ⴱⴱ, p ⬍ 0.05 compared with iMTbRv ⫹ OA.

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FIGURE 3. Preserved TLR2 and TLR4 surface expression, but impaired TLR-mediated TNF-␣ release and signaling in HIV⫹ human macrophages. A, Differentiated U937 and HIV⫹ U1 macrophages incubated on low-binding tissue culture plates were stained with PE-labeled receptor Ab, and cell surface fluorescence was measured by flow cytometry as described in Materials and Methods. Receptor density was measured as RFU. For each histogram panel, the right-sided tracing represents anti-TLR labeling, and the left-sided tracing represent isotype control Ab labeling. Representative flow cytometry histograms of three independent experiments with similar results. Specific TLR2 agonist (Aralam 1 ␮g/ml) and TLR4 agonist (Lipid A 0.1 ␮g/ml) show decrease TNF-␣ release at 24 h in differentiated HIV⫹ U1 cells compared with U937 cells. B, Reduced NF-␬B nuclear translocation in HIV⫹ U1 macrophages in response to virulent MTb. Differentiated human U937 and HIV⫹ U1 macrophages were incubated with irradiated virulent MTb (iMTbRv; MOI 10:1) for 0 –120 min. NF-␬B nuclear translocation in nuclear extracts was measured by ELISA. Date are expressed as mean OD ⫾ SEM. C, Reduced IRAK phosphorylation in HIV⫹ U1 macrophages in response to virulent MTb. Differentiated human U937 and HIV⫹ U1 macrophages were incubated with irradiated virulent MTb (iMTbRv; MOI 10:1) for 0 –20 min or iMTbRv plus okadaic acid (OA, 50 ng/ml). Cellular lysates were resolved by Western blot using specific Abs to IRAK and phosphorylated IRAK (p-IRAK). Representative blot of three independent experiments with similar results.

significantly induced macrophage apoptosis in response to virulent MTb (Fig. 2D). The effect of stimulating endogenous TNF-␣ release on apoptosis was abrogated in the presence of neutralizing anti-TNF-␣ Abs (Fig. 2D), supporting a central role for TNF-␣. Taken together, these data demonstrate that MTb-induced U937 apoptosis was dependent in part on TNF-␣, and that reduced MTbmediated apoptosis in HIV⫹ U1 macrophages was associated with reduced TNF-␣ release which could in part be rescued by exogenous TNF-␣ or stimulation of endogenous TNF-␣ release.

in a time dependent increase in phosphorylated IRAK (Fig. 3C). In contrast, no significant IRAK phosphorylation was observed in HIV⫹ U1 cells in response to MTb. Recognizing that okadaic acid can restore IRAK activity in LPS-tolerant macrophages (38), incubation of

Preserved TLR expression but impaired TLR-mediated signaling in HIV-infected human macrophages TLR2 and TLR4 mediate proinflammatory cytokine release (including TNF-␣) in response to MTb (32). Surface expression of TLR2 and TLR4 were similar comparing U937 to HIV⫹ U1 macrophages (Fig. 3A), suggesting that differences in TNF-␣ release were not associated with loss or absence of surface TLR2 or TLR4. However, TNF-␣ release in HIV⫹ U1 cells in response to the TLR2 agonist mycobacterial cell wall glycolipid lipoarabinomannan (Aralam) (32) or the TLR4 agonist Lipid A) (33) was markedly lower compared with U937 cells, suggesting that TLR signaling may be altered. Recognizing the importance of NF-␬B in regulating TNF-␣ release (34), and the importance of NF-␬B in TLR signaling (35), iMTbRv induced NF-␬B nuclear translocation in a time dependent manner (Fig. 3B) in U937 macrophages. In contrast, iMTbRv-mediated NF-␬B nuclear translocation was significantly lower in HIV⫹ U1 macrophages over the same time course. Experiments next measured IRAK phosphorylation, an important step in TLR-mediated NF-␬B activation (35, 36). Autophosphorylation at threonine 209 is a necessary early event in the activation of IRAK (37). Incubation of U937 cells with MTb resulted

FIGURE 4. Specific enhanced phagocytosis of virulent MTb by HIV⫹ U1 human macrophages. Differentiated human U937 and HIV⫹ U1 macrophages were incubated with either FITC-labeled irradiated virulent MTb (FITC-iMTb) or FITC-labeled Pneumocystis (FITC-Pc) organisms for 60 min. Panels provide representative microscopic fields by Nomarski or epiflourescence methods (FITC-labeled microbes viewed as apple-green color; macrophages appear faint yellow-green by autofluorescence). Phagocytosis was quantified by flow cytometry, and data are expressed as the Phagocytic Index in RFU as described in Materials and Methods. All quantitative data values represent mean ⫾ SEM. ⴱ, p ⬍ 0.05.


