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Published ahead of print on August 18, 2005, doi:10.1165/rcmb.2004-0341OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 33, pp. 610-621, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2004-0341OC

HIV Impairs TNF-{alpha} Release in Response to Toll-Like Receptor 4 Stimulation in Human Macrophages In Vitro

Souvenir D. Tachado, Jianmin Zhang, Jinping Zhu, Naimish Patel and Henry Koziel

Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts

Correspondence and requests for reprints should be addressed to Henry Koziel, M.D., Department of Pulmonary, Critical Care and Sleep Medicine, Kirstein Hall, Room E/KSB-23, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215. E-mail: hkoziel{at}bidmc.harvard.edu


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The molecular mechanisms for increased risk of bacterial pneumonia in HIV+ persons remain incompletely understood. Recognizing the critical role of Toll-like receptor (TLR) signaling in host defense, this study showed that human U937 macrophage stimulation by the TLR4-specific ligand, lipid A (biologically active component of bacterial LPS), promoted TNF-{alpha} release through extracellular regulated kinase (ERK)1/2 mitogen-activated protein (MAP) kinase phosphorylation. In contrast, HIV+ U1 macrophages had significantly reduced TNF-{alpha} release (despite preserved TLR4 expression) and reduced ERK1/2 phosphorylation, whereas TNF-{alpha} release was intact via a TLR4-independent pathway. In HIV+ U1 cells, reduced ERK1/2 phosphorylation was not due to reduced upstream MEK1/2 activation, but was associated with a reciprocal induction of MAP kinase phosphatase-1 (MKP-1). HIV nef protein was sufficient to reduce TNF-{alpha} release and induce MKP-1 in healthy macrophages. Pharmacologic inhibition of endogenous cellular phosphatases increased ERK1/2 phosphorylation and partially restored TLR4-mediated TNF-{alpha} release in HIV+ macrophages. Furthermore, targeted gene silencing of MKP-1 partially restored lipid A–mediated TNF-{alpha} release in HIV+ U1 cells. Similar results were observed using clinically relevant human alveolar macrophages, comparing healthy to asymptomatic HIV+ persons at clinical risk for bacterial pneumonia. Thus, reduced TLR4-mediated TNF-{alpha} release through altered ERK1/2 regulation by HIV may impair an effective innate immune response to bacterial challenge. Inhibition of cellular phosphatases may serve as a potential therapeutic target in the management of bacterial pneumonia in HIV+ persons.

Key Words: alveolar macrophages • innate immunity • MAP kinase • MAP kinase phosphatase-1 • lipid A


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Respiratory tract infections remain a frequent complication in HIV+ persons (1) and represent a major global health problem despite advances in AIDS management and the development of more effective antiretroviral therapy. Bacterial pneumonia occurs with increased frequency in HIV+ individuals, even among persons with relatively preserved peripheral CD4+ T-lymphocyte counts (2). Recent international data suggest that the incidence of bacterial pneumonia may be unchanged despite the introduction of highly active antiretroviral therapy (HAART) (3, 4) in contrast to the concurrent decline in opportunistic infections such as Pneumocystis pneumonia (5). Bacterial pneumonia attributed to Gram-negative organisms such as Pseudomonas aeruginosa, Haemophilus influenza, and Klebsiella pneumoniae is associated with a high mortality (6), especially when complicated by bacteremia (7). Although the increased incidence and severity may in part be attributed to deficiencies of B-lymphocyte function (8), the molecular mechanisms accounting for the higher risk of bacterial pneumonia in HIV+ persons remains incompletely understood.

The family of mammalian Toll-like receptors (TLRs), including TLR1 to TLR11 (9, 10), serves an important role in the early host defense response of innate immunity. Through recognition of conserved molecules derived from microbial pathogens (such as lipoteichoic acid, peptidoglycan, bacterial lipoproteins, and lipopolysaccharide [LPS]), TLRs serve a critical role in the discrimination of "self" from "non-self" in the early response to infectious challenge (11). Expressed on cells near mucosal portals of entry, including macrophages (12), dendritic cells (13), and lung epithelial cells (14), TLRs represent critical molecules in the first line of host defense to microbes such as pathogenic bacteria. TLR4, the best-characterized member of the TLR family (15, 16), recognizes Gram-negative bacteria through the essential cell wall components LPS or lipid A. Functional deficiency or genetic deletion of TLR 4 increase susceptibility to H. Influenza, Streptococcus pneumoniae, and K. pneumoniae respiratory tract infection in murine models (16, 17). In vitro ligation of TLR4 by LPS directs an immune response in part through activation of MAP kinases (18) and NF-{kappa}B (19), with the subsequent release of critical host defense molecules such as TNF-{alpha} (20). However, the expression and function of TLRs in pathogenesis of bacterial pneumonia in HIV-infected persons has not been investigated.

Alveolar macrophages represent the predominant resident innate immune cell in the lungs (21) and may modulate the inflammatory response to pneumonia (22). Human alveolar macrophages express a variety of innate immune receptors involved in pathogen recognition (23) and respond to TLR2 and TLR4 agonists (12), although the expression and specific function of human alveolar macrophage TLRs in health has not been fully investigated. HIV can infect alveolar macrophages (24, 25), and HIV infection is associated with specific defects of macrophage innate immune function—such as mannose receptor-mediated phagocytosis (26) and NF-{kappa}B activation (10)—that may contribute to the pathogenesis of opportunistic pneumonia. Alveolar macrophage phagocytosis of bacterial pathogens such as S. pneumoniae and Escherichia coli may be preserved in asymptomatic HIV-infected persons (27, 28), although whether macrophage receptor–mediated signal transduction in response to bacteria remains intact has not been investigated.

