Genome-Wide Innate Immune Responses in HIV-1-Infected Macrophages Are Preserved Despite Attenuation of the NF-κB Activation Pathway

Mahdad Noursadeghi, Jhen Tsang, Robert F. Miller, Sarah Straschewski, Paul Kellam, Benjamin M. Chain and David R. Katz
J Immunol January 1, 2009, 182 (1) 319-328; DOI: https://doi.org/10.4049/jimmunol.182.1.319

Abstract

Macrophages contribute to HIV-1 infection at many levels. They provide permissive cells at the site of inoculation, augment virus transfer to T cells, generate long-lived viral reservoirs, and cause bystander cell apoptosis. A body of evidence suggests that the role of macrophages in cellular host defense is also compromised by HIV-1 infection. In this respect, macrophages are potent cells of the innate immune system that initiate and regulate wide-ranging immunological responses. This study focuses on the effect of HIV-1 infection on innate immune responses by macrophages at the level of signal transduction, whole genome transcriptional profiling, and cytokine secretion. We show that in an ex vivo model, M-CSF-differentiated monocyte-derived macrophages uniformly infected with replicating CCR5-tropic HIV-1, without cytopathic effect, exhibit selective attenuation of the NF-κB activation pathway in response to TLR4 and TLR2 stimulation. However, functional annotation clustering analysis of genome-wide transcriptional responses to LPS stimulation suggests substantial preservation of gene expression changes at the systems level, with modest attenuation of a subset of up-regulated LPS-responsive genes, and no effect on a selection of inflammatory cytokine responses at the protein level. These results extend existing reports of inhibitory interactions between HIV-1 accessory proteins and NF-κB signaling pathways, and whole genome expression profiling provides comprehensive assessment of the consequent effects on immune response gene expression. Unexpectedly, our data suggest innate immune responses are broadly preserved with limited exceptions, and pave the way for further study of the complex relationship between HIV-1 and immunological pathways within macrophages.

Macrophages are permissive to CCR5-tropic HIV-1, and in most models can host active viral replication without significant cytopathic effect (1, 2). This interaction may contribute to HIV-related disease during HIV-1 transmission when R5-tropic virus predominates (3), by transfer of virus to T cells (4), as a long-lived viral reservoir (2) and by provoking bystander cell apoptosis that may be involved in T cell or neuronal death (5, 6). In addition, the effect of HIV-1 infection on macrophage function is of significant interest. HIV-1-mediated inhibition of complement and FcR phagocytosis (7, 8) and attenuated intracellular killing of Leishmania (9) suggest that the role of macrophages as host defense effector cells may be compromised. Importantly, macrophages also function as sentinel cells of innate immunity involved in recognition of microbial pathogens that leads to both activation and regulation of host immune responses to wide-ranging microbial pathogens (1). Innate immune stimulation of monocyte-derived macrophages (MDM)4 and related monocyte-derived dendritic cells from HIV-infected subjects has frequently been reported to show attenuated or altered host cell responses (1), but these observations are unlikely to represent direct effects because the extent of HIV-1 infection of monocytes in vivo is estimated to be very low (10, 11). The same may be true of reports of deficient innate immune responses in alveolar macrophages from HIV-1-infected subjects (12, 13, 14, 15, 16). Nonetheless, two additional lines of evidence support the hypothesis that HIV-1 inhibits innate immune responses in macrophages. First, the latently infected myeloid leukemia cell line, U1, in which HIV-1 replication can be activated by PMA-induced differentiation into a macrophage-like adherent phenotype, shows reduced inflammatory cytokine production compared with the parental noninfected U937 cell line (13, 14). This effect has been attributed to up-regulation of mitogen-activated protein kinase phosphatase-1 by the HIV-1 accessory protein nef, and consequent inhibition of the innate immune signaling cascade through ERK1/2 (14). Second, in a variety of models, other interactions between HIV accessory proteins and components of the innate immune signaling pathway have also been reported to inhibit the function of the NF-κB family of transcription factors. The HIV accessory protein vpu can interact with β-transducin repeat-containing protein, involved in E3 ubiquitin ligase complex-mediated degradation of intracellular proteins (17, 18). This interaction competitively inhibits transducin repeat-containing protein-dependent degradation of IκBα, and in Drosophila, transgenic expression of vpu inhibits Toll-mediated degradation of the IκB homologue, cactus, and consequent activation of the NF-κB homologues, dorsal and dif (19). In addition, another HIV-1 accessory protein, vpr, interacts with the glucocorticoid receptor and generates a novel biological effect, inhibiting nuclear translocation of the NF-κB-coactivating factor poly(ADP-ribose) polymerase-1 (20). The NF-κB family of heterodimeric transcription factors is a major target for innate immune receptor-mediated signaling in general (21, 22). The most abundant component of this family is the p65 (RelA)/p50 heterodimer in which p50 principally functions as a regulatory element interacting with IκBα. This complex shuttles between nucleus and cytoplasm, but in unstimulated cells shows relative cytoplasmic sequestration. Activation of the classical NF-κB pathway induces phosphorylation and degradation of IκBα and nuclear translocation of NF-κB, in which RelA exerts potent transcription factor activity (23). Inhibition of this pathway by microbial pathogens is increasingly evident (24) and currently a key focus of research on host-pathogen interactions that may provide opportunities for novel therapeutic interventions in the future.