FIGURE 5. Reduced MTb-mediated apoptosis of human alveolar macrophages from HIV⫹ persons or alveolar macrophages from healthy persons following in vitro HIV infection. Isolated human alveolar macrophages (AMs) from healthy persons, (Healthy), asymptomatic HIV⫹ persons (HIV⫹), or healthy persons following in vitro HIV infection (in vitro HIV) were incubated with increasing MOI of virulent irradiated MTb (iMTbRv) for 5 days at 37°C. Positive control for TLR-independent apoptosis included incubation of AM with staurosporine (SS; 5 ␮M) for 24 h. Apoptosis was measured by ELISA. Data are presented as mean OD ⫾ SEM. (n ⫽ 3 subjects for each group) ⴱ, p ⬍ 0.05 compared with healthy alveolar macrophages.

HIV⫹ U1 macrophages with okadaic acid induced phosphorylation of IRAK (Fig. 3C), demonstrating that intrinsic IRAK activity was not globally impaired in HIV⫹ macrophages. Increased MTb phagocytosis by HIV⫹ human macrophages Recognizing that phagocytic receptors participate in or modulate TLR signaling (39 – 43), experiments next measured MTb

6977 phagocytosis. Qualitative assessment demonstrated that FITClabeled MTb was phagocytosed by U937 and HIV⫹ U1 macrophages (Fig. 4). Quantitative assessment demonstrated that MTb phagocytosis was significantly greater in HIV⫹ U1 macrophages (Fig. 4). In contrast, phagocytosis of the opportunistic pathogen Pneumocystis was lower in HIV⫹ U1 cells compared with U937 macrophages suggesting that the increased MTb phagocytosis was pathogen specific (Fig. 4). These data suggest that decreased TNF-␣ release in HIV⫹ U1 cells was independent of phagocytosis, as MTb phagocytosis was higher in HIV⫹ U1 macrophages. Reduced apoptosis in AM from HIV⫹ persons in response to MTb To determine the clinical relevance of the results using cell lines, experiments were performed comparing primary human AM from healthy individuals to asymptomatic HIV⫹ persons (not currently on HAART therapy) who are at elevated clinical risk for MTb infection. Similar to experiments with human macrophage cell lines, virulent MTb induced apoptosis in AM from healthy individuals, but MTb-induced macrophage apoptosis was significantly lower in AM from asymptomatic HIV⫹ persons (Fig. 5). Importantly, in vitro HIV infection of AM from healthy persons significantly impaired apoptosis similar to that observed for AM from HIV⫹ persons (Fig. 5). Importantly, macrophage apoptosis was induced equally in all three macrophage populations in response to staurosporine, indicating that apoptosis pathways were not globally disrupted in HIV⫹ AM.

FIGURE 6. Reduced MTb-mediated TNF-␣ release in human alveolar macrophages from HIV⫹ persons or alveolar macrophages from healthy persons following in vitro HIV infection. A, Isolated human alveolar macrophages (AMs) from healthy persons (Healthy; n ⫽ 3) and asymptomatic HIV⫹ persons (HIV⫹; n ⫽ 3) were incubated with increasing MOI of virulent irradiated MTb (iMTbRv), LPS (1000 ng/ml), or okadaic acid (OA; 100 ng/ml) for 24 h. Cell-free culture supernatants were harvested and TNF-␣ was measured by ELISA. B, Impaired IRAK phosphorylation of human AM from HIV⫹ persons in response to virulent MTb. Adherent human AM from healthy (Healthy) or asymptomatic HIV⫹ persons (HIV⫹) were incubated with virulent MTb for 0 or 20 min, and the cell lysates were resolved by SDS-PAGE and Western blot for total IRAK and phosphorylated IRAK (p-IRAK). Representative Western blot of at least three experiments performed on AM from each group. C, In vitro HIV infection of AM from healthy persons reduced MTb-mediated TNF-␣ release. Isolated human AM from healthy persons (Healthy; n ⫽ 3) and AM from healthy persons following in vitro HIV infection (in vitro HIV; n ⫽ 3) were incubated with virulent irradiated MTb (iMTbRv; MOI of 10:1) for 5 days, or LPS (1000 ng/ml), or okadaic acid (OA; 100 ng/ml) for 24 h at 37°C. Cell-free culture supernatants were harvested and TNF-␣ was measured by ELISA. D, Elevated levels of sTNFR1 and sTNFR2 in BAL fluid from HIV⫹ persons. Archived cell-free BAL samples from healthy (n ⫽ 15) and asymptomatic HIV⫹ persons (n ⫽ 25) were assayed for sTNFR1 and sTNFR2, by ELISA. Measurements were normalized for BAL dilution as described in Materials and Methods. Data expressed as mean values ⫾ SEM. ⴱ, p ⬍ 0.05 compared with healthy AM.