The expression and function of human macrophage TLR4 in HIV infection has not been previously examined. TLR4 activation by LPS enhances HIV replication (29), although the influence of HIV infection on TLR4 expression and function in the context of host defense has not been investigated. To test the hypothesis that HIV infection is associated with impaired human macrophage TLR-mediated host defense response to bacterial pathogens, this study examined the mechanism of TNF-{alpha} release in response to the specific TLR4 ligand lipid A (biologically active component of LPS), comparing human macrophage U937 cell line to human macrophage U1 cell line (HIV-infected subclone of U937 cells). Furthermore, the significance of the results were examined by comparing TNF-{alpha} release in clinically relevant alveolar macrophages from healthy individuals to asymptomatic HIV+ persons at clinical risk for bacterial pneumonia.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Reagents
Lipid A from E. coli F583 Rd mutant (the biologically active component of LPS, and specific TLR4 ligand), protease inhibitor cocktail, phorbol myristic acid (PMA), okadaic acid (general cellular phosphatase inhibitor), and okadaone (inactive analog of okadaic acid) were purchased from Sigma Chemical Co. (St. Louis, MO); MEK inhibitor UO126 and p38 inhibitor SB203580, protein A/G beads from Promega (Madison, WI); Thermoscript RT PCR kit from Invitrogen (Carlsbad, CA); recombinant nef (HIV-1) from Trinity Biotech Plc, (IDA Business Park, Bray, Co Wicklow, Ireland); and analytical or HPLC grade chloroform, methanol, diethyl ether from Fisher Scientific (Pittsburgh, PA).

Antibodies
Anti-TLR4, MKP-1, MEK, and pMEK were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); anti–phospho- and non–phospho-extracellular regulated kinase (ERK) p44/42, p38, and c-Jun N-terminal kinase (JNK) from Cell Signaling (Beverly, MA); and anti–{beta}-actin from Sigma Chemical.

Human Macrophages
Macrophages were differentiated from human promonocytic U937 (American Tissue and Cell Co., ATCC, Manassas, VA) and HIV-infected U1 cell lines (AIDS Research and Reference Reagent Program, Bethesda, MD). U1 cells (HIV-infected subclone of U937 cells) contain two integrated copies of HIV-1 proviral DNA, and are characterized by low levels of constitutive virus expression that can be modulated by cytokines and pharmacologic agents (30). For experiments, U937 and U1 cells were harvested during exponential growth phase, washed, and then incubated in complete medium (RPMI 1640 containing 10% heat-inactivated fetal calf serum [FCS], 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin). To allow differentiation to macrophages, U937 and U1 cells were incubated with PMA 100 nM at 37°C in a humidified atmosphere containing 5% CO2 for 24 h. Adherent cells were then washed three times with PBS (to remove PMA), and incubated in complete media (without PMA) for an additional 24 h before use in experiments.

To determine the clinical relevance, select experiments were performed using human alveolar macrophages. Recruited healthy and asymptomatic HIV+ individuals were without evidence for active pulmonary disease and had normal spirometry. Healthy individuals were confirmed to be HIV seronegative by ELISA and had no known risk factors for HIV infection. For the HIV+ subjects, peripheral blood CD4 lymphocyte counts were > 200 cells/mm3, HIV risk factors included IVDU and homosexual exposures, all were prescribed highly active antiretroviral therapy (HAART), all had undetectable serum viral load (< 50 HIV-1 RNA copies/ml), and none experienced a prior opportunistic pneumonia. Using standard techniques, bronchoalveolar lavage (BAL) was performed to obtain lung immune cells (26). All procedures were performed on consenting adults following protocols approved by Beth Israel Deaconess Medical Center institutional review board and Committee for Clinical Investigations. The cells were separated from the pooled BAL fluid as described previously (26). Cells were then counted on a hemacytometer with light microscopy. Alveolar macrophages were isolated by adherence to culture plates, and yielded cells that were > 98% viable as determined by trypan blue dye exclusion, and demonstrated > 95% positive nonspecific esterase staining.

Immunoblotting and Immunoprecipitation
Adherent human macrophages were treated with indicated dose of lipid A and placed into lysis buffer containing 1% Triton-X 100, 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 2 mM EDTA, and protease inhibitor cocktail, placed on ice for 20 min, and the Triton-X 100-soluble protein was separated by centrifugation at 10,000 x g for 15 min at 4°C. Lysates were subjected to immunoblotting and immunoprecipitation as described (31).

Total RNA Isolation and RT-PCR
Total cellular RNA was extracted from human macrophages with Trizol reagent following manufacturer's instructions. The cDNA was synthesized from 1 µg of total RNA by extension of the oligo dT primer with 200 units of Thermoscript (Invitrogen). Reverse transcription (RT) and PCR were conducted using avian myeloblastosis virus transcriptase and Taq transcriptase, respectively. PCR assays were conducted for 35 cycles with Perkin Elmer Geneamp PCR system using primers and conditions described. As negative control, non–reverse-transcribed samples were amplified by PCR. The oligonucleotide primers are listed in Table 1. After PCR, 10 µl of the total amplified product was electrophoresed on ethidium bromide-stained 1% agarose gels visualized under ultraviolet light.


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  		TABLE 1. HUMAN PRIMER SEQUENCES USED FOR RT-PCR

 
Quantitative Analysis of MKP-1 mRNA by Real-Time PCR
Total RNA was extracted from human macrophages with Trizol reagent following manufacturer's instructions (Invitrogen). The cDNA was synthesized from 1 µg of total RNA by extension of the oligo (dT) 20 and Ready-to-Go reverse transcription kit (Amersham, Foster City, CA). SYBR Green I-based real-time PCR was performed on Opticon Monitor (MJ Research, Inc., Waltham, MA) with cDNA templates (1/60 of the RT reaction) using Taq polymerase (Promega) and buffer [2 mM MgCl2, 400 µM deoxy-NTP (Roche, Nutley, NJ) 0.5x SYBR Green I, 0.8 µM of each PCR primer (Operon, Alameda, CA)] in a 25-µl final volume reaction. The samples were loaded onto wells of Low Profile 96-well microplates. After an initial denaturation step at 95°C for 5 min, conditions for cycling were 40 cycles of denaturation (95°C for 30 s), annealing (for 30 s), and extension (72°C for 1 min). To minimize possible primer dimer detection, the fluorescence signal was measured immediately after incubation at 79°C for 5 s after each extension step. A melting curve was generated to identify specificity of PCR products. Serial dilutions of human GAPDH plasmids were used as standards for quantitative measurement of amplified cDNA. All PCR reactions were run in duplicate. Comparative CT method was used to quantify the amplified transcripts.