In this study, in view of the limited evidence showing HIV-1 induced inhibition of innate immune cellular activation and in particular, possible inhibition of the NF-κB activation pathway that plays a central role in mediating innate immune cellular responses, we used ex vivo replication-competent HIV-1 infection of human MDM to investigate the effect of HIV-1 on activation of selected innate immune signaling pathways and downstream immune responses. Importantly, innate immune signaling in macrophages induces complex and wide-ranging transcriptional responses (25) that include expression of cytokines, inducible intracellular enzymes, cell surface molecules, plasma proteins, cytoskeletal components, and factors that regulate cell cycle or apoptosis. Despite previous mechanistic reports of HIV-1-mediated inhibition of innate immune signaling, the effect of HIV-1 infection in macrophages on the broad repertoire of innate immune response elements has not previously been assessed. Therefore, in addition to testing the hypothesis that HIV-1 inhibits innate immune signaling in a more physiological macrophage model, we have extended the assessment of effects on downstream immune response genes using whole genome transcriptional profiling.

Materials and Methods

PBMC and MDM

Human blood samples were obtained from healthy volunteers for isolation of PBMC and production of MDM cultures. The study was approved by the joint University College London/University College London Hospitals National Health Service Trust Human Research Ethics Committee, and written informed consent was obtained from all participants. PBMC were prepared by density-gradient centrifugation of heparinized blood with Lymphoprep (Axis-Shield), according to the manufacturer’s instructions, and MDM were prepared, as previously described (26). PBMC were seeded (2 × 106/cm2) for adhesion onto tissue culture plastic (Nunc). After 1 h at 37°C, nonadherent cells (lymphocytes) were removed and adherent monocytes were incubated in RPMI 1640 (Life Technologies Invitrogen) with 10% autologous heat-inactivated human serum (HS) supplemented with 20 ng/ml M-CSF (R&D Systems) for 3 days. The medium was then refreshed (without additional M-CSF), removing any remaining nonadherent cells. Typically, this protocol yields 105 MDM/cm2. After 6-day culture, 10% autologous HS was replaced with 5% normal HS (NHS; Sigma-Aldrich).