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Reduced TNF-␣ release in HIV⫹ human AMs in response to MTb Similar to experiments with human macrophage cell lines, virulent MTb induced TNF-␣ release in primary AM from healthy individuals, but TNF-␣ release was significantly lower in AM from asymptomatic HIV⫹ persons (Fig. 6A). Importantly, TNF-␣ release was preserved in AM from asymptomatic HIV⫹ persons in response to MTb-independent stimuli LPS and okadaic acid (Fig. 6A). Similar to results comparing U937 to HIV⫹ U1 cells, MTb-mediated IRAK phosphorylation was also impaired in AM from HIV⫹ persons (Fig. 6B). Importantly, in vitro HIV infection of AM from healthy persons impaired TNF-␣ release in response to iMTbRv (Fig. 6C) whereas response to okadaic acid or LPS was preserved suggesting a targeted and direct effect of HIV on macrophage response to MTb. To identify other factors that may influence TNF-␣ activity in the lungs, experiments measured levels of soluble TNF receptor 1 (sTNFR1) and sTNFR2 in BAL fluid from healthy and HIV⫹ persons. Prior studies demonstrated that sTNFR1 and sTNFR2 can neutralize TNF-␣-mediated cytotoxicity and apoptosis (44), and sTNFR2 in particular may play a role in down regulating virulent H37Rv-mediated apoptosis of AM (14). In the current study, levels of sTNFR1 and sTNFR2 in cell-free BAL fluid samples from asymptomatic HIV⫹ persons were significantly higher compared with healthy individuals (Fig. 6D).

Discussion These data demonstrated that HIV infection significantly reduced macrophage apoptosis in response to virulent MTb, and reduced apoptosis was associated with reduced MTb-mediated TNF-␣ release in HIV⫹ macrophages. Furthermore, whereas expression of major host defense molecules TLR2 and TLR4 was similar in healthy and HIV⫹ macrophages, TNF-␣ release in response to specific TLR agonists, intracellular signaling through IRAK-1, and NF-␬B nuclear translocation was significantly reduced in HIV⫹ macrophages. These abnormalities were specific, as apoptosis, TNF-␣ release, and TLR-mediated intracellular signaling were preserved in response to MTb-independent stimuli, and apoptosis was independent of MTb phagocytosis. Importantly, induction of TNF-␣ restored MTb-mediated apoptosis in HIV⫹ macrophages. Similar findings were observed using clinically relevant AM from healthy and HIV⫹ individuals at high clinical risk for MTb infection. Furthermore, in vitro HIV infection of AM from healthy individuals was sufficient to reduce MTb-mediated apoptosis and TNF-␣ release. The observed reduction in MTb-mediated apoptosis was independent of other immune cells such as CD4⫹ T-lymphocytes, as the experiments were conducted on isolated macrophages. These data suggest that HIV infection promotes specific intrinsic abnormalities in macrophage host defense function and these abnormalities are evident in the early clinical stages of HIV infection. The current study is the first to investigate the influence of HIV infection human macrophage apoptosis in response to virulent MTb. Macrophage apoptosis, an important mechanism for the elimination of intracellular mycobacteria (7, 8, 45), is evident in in vivo studies of MTb infection (9, 12, 46 – 48). In studies with human macrophages, apoptosis, mediated in part by autocrine TNF-␣ function (9) is associated with reduced mycobacterium growth (10, 11, 13), enhanced phagosomal maturation (15), and enhanced Ag presentation via vesicle formation and crosspriming of CD8⫹ T cells (16, 49). The current study identifies for the first time evidence for reduced macrophage apoptosis in response to virulent MTb in AM from HIV⫹ persons.