Flow Cytometry Analysis
Cell surface expression of TLRs was determined by Epics XL flow cytometer (Beckman/Coulter, Miami, FL) with laser power of 5.76 mW. The instrument was calibrated before each measurement with standardized fluorescent particles (Immunocheck; AMAC, Inc., Westbrook, ME). Fluorescent signals of the cells were measured simultaneously by three photomultiplier tubes and optical filters and shown as the mean of the log fluorescence intensity of the cell population within gate. Macrophages were incubated with an anti-TLR4 antibody on ice for 60 min, washed three times, incubated with a FITC-conjugated secondary antibody for 30 min on ice protected from light, fixed in Optilyse (Beckman/Coulter) at room temperature for 5–10 min, and analyzed by flow cytometry. Human macrophages were first identified by the characteristic forward and side scatter parameters on unstained cells, and confirmed by staining with PE-conjugated primary anti-human HLA-DR (Beckman/Coulter). Data were expressed as a mean relative fluorescence units (RFU) and the percentage of cells staining positive. Isotype primary conjugated antibodies served as a negative control. Samples were prepared and analyzed in duplicate, and a minimum of 5,000 cells was counted for each sample.

ELISA
Cultured supernatants were collected, centrifuged to remove cellular debris, and stored at –80°C until assayed. Cytokine measurements were performed using commercially available ELISA (R&D Systems, Minneapolis, MN) following manufacturer's instructions, and absorbance measured at 450 nm on a Bio kinetic Elisa reader (Bio-Tek Instruments, Winooski, VT). The detection limit for TNF-{alpha} was 4.4 pg/ml. All measurements were performed in duplicate, and mean values of the two measurements were used for statistical analysis.

Functional Gene Silencing of Macrophage MKP-1 Using Specific Short Interfering RNA
Functional gene silencing was performed on HIV+ U1 macrophages using short interfering RNA (siRNA) targeting MKP-1 following the manufacturer's instructions (Santa Cruz Biotechnology). Briefly, 5.0 µl of desired 10 µM siRNA was added to 40 µl siRNA transfection medium, mixed gently, and incubated at room temperature for 5 min. In a separate tube 5.0 µl of siRNA transfection reagent was added to 40 µl of siRNA transfection medium, mixed gently and incubated at room temperature for 5 min, combined with transfection medium mixture, and incubated at room temperature for 20 min. After incubation, the mixture complex was diluted with 320 µl of siRNA transfection medium, gently mixed, and then overlaid onto washed U1 cells for each transfection and incubated at 37°C for 5–7 h. After incubation, 400 µl of complete growth medium containing twice the normal concentrations of serum and antibiotics were added to each well and incubated further for 30 h at 37°C. Control conditions for siRNA included an irrelevant 21-mer duplex siRNA. In select wells, functional MKP-1 gene suppression was confirmed by Western blot. Transfection efficiency was assessed using fluorochrome-labeled nontargeted siRNA. To determine the specificity of siRNA-targeted MKP-1 gene silencing, assay for cellular phosphatases PP1 and PP2A protein levels were determined by Western blot. siRNA-treated and untreated U1 cells were incubated in the presence of Lipid A and TNF-{alpha} release was measured by ELISA.

Statistical Analysis
Group comparisons were performed using Student's t test (two sample test) or one-way ANOVA. Calculations were performed with StatView (SAS Institute, Inc., Cary, NC) and INSTAT2 (GraphPad Software, San Diego, CA) software package. Results are given as mean ± SEM. Statistical significance was accepted for P < 0.05.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Reduced TLR4-Mediated TNF-{alpha} Release in HIV-Infected Human Macrophages
To examine the function of TLR4 in human macrophages, experiments measured TNF-{alpha} release in response to the specific TLR4 agonist, lipid A, comparing U937 cells to HIV-infected U1 cells. Incubation of U937 macrophages with lipid A resulted in a dose-dependent release of TNF-{alpha} (Figure 1). In comparison, incubation of U1 macrophages with lipid A also resulted in a dose-dependent increased release of TNF-{alpha}, although the absolute amount of TNF-{alpha} released was significantly reduced over a range of lipid A concentrations (0.1–5.0 µg/ml). The pattern of IL-1{beta} and IL-6 release was similar to TNF-{alpha} for both cell lines (data not shown). In contrast to lipid A, the release of TNF-{alpha} through a TLR4-independent pathway was comparable in U937 and U1 cells (Figure 1, insert). The observed differences in macrophage responses to lipid A were not associated with alterations in TLR4 expression, as surface receptor expression, cellular protein content, and mRNA transcripts were similar comparing U937 and U1 cells (Figure 2).



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  		Figure 1. Reduced TNF-{alpha} release by HIV-infected human macrophages in response to TLR4 activation. Differentiated adherent human macrophage U937 and HIV-infected U1 cell lines were incubated with lipid A (0–5.0 µg/ml) for 18–24 h, and cell-free cultured supernatants were assayed for TNF-{alpha} by ELISA. To examine TLR4-independent release of TNF-{alpha}, inset depicts differentiated adherent human U937 and HIV-infected U1 macrophages in the absence (control) and presence of acute exposure to PMA (100 nM). Data presented are means ± SEM for an assay performed in duplicate for a representative experiment. Similar results were obtained in at least three independent experiments.