HIV-1 strains and cell culture infections

The CCR5-tropic HIV-1 strain, Ba-L, was propagated in PBLs. Nonadherent PBLs from MDM preparations were cultured for 3 days in RPMI 1640 with 20% FCS and 0.5 μg/ml PHA (Sigma-Aldrich) to generate activated T cells. These cells were then inoculated with HIV-1 Ba-L, using a multiplicity of infection (MOI) of 1, and subsequently cultured in RPMI 1640 with 20% FCS and 20 U/ml IL-2 (PeproTech). At 3- to 4-day intervals, the cell culture supernatants were collected and additional PHA-stimulated PBMC were added to maintain the cell density at 1 × 106/ml. Cell culture supernatants containing PBMC-derived HIV-1 were filtered through 0.45-μm filters (Millipore) and used to inoculate 6-day-old MDM cultures overnight (MOI 1), refreshing the medium on the following day. Culture supernatants from infected MDM, containing MDM-derived HIV-1 Ba-L, were collected at weekly intervals, centrifuged at 400 × g for 5 min, and filtered (0.45-μm Millipore filter) to remove cellular debris. The CCR5/CXCR4 dual-tropic HIV-1 strain, 89.6, and the CXCR4-tropic HIV-1 strain, NL4-3, were derived from infectious clones by transient transfection of HEK293t producer cell cultures using Fugene 6 transfection reagent (Roche), according to manufacturer’s instructions, and collecting culture supernatants 72 h later. All virus suspensions were ultracentrifuged through a 20% sucrose buffer and resuspended in RPMI 1640 with 5% NHS, for subsequent infection of MDM. All virus preparations were titrated on the NP2 astrocytoma cell line stably transfected with CD4 and CCR5 or CXCR4, as previously described (27). Briefly, adherent NP2/CD4/CCR5 and NP2/CD4/CXCR4 cells, cultured in DMEM (Life Technologies Invitrogen) with 5% FCS, 1 μg/ml puromycin (Sigma-Aldrich), and 100 μg/ml G418 (Sigma-Aldrich), were inoculated with serial log-fold dilutions of viral stocks. The cells were incubated with the virus for 2 h at 37°C before removing the inoculum and replacing the medium. Infection was detected by p24 immunostaining 72 h later (described below). MDM-derived HIV-1 Ba-L, and HEK293t-derived HIV-1 NL4-3 and 89.6 strains were used for overnight inoculation (MOI 3–5) of 6-day-old MDM cultures in the experimental model presented in this study.

Intracellular p24 staining

Cell cultures were fixed with ice-cold methanol:acetone (1:1) for 5 min, then incubated with mouse anti-HIV-1 gag (p24) mAbs (E365/366; National Institute for Biological Standards and Control), followed by goat anti-mouse Ig Ab conjugated to β-galactosidase (Southern Biotechnology Associates) for 1 h each at room temperature. The β-galactosidase substrate solution (0.5 mg/ml 5-bromo-4-chloro-3-indolyl-β-galactopyranoside in PBS containing 3 mmol/L potassium ferricyanide, 3 mmol/L potassium ferrocyanide, and 1 mmol/L magnesium chloride) was then added and incubated overnight at 37°C to develop a blue stain. Positively staining cells were counted microscopically to provide a virus titer (focus-forming U/ml) or to calculate the number and proportion of infected cells.

RT-PCR detection of HIV-1 transcripts

MDM culture lysates collected in RLT buffer (Qiagen) were used to purify total RNA with RNAeasy spin columns (Qiagen), according to manufacturer’s instructions. A quantity amounting to 1 μg of total RNA was used to generate cDNA with the First Strand cDNA synthesis kit (New England Biolabs) using oligo(dT) primers. The product was then heat inactivated (95°C, 5 min) and subjected to PCR (95°C denaturing temperature, 55°C annealing temperature, and 72°C extension temperature for 30 cycles) using primers (Table I⇓) for a conserved unspliced HIV-1 gag product (SK38 and SK39) (28) and spliced HIV-1 tat, rev, nef product (413MOD and P659) (29). Samples from HIV-1 Ba-L-infected MDM were also subjected to sequencing reactions (Lark Technologies) for the HIV accessory genes nef, vpu, and vpr using primers (Table I⇓) based on the published HIV-1 Ba-L sequence (accession no. AB253432). All three full-length accessory genes were sequenced successfully and had 95–100% amino acid homology to the expected sequences (data not shown).

View this table:
Table I.