The mechanism for reduced MTb-mediated apoptosis observed in HIV⫹ macrophages in the current study was attributed to reduced TNF-␣ bioavailability and reduced TNF-␣ release by HIV⫹ macrophages in response to virulent MTb. Similar to prior studies (9, 14), healthy macrophage apoptosis was mediated in part by TNF-␣, as neutralization of macrophage-secreted TNF-␣ significantly reduced macrophage apoptosis in response to virulent MTb. In HIV⫹ macrophages, reduced MTb-mediated apoptosis was related to reduced TNF-␣ release and was rescued by exogenous TNF-␣ or stimulation of endogenous macrophage TNF-␣. Furthermore, the observed elevation of levels of sTNFR1 and sTNFR2 in the lung lavage samples from HIV⫹ persons could potentially influence TNF-␣ bioavailability in the lungs. As TNFRI signaling leads to the initiation of apoptosis (50), enhanced release of sTNFR1 in the lungs of asymptomatic HIV⫹ persons may reduce cell surface expression of TNFR1 and TNFR2, attenuate TNF-␣ activity by competing with cell surface TNF receptors, and thus desensitize cells to TNF-␣ and reduce apoptosis in cells such as macrophages. Taken together, these data support the concept that altered TNF-␣ trafficking and/or bioavailability in the lungs of HIV⫹ persons may account for reduced macrophages apoptosis in response to MTb. Findings of altered AM host defense function to virulent MTb in AM from HIV⫹ persons in the current study are consistent with studies reported by other investigators. Prior studies of AM from HIV⫹ persons demonstrated decreased release of MIP-1␣ and RANTES in response to virulent MTb (25), and a trend toward reduced TNF-␣ release (25). Other investigations showed increased MTb phagocytosis by AM from HIV⫹ persons compared with AM from healthy individuals (51), similar to findings in the current study. These observations support the concept that AM from HIV⫹ persons demonstrate consistent and reproducible alterations in innate response to virulent MTb. Findings in the current study support the general concept that HIV infection of macrophages promotes specific and targeted intrinsic defects in macrophage innate immune function. Examining AM from asymptomatic HIV⫹ persons, prior investigations demonstrated impaired phagocytosis (22), NF-␬B nuclear translocation (21), and respiratory burst response (23) to Pneumocystis, whereas phagocytosis (IgG opsonized erythrocytes), NF-␬B activation (LPS), and respiratory burst response (zymosan) to control particles or agonists was intact. In the current study, reduced MTbmediated macrophage apoptosis and TNF-␣ release in HIV⫹ AM was intact to non-MTb stimuli. Furthermore, MTb phagocytosis was significantly increased in HIV⫹ macrophages, whereas Pneumocystis phagocytosis was significantly decreased in HIV⫹ macrophages. Taken together, these results demonstrate that HIV infection targets specific macrophage host defense functions and pathways. The importance of these observations relate to the potential for therapeutic immunomodulation for the purpose of restoring immune function of macrophages from HIV⫹ persons. This potential application is supported in the current study as apoptosis was rescued in HIV⫹ macrophages though stimulation of endogenous TNF-␣ through TLR-independent mechanism. In the current study, although apoptosis of AM from HIV⫹ persons was reduced in response to virulent MTb, the consequences of reduced apoptosis on mycobacterial growth was not specifically investigated. One investigation suggests that MTb growth may not be altered in AM from HIV⫹ persons (51), although the study was limited to HIV⫹ subjects with evidence of clinical control of HIV. The influence of TNF-␣ on MTb viability was not examined in the current study. Other studies have shown that TNF-␣ may actually promote the growth of virulent MTb (52, 53), although the ability to suppress MTb growth may be more directly related to TNF-␣

The Journal of Immunology bioactivity (13). As only 1–10% of isolated AMs from HIV⫹ individuals are infected with HIV (18, 21), the impaired AM response to MTb observed in the current study is likely due to endogenous soluble factor(s) released by HIV⫹ AM or exogenous factor(s), which in turn globally influence function of bystander AMs, although this was not specifically investigated. A candidate soluble factor may be IL-10 which is released by monocytes exposed to specific HIV proteins such as nef (54) and tat (55) and which can depress macrophage apoptotic response to mycobacteria (13, 56). Whereas the current study focused on isolated cultured human macrophages, experiments did not examine the possible specific contribution of altered lymphocyte populations on macrophage function that may occur at mucosal sites such as gut and lung (57, 58). Other limitations of the current study include the fact that specific macrophage receptor(s) mediating signal transduction were not identified, although results implicate a TLR-mediated mechanism. Although these experiments included pathogenic MTb laboratory isolates, whether similar findings would apply to clinical MTb isolates was not tested. Finally, although these in vitro experiments may not accurately reflect function and behavior of macrophages in the lungs of persons, the use of primary human AM may allow for more direct extrapolation of experimental findings to human disease. In conclusion, this study demonstrates that HIV infection is associated with significant reduction of macrophage apoptosis in response to virulent MTb in vitro. This study supports the concept that depletion of circulating peripheral blood CD4⫹ T lymphocyte may not be the exclusive immunological abnormality that impairs host defense responses and predisposes HIV⫹ persons to MTb infection and disease. Crippling of macrophage innate immune apoptotic response combined with impairment of adaptive immunity associated with reduction in the function or numbers of circulating peripheral blood and lung CD4⫹ T-lymphocytes, may together contribute to the exceptionally high rates of MTb disease that characterizes the clinical course of HIV infection. Recognizing that suppressed macrophage function can be re-established by drugs that modulate signaling pathways (59) macrophage apoptosis may represent a viable therapeutic target in the management of MTb disease in HIV⫹ persons.

Acknowledgments We thank all study participants who consented to research bronchoscopy, and Robert Garland, Lorraine Gryniuk, Renee Andwood, and Darren Tavernelli for technical assistance. We also thank Melanie Cushion (VAMC, University of Cincinnati, Cincinnati, Ohio) for providing the Pneumocystis organisms.

Disclosures The authors have no financial conflict of interest.

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