 


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  		Figure 2. Human macrophage TLR 4 expression was not altered by HIV infection. (A) TLR4 surface expression on human U937 and HIV+ U1 macrophages by flow cytometry. For each panel, left-sided curve represents isotype control (which was similar to background autofluorescence), and right-sided curve represent PE-conjugated anti-TLR4 labeling. Representative profiles were similar of three independent experiments. (B) Total cellular TLR4 protein determination of whole cell extracts by Western blot analysis. Cellular extracts were obtained with and without PMA differentiation, comparing U937 and HIV+ U1 cells. TLR4 protein expression was low in undifferentiated promonocytic macrophages, but markedly enhanced following macrophage differentiation by PMA (conditions used for all experiments). Representative blot was similar of three independent experiments. (C) RT-PCR determination of TLR4 mRNA transcripts were similar comparing U937 to U1 cells, and mRNA levels increased in response to lipid A. {beta}-actin served as an internal control for mRNA loading. Representative gel was similar of three independent experiments.

 
HIV Impaired ERK 1/2 and p38 MAPK Phosphorylation in Response to TLR4 Ligation
To examine whether differences in human macrophage TNF-{alpha} release were due to differences in MAP kinase activation (32, 33) experiments next measured ERK1/2, JNK, and p38 kinase phosphorylation (activation) in response to TLR4 stimulation comparing U937 to U1 cells. In unstimulated U937 macrophages, constitutive expression of activated ERK1/2 (ppERK1/2) was absent, but increased in a dose-dependent manner in response to lipid A (Figure 3A). Unstimulated HIV-infected U1 cells also did not have constitutive expression of activated ERK1/2, but the response to lipid A was markedly reduced compared with U937 cells. Levels of activated JNK (ppJNK) were present constitutively and did not increase in response to lipid A in either U937 or U1 cells. Constitutive levels of phosphorylated p38 (pp38) were readily detected in unstimulated U937 cells, and levels increased in a dose-dependent manner in response to lipid A. In comparison, constitutive phosphorylated p38 levels were easily detected in unstimulated U1 cells, and the levels decreased in a dose-dependent manner in response to lipid A. Thus, HIV infection impaired macrophage ERK1/2 and p38 phosphorylation in response to lipid A.



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  		Figure 3. Impaired ERK 1/2 and p38 MAPK phosphorylation in response to TLR4 ligation was not associated with reduced MEK activity. (A) Adherent human macrophage U937 and HIV+ U1 cells were incubated with lipid A for 15 min at 37°C and 5% CO2, cell lysates collected, and kinase activity detected using specific phosphoantibodies as detailed in MATERIALS AND METHODS. Total protein was detected by Western blot using specific antibody. Immunoreactive bands were detected using horseradish peroxidase–conjugated anti-IgG and visualized by ECL Western blotting system. Total cellular ERK1/2, JNK and p38 protein were used to control for loading. Data are representative of three experiments with similar results. (B and C) TNF-{alpha} release by adherent differentiated U937 macrophages after incubation with lipid A (1.0 µg/ml) in the absence and presence of (B) MEK inhibitor (UO126), and (C) p38 inhibitor (SB 203580). After 18–24 h, cell cultured supernatants assayed for TNF-{alpha} by ELISA. Data presented are means ± SEM for assays done in duplicate. Similar results were obtained in at least three independent experiments. (D) MEK phosphorylation (activation) in U937 and HIV+ U1 cells in response to lipid A. Data are representative of three experiments with similar results.

 
To determine whether ERK1/2 and p38 MAP kinase activation were necessary for TLR4-mediated TNF-{alpha} release in U937 cells, experiments were performed using pharmacologic inhibitors. In the presence of a specific MEK inhibitor (UO 126; inhibits upstream of ERK1/2), lipid A–mediated TNF-{alpha} release was markedly reduced in a dose-dependent manner (Figure 3B). In contrast, in the presence of a specific p38 inhibitor (SB203580), lipid A–mediated TNF-{alpha} release was not reduced (Figure 3C). These data demonstrate that in U937 macrophages, TLR4-mediated TNF-{alpha} release was dependent on ERK1/2 phosphorylation, and suggest that reduced TLR4-mediated TNF-{alpha} release in HIV-infected U1 macrophages was in part attributed to impaired ERK 1/2 phosphorylation.

Reduced ERK 1/2 Phosphorylation in HIV-Infected Macrophages Is Not Due to Impaired Upstream MEK Activation
Phosphorylation of the upstream molecule MEK (mitogen- activated protein kinase/extracellular signal-regulated kinase kinase) activates ERK1/2, and the observed reduction in TNF-{alpha} release in the presence of the MEK inhibitor UO 126 identified MEK as a critical molecule for TLR4-mediated TNF-{alpha} release (Figure 3B) and possible target for HIV. Experiments next measured MEK activation, and demonstrated absent constitutive MEK phosphorylation in unstimulated U937 and U1 macrophages, with comparable dose-dependent increases in response to lipid A (Figure 3D). Thus, the reduced ERK1/2 phosphorylation observed in HIV-infected U1 cells was not due to reduced MEK activation, and suggested the possibility of altered molecular signaling events downstream of MEK.

Reduced ERK1/2 Phosphorylation in Human Macrophages Was Associated with Reciprocal Induction of MKP-1
ERK1/2 activation requires dual phosphorylation of conserved threonine and tyrosine residues within the TGY motif (34). Phosphorylation of MAP kinases is regulated in part by a family of dual specificity phosphatases such as MAP kinase phosphatase-1 (MKP-1). MKP-1 mediates inhibition of MAP kinases in vivo, and is a critical regulator of macrophage signaling in response to LPS stimulation because it terminates production of proinflammatory cytokines such as TNF-{alpha} (35). To examine the possible role of cellular phosphatase in the regulation of ERK1/2 in TLR4-mediated TNF-{alpha} release in human macrophages, experiments next examined the kinetics of ERK1/2 phosphorylation and MKP-1 induction in U937 cells.