Primer sequences

Cell culture viability testing

Relative viability of HIV-infected and uninfected MDM cultures was compared by using a MTT assay. Cell culture supernatants were removed and replaced with 1 mg/ml MTT (Sigma-Aldrich) in serum-free medium (1 ml/106 cells) and incubated for 3 h at 37°C. This was then replaced with DMSO (Sigma-Aldrich) to permeabilize cells in a plate shaker for 5 min (room temperature). Relative formazan concentration was quantified by spectrophotometry (OD540 nm) within 0.1–1.8 linear range.

Innate immune cellular activation of MDM

Ultrapure LPS from Escherichia coli O111:B4 (InvivoGen) and N-palmitoyl-S-[2,3-bis(palmitoloxy)-(2RS)-propyl]-Cys-Ser-Lys4 (Pam3CSK4; Axis-Shield) were used as minimal innate immune stimuli of TLR4 and TLR2, respectively. Both stimuli were prepared in RPMI 1640 with 5% NHS. MDM cultures were stimulated for 0–60 min for detection of innate immune cellular activation and 3–24 h for detection of transcriptional responses and cytokine production. In selected experiments, MDM were stimulated with 10 ng/ml human rIFN-γ (PeproTech) 24 h before or after infection with HIV-1.

Western immunoblotting analysis of HIV-1 gag expression and innate immune signaling

Cell lysates from MDM cultures were collected directly into SDS sample buffer (62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, 0.01% bromphenol blue, and 5% 2-ME), sonicated, and heated (100°C for 5 min) before PAGE (4–12% gradient gels) and transfer onto Amersham Hi-bond membranes (GE Healthcare). Membranes were blocked for 1 h in 5% milk powder in TBS with 0.05% Tween 20 (Sigma-Aldrich) and then immunoblotted sequentially with primary Ab overnight (4°C), biotin-conjugated secondary Ab for 2 h (room temperature), and HRP-conjugated streptavidin for 1 h (room temperature), all prepared in TBS/Tween 20 with 1% milk powder. Membranes were washed with TBS/Tween 20 after each step. Immunostains were developed with Amersham ECL reagent (GE Healthcare) and visualized on Amersham Hyperfilm ECL (GE Healthcare), according to manufacturer’s instructions. Mouse anti-HIV-1 gag (p24) Ab (E365/366; NIBSC), rabbit anti-IκB-α (Cell Signaling Technology), rabbit antiphosphorylated ERK1/2 (clone 197G2; Cell Signaling Technology), rabbit antiphosphorylated p38 MAPK (Cell Signaling Technology), and rabbit anti-actin (Sigma-Aldrich) were used as 1° Abs. Biotin-conjugated sheep anti-mouse IgG and sheep anti-rabbit IgG (DakoCytomation) were used as secondary Abs. HRP-conjugated streptavidin was obtained from R&D Systems.

Quantitative confocal immunofluorescence analysis of NF-κB nuclear translocation

Immunofluorescence staining of NF-κB RelA (p65) was performed, as described previously (26). HIV-1-infected and control MDM cultured on glass coverslips were subjected to immunofluorescence staining with rabbit polyclonal anti-NF-κB RelA (C-20) (Santa Cruz Biotechnology) with Alexa- Fluor (AF)655-conjugated goat anti-rabbit IgG (Invitrogen), mouse anti-HIV-1 Gag (p24) Ab (E365/366; NIBSC) with AF488-conjugated goat anti-mouse IgG (Invitrogen), and nuclear counter staining with 4,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Subsaturating fluorescence images were captured on a Leica SP2 confocal microscope with a pin hole of 1 Airy (114.5 μm), scan speed of 400 Hz, and four-frame averaging. Sequential image acquisition was used to give separate image files for DAPI (excitation 405 nm, emission 400–450 nm), AF488 (excitation 488 nm, emission 510–530 nm), and AF555 (excitation 543 nm, emission 560–580 nm). Image analysis was performed with Metamorph v7.17 (Molecular Devices) to quantify nuclear:cytoplasmic ratios NF-κB RelA staining and proportion of cells demonstrating positive colocalization of DAPI/RelA (AF655) staining