Phosphorylated (activated) ERK1/2 was absent or very low in unstimulated adherent human U937 macrophages, and increased in response to lipid A in a time-dependent manner with maximum detection by 15 min, followed by a rapid decline to unstimulated levels by 30–60 min (Figure 4A). MKP-1 was also induced in these same cells in response to lipid A, with increased levels up to 60 min (Figure 4A). Quantitative analysis comparing phosphorylated ERK1/2 and total MKP-1 levels demonstrated that both were detected by 15 min, but further increase in MKP-1 induction was associated with reciprocal decline (inactivation) of ERK1/2 phosphorylation (Figure 4B). The observed time course induction of MKP-1 mRNA and protein was consistent with reports using murine macrophages (36). Immunoprecipitation experiments demonstrated direct interaction of ERK1/2 with MKP-1 in U937 cells in response to lipid A (Figure 4C) and suggested the possibility that MKP-1 directly regulated ERK1/2 activity. The temporal characteristics of these data suggest that TLR4-mediated control of ERK1/2 phosphorylation in U937 macrophages may be modulated in part by MKP-1 induction.



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  		Figure 4. Role of MKP-1 in the regulation of ERK1/2 phosphorylation in human macrophages. (A) Adherent differentiated U937 macrophages were incubated with lipid A (1.0 µg/ml), and Western blots of cell lysates prepared using specific antibodies against phospho-ERK, total ERK, and anti–total MKP-1. Data are representative of three experiments with similar results. (B) Densitometric analysis of MKP-1 and ppERK data from blots presented in A. (C) Demonstration of physical interaction between ERK1/2 and MKP-1 in response to lipid A, by immunoprecipitation of U937 cell lysates with anti–MKP-1 antibody followed by Western blot analysis with anti-phosphoMEK and total MEK antibodies. Data are representative of three experiments with consistent results. (D) Induction of MKP-1 comparing adherent differentiated U937 and HIV+ U1 cells in response to lipid A for 15 min. Detergent soluble proteins were separated on 10% SDS-PAGE, transferred to nitrocellulose membrane, and probed with monoclonal anti–MKP-1. Immunoreactive band were detected with horseradish peroxidase–conjugated mouse anti-rabbit and visualized by ECL Western blotting system. Western blot is a representative experiment of three independent experiments with consistent results. Densitometric analysis of MKP-1 presented as fold increase (n = 3).

 
HIV Infection of Human Macrophages Associated with MKP-1 Induction
To determine the mechanism for impaired ERK1/2 phosphorylation in HIV+ U1 cells, experiments measured MKP-1 induction in response to lipid A. In unstimulated adherent U937 macrophages, MKP-1 was low or undetectable, but increased in a dose-dependent manner to lipid A (Figure 4D). In comparison, unstimulated adherent HIV+ U1 cells demonstrated higher constitutive MKP-1 levels compared with unstimulated adherent U937 cells, with further increase of MKP-1 in a dose-dependent manner to lipid A. In general, MKP-1 induction was higher in HIV+ U1 cells. Other cellular phosphatases PP1 and PP2A were not detected (data not shown). Taken together, these data demonstrated that reduced ERK1/2 phosphorylation in HIV+ macrophages was associated with increased induction of MKP-1 compared with U937 cells.

Inhibition of Cellular Phosphatases Enhanced TLR4-Mediated TNF-{alpha} Release in HIV-Infected Human Macrophages
To further define the role and determine the biological significance of increased cellular phosphatase activity (such as MKP-1) on the regulation of ERK1/2 phosphorylation and TLR4-mediated TNF-{alpha} release in HIV+ macrophages, experiments were performed in the absence and presence of the general cellular phosphatase inhibitor, okadaic acid. Although not a specific inhibitor, okadaic acid is known to inhibit MKP-1 in human bronchial epithelial cells (37). In HIV+ U1 macrophages, phosphorylated ERK1/2 was constitutively expressed at low levels with minimal increase in response to lipid A alone, but increased significantly in response to lipid A in the presence of okadaic acid (Figure 5A). Similarly, unstimulated adherent HIV+ U1 cells released low levels of TNF-{alpha}, without significant increase in response to lipid A alone, but TNF-{alpha} release was greatly enhanced after preincubation with okadaic acid in a dose-dependent manner in these same cells (Figure 5B). TNF-{alpha} release was not influenced by the inactive okadaic acid analog, okadaone (data not shown). Taken together, these data demonstrated that inhibition of endogenous protein phosphatases (such as MKP-1) increased ERK1/2 phosphorylation and normalized TLR4-mediated TNF-{alpha} release in HIV+ U1 macrophages.



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  		Figure 5. General pharmacologic inhibition of cellular phosphatases or targeted gene silencing of MKP-1 partially restored TLR4-mediated TNF-{alpha} release in HIV+ human macrophages. (A) Adherent HIV+ U1 macrophages were incubated with lipid A for 15 min in the absence or presence of a general cellular phosphatase inhibitor (okadaic acid; pretreatment for 1 h), cells lysates prepared for Western blotting with phosphoERK1/2-specific mAb. Immunoreactive bands were detected with horseradish peroxidase–conjugated anti-rabbit IgG and visualized by ECL. Data are representative of three experiments with similar results. Densitometry performed and data expressed as ERK volume analysis (n = 3). (B) TNF-{alpha} release by adherent HIV+ U1 macrophages incubated with lipid A in the absence and presence of okadaic acid (10 nM; pretreatment for 1 h). After 18–24 h, cultured supernatants were assayed for TNF-{alpha} by ELISA. Data are representative of three experiments with similar results. (C) Targeted MKP-1 functional gene silencing of HIV+ U1 cells using siRNA (siRNA-MKP-1) reduced MKP-1 protein, but not ERK1/2 protein. Data are representative Western blot of three experiments with similar results. (D) TNF-{alpha} release by HIV+ U1 macrophages after targeted MKP-1 gene silencing. After incubation with lipid A (10 µg/ml) for 24 h, cell-free cultured supernatants were assayed for TNF-{alpha} by ELISA (n = 3).

 
Recovery of TLR4-Mediated TNF-{alpha} Release after Functional Gene Silencing of MKP-1 in HIV+ Macrophages
To determine the specific role of MKP-1 on TLR4-mediated TNF-{alpha} release, experiments compared TNF-{alpha} release by HIV+ U1 macrophages following functional gene silencing of MKP-1 using siRNA (Figure 5C). TNF-{alpha} release was significantly increased in HIV+ U1 cells pretreated with siRNA specifically targeted to MKP-1 compared with U1 cells treated with irrelevant siRNA (Figure 5D). These studies demonstrate that functional gene silencing of MKP-1 expression enhanced TNF-{alpha} release in response to lipid A, suggesting an important regulatory role for MKP-1 in TLR4-mediated signal transduction in HIV+ macrophages.

Impaired TLR4-Mediated TNF-{alpha} Release by Human Alveolar Macrophages from Asymptomatic HIV+ Persons
To determine the clinical relevance of these findings, experiments next focused on primary human alveolar macrophages, comparing alveolar macrophages from healthy individuals to alveolar macrophages from asymptomatic HIV+ persons at clinical risk for bacterial pneumonia. TLR4 surface expression was relatively preserved comparing alveolar macrophages from healthy to HIV+ persons, with mean fluorescence intensity of 3.85 ± 0.24 and 3.03 ± 0.34, respectively (Figure 6A). For both groups, TNF-{alpha} release demonstrated a dose-dependent increase in response to lipid A, but the absolute amount was significantly reduced in alveolar macrophages from asymptomatic HIV+ persons over a range of lipid A concentrations (Figure 6B). Compared with HIV+ U1 cells, the differences in the magnitude of TNF-{alpha} release may in part reflect different levels of HIV infection of macrophages, as 100% of U1 cells are HIV-infected compared with < 10% of human alveolar macrophages (38).



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  		Figure 6. Reduced TNF-{alpha} release by human alveolar macrophages from asymptomatic HIV+ persons was associated with impaired ERK1/2 phosphorylation. The significance of findings in U937 and HIV+ U1 cells was investigated using clinically relevant human alveolar macrophages. (A) Flow cytometry determination of TLR4 surface expression on human alveolar macrophages comparing healthy to asymptomatic HIV-infected persons. For each panel, the profile on the left represents cell staining to PE-conjugated control idiotype antibody (which was similar to background autofluoresence). Average mean fluorescence intensity healthy: 3.85 ± 0.24; HIV+: 3.03 ± 0.34. Representative profiles for cells from at least five different individuals for each group. (B) Adherent human alveolar macrophages from healthy and HIV+ subjects were incubated with lipid A for 18–24 h, and cultured supernatants were assayed for TNF-{alpha} by ELISA. Data presented are means ± SEM for assays done in duplicate. Similar results were obtained in at least three independent experiments using alveolar macrophages from different individuals. (C) ERK1/2 activation in human alveolar macrophages from healthy and HIV+ subjects in the presence and absence of lipid A (10 µg/ml x 15 min), with and without pretreatment with the general cellular phosphatase inhibitor, okadaic acid (10 nM x 1 h). Detergent soluble proteins were probed with monoclonal anti-phosphoERK1/2 and anti-ERK1/2, immunoreactive 42-kD and 44-kD bands were detected with HPO-conjugated mouse anti-rabbit. Data demonstrate reduced ERK1/2 bands in HIV+ cells, with complete absence of the 42-kD band. Western blot is a representative experiment of three independent experiments with consistent results. (D) TNF-{alpha} release by adherent alveolar macrophages from asymptomatic HIV+ persons in response to lipid A (10 µg/ml x 15 min), in the presence and absence of okadaic acid (10 nM; pretreatment for 1 h). After 24 h, TNF-{alpha} release into cell-free cultured supernatants was measured by ELISA. Data are representative of two experiments with similar results.

 
Inhibition of Cellular Phosphatases Enhanced TNF-{alpha} Release by Alveolar Macrophages from Asymptomatic HIV+ Persons
To determine the molecular mechanism for reduced TLR4- mediated TNF-{alpha} release in primary human alveolar macrophages, experiments next examined ERK1/2 phosphorylation in response to lipid A. Similar to HIV+ U1 cells, ERK1/2 activation was impaired in alveolar macrophages from asymptomatic HIV+ persons and phosphorylated ERK1/2 was increased in the presence of the general cellular phosphatase inhibitor, okadaic acid (Figure 6C). In addition, TLR4-mediated TNF-{alpha} release was restored in the presence of an inhibitor of cellular phosphates, okadaic acid (Figure 6D). Taken together, these responses were similar to the responses observed above with U937 and HIV+ U1 macrophages. Thus, with similar patterns of impaired TLR4 signal transduction (including impaired ERK1/2 phosphorylation, increased MKP-1 induction, and impaired TNF-{alpha} release in response to lipid A) in both HIV+ U1 cells and alveolar macrophages from asymptomatic HIV+ persons in part validate the use of U937 and U1 cells as an in vitro model for the investigation of TLR4-mediated function in human HIV disease.

Increased MKP-1 mRNA and Protein Expression in Alveolar Macrophages from HIV+ Persons
To determine whether reduced TNF-{alpha} release was in part due to increased cellular phosphatase expression in alveolar macrophages from asymptomatic HIV+ persons, experiments measured MKP-1 mRNA transcripts in response to lipid A. MKP-1 mRNA levels were consistently higher in unstimulated and in lipid A–stimulated alveolar macrophages from HIV+ persons compared with healthy individuals (Figure 7A). In alveolar macrophages from healthy persons, MKP-1 was absent in unstimulated cells and did not increase in response to lipid A. Constitutive expression of MKP-1 was higher in HIV+ macrophages and further increased in response to lipid A in a dose-dependent manner (Figure 7B). These data demonstrated that MKP-1 mRNA and protein activation were increased in alveolar macrophages from asymptomatic HIV+ persons.



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  		Figure 7. MKP-1 is induced in alveolar macrophages from asymptomatic HIV+ persons. (A) PCR of MKP-1 and {beta}-actin mRNA in alveolar macrophages from healthy and asymptomatic HIV+ persons in the absence and presence of lipid A (0.1 ug/ml x 60 min). Quantitative analysis of MKP-1 mRNA was performed by real-time PCR and data expressed as fold increase. Data are representative of two experiments with similar results. (B) Western blot of MKP-1 and ERK1/2 protein in alveolar macrophages from healthy and asymptomatic HIV+ persons in the presence and absence of lipid A x1 h Detergent soluble proteins were separated on 10% SDS-PAGE, probed with monoclonal antibodies against MKP-1 or ERK1/2, and immunoreactive bands were detected with HPO-conjugated mouse anti-rabbit antibodies. Western blot is a representative experiment of three independent experiments with consistent results. Quantitaive analysis was performed by densitometry and data expressed as fold increase (n = 3).

 
HIV nef Impaired TNF-{alpha} Release by Human Macrophages and Increased MKP-1 mRNA
To investigate the possible mechanism for HIV-associated increased MKP-1 activation and decreased TNF-{alpha} release, experiments were performed with exogenous HIV nef protein. HIV nef is important for viral replication and HIV pathogenicity, and may be especially important in macrophages (39). Using human U937 macrophages, TNF-{alpha} release was low in unstimulated adherent cells, but increased in response to lipid A. However, incubation with lipid A in the presence of recombinant nef significantly reduced TNF-{alpha} in a dose-dependent manner (Figure 8A). In addition, measurement of MKP-1 mRNA was undetectable in unstimulated adherent U937 macrophages, but induced in response to HIV nef (Figure 8B). These data demonstrated that HIV nef was sufficient to reduce TLR4-mediated TNF-{alpha} release and promote MKP-1 induction.



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  		Figure 8. HIV nef reduced TNF-{alpha} release by human macrophages and increased MKP-1 mRNA. (A) TNF-{alpha} release by adherent differentiated human U937 macrophages were incubated with lipid A in the absence and presence of increasing concentrations of HIV nef protein. After 18–24 h, cultured supernatants were assayed for TNF-{alpha} release by ELISA. Data are representative of three experiments with similar results. (B) MKP-1 mRNA detection by RT-PCR on adherent differentiated U937 macrophages following incubation with lipid A or HIV-1 nef for 60 min {beta}-actin mRNA served as PCR control conditions. Data are representative of three experiments with similar results.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
This study demonstrated that TNF-{alpha} release by HIV+ macrophages was significantly decreased (compared with non-HIV macrophages) in response to TLR4 stimulation. Reduced TLR4-mediated TNF-{alpha} release was not due to reduced cellular TLR4 expression, and did not represent a global impairment of macrophage function as stimulation through a TLR4-independent pathway released similar levels of TNF-{alpha} in both HIV-infected and uninfected macrophages. Reduced TNF-{alpha} release was associated with reduced phosphorylation of ERK1/2 in HIV+ macrophages and a reciprocal increase in a regulatory cellular phosphatase, MKP-1. Furthermore, targeted gene silencing and pharmacologic inhibition of endogenous phosphatases increased ERK1/2 phosphorylation and partially restored TNF-{alpha} release in HIV+ macrophages. Importantly, similar results were observed using clinically relevant human alveolar macrophages from healthy and asymptomatic HIV+ persons at risk for bacterial pneumonia. These data identified a specific impairment in the regulation of critical TLR4-mediated signaling in HIV+ macrophages that may in part contribute to an ineffective host cell response to bacterial products.

The mechanism for reduced TLR4-dependent TNF-{alpha} release by HIV+ macrophages involved regulation of ERK1/2 MAP kinase. TLR4-mediated TNF-{alpha} release by healthy macrophages was dependent on ERK1/2 phosphorylation (and not p38 or JNK phosphorylation), since pharmacologic inhibition of ERK1/2 but not p38 inhibited TLR4-mediated TNF-{alpha} release. Reduced ERK1/2 phosphorylation was not due to reduced activation of the upstream regulatory molecule MEK, but was associated with a reciprocal induction of the MAP kinase phosphatase MKP-1. Furthermore, induction of cellular phosphatases in HIV+ macrophages may be in part related to the influence of specific HIV proteins, as HIV nef protein was sufficient to induce MKP-1 and reduce macrophage TNF-{alpha} release. These findings support the mechanism that HIV infection or an HIV protein (such as HIV nef) induced cellular phosphatases (such as MKP-1 or other phosphatases), which in turn reduced ERK1/2-dependent release of TNF-{alpha}.

In the current study, regulation of ERK1/2 phosphorylation in human macrophages was mediated in part by MKP-1. The family of MAP kinase phosphatases regulates the activities of MAP kinases (40) via reversible dephosphorylation of conserved MAP kinase threonine and tyrosine residues in tripeptide TXY signature motifs (41). MKP-1 (the archetype phosphatase) is an immediate early gene whose expression is rapidly induced by a variety of extracellular stimuli (40). In macrophages, MKP-1 is a critical negative regulator in the signaling response to LPS by switching off production of proinflammatory cytokines such as TNF-{alpha} (35). An important regulatory role for MKP-1 in human macrophage MAP kinase activation is supported in part by prior investigations (42), and in the current study is supported by the findings of direct physical interaction between ERK1/2 and MKP-1, reduced ERK1/2 phosphorylation associated with a reciprocal induction of MKP-1, elevated levels of MKP-1 in HIV+ macrophages exhibiting reduced ERK1/2 phosphorylation, and the partial restoration of TLR4-mediated TNF-{alpha} release with pharmacologic inhibitor as well as targeted gene silencing of MKP-1.

The current study represents the first to examine the influence of HIV on TLR4-mediated signal transduction in human alveolar macrophages in the context of host defense. Prior studies examined the influence of TLR2, TLR4, and TLR9 ligands on HIV replication in human mast cells or HIV-transgenic murine spleen cells (29, 43, 44). Investigators noted TNF-{alpha} release by HIV-1 transgenic mouse spleen cells in response to LPS was similar to control cells (29), although macrophages were not specifically investigated. In the current study, the finding of reduced TNF-{alpha} release by HIV+ macrophages in response to the LPS component lipid A was consistent with prior studies (45). Recognizing that an effective host response to infection requires a balanced regulation of TNF-{alpha} (the absence of TNF-{alpha} resulted in increased morbidity and mortality [46–48], whereas too vigorous a TNF-{alpha} response was associated with septic shock and death [49]) data from the current study support the concept that alveolar macrophage TNF-{alpha} response to TLR4 stimulation may be insufficient or only partially protective in the setting of HIV infection.

The current study suggested HIV nef induction of MKP-1 may in part provide the mechanism for reduced ERK1/2 phosphorylation in HIV infected macrophages. Specific HIV gene products are known to influence cellular receptor expression and signal transduction pathways (50). Prior studies reported that HIV-1 nef altered ASK1 signal transduction pathways (51) and downregulated NF-{kappa}B signaling in T cell lines (52), reduced mannose receptor surface expression on dendritic cells (53) and downmodulated Fc{gamma}-R expression on monocyte-derived macrophages (4). The current study extends the influence of HIV nef to the macrophage MAP kinase signaling pathway. Although HIV nef was sufficient to induce MKP-1 and impair TNF-{alpha} release, the specific mechanism of HIV nef-mediated MKP-1 induction was not determined, and the role of other MAP kinase phosphatases was not evaluated. Although other HIV proteins may influence cell signal transduction, such as HIV tat downregulation of mannose receptor transcription (54) and HIV vpu inhibition of NF-{kappa}B activation (55), the influence of these other HIV proteins on macrophage MAP kinase activation was not specifically investigated, and remain the focus of active investigation.

The clinical relevance of the findings in the current study is supported by the use of primary alveolar macrophages from HIV+ persons at increased clinical risk for bacterial pneumonia. The finding that impaired TNF-{alpha} release was observed in asymptomatic HIV+ persons with relatively preserved CD4 T-lymphocyte counts (> 200 cells/mm3) and undetectable serum viral load suggests that specific impairment of innate immune function may be evident even during clinically latent period of HIV infection in subjects. The finding of impaired TLR4-mediated TNF-{alpha} release may be evident even in patients with apparent good clinical responses to HAART may in part provide a mechanism for the increased incidence of bacterial pneumonia in persons with relatively preserved CD4 counts (56).

The role of other TLRs was not specifically investigated, so conclusions regarding the specificity or selectivity of impaired responses by other TLRs were not possible. The role of other pathways activated by TLRs, such as NF-{kappa}B, was not specifically investigated. Other limitations of the current study include the use of okadaic acid, which may inhibit other cellular phosphatases in addition to MKP-1. However, okadaic acid, known to inhibit MKP-1 in other investigations (37), was sufficient to inhibit MKP-1 induction and at least in part contributed to partially restore TNF-{alpha} release, although the involvement of other cellular phosphatases in TNF-{alpha} restoration in HIV+ macrophages could not be excluded. Recognizing that TNF-{alpha} expression is regulated via both transcriptional and posttranscriptional mechanisms (57), other factors may contribute to the differences in TNF-{alpha} release by HIV+ cells, such as post-transcriptional regulation by an AU-rich element (ARE) residing in the 3' untranslated region of TNF-{alpha} mRNA (57, 58), although these were not specifically investigated. Finally, the possibility that in vitro observations may not accurately reflect events in vivo should be considered, although the use of clinically relevant human macrophage cell lines and primary human alveolar macrophages may allow for more direct application to human disease.

In summary, these data demonstrated that HIV infection of human macrophages impaired TLR4-mediated TNF-{alpha} release. The mechanism for impaired TNF-{alpha} release was downstream of MEK activation, and may in part represent HIV-mediated induction of cellular phosphatases (such as MKP-1) and reduced ERK1/2 phosphorylation. Impairment of the MAP kinase pathway may be in part attributed to specific HIV proteins such as nef. The significance of these findings relate to an effective or adequate host cell response to infection. Recognizing that TLR4 is a critical molecule for the recognition of Gram-negative bacterial products, impairment in the alveolar macrophage TLR4-mediated MAP kinase pathway may impair an effective host response to infectious challenge in the lungs and in part contribute to pathogenesis of bacterial pneumonia in HIV+ persons. Furthermore, the observation that inhibition of cellular phosphates (including MAP kinase phosphatases such as MKP-1) partially restored the macrophage TNF-{alpha} release in HIV infected macrophages identified this pathway as a potential therapeutic target to augment the host cell response to bacterial challenge in HIV+ persons.


   Acknowledgments
 
The authors gratefully acknowledge the participation of all persons who consented to bronchoscopy, and the expert technical assistance of Robert Garland, Lorraine Gryniuk, and Rene Andwood.


   Footnotes
 
Originally Published in Press as DOI: 10.1165/rcmb.2004-0341OC on August 18, 2005

Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form November 3, 2004

Accepted in final form August 9, 2005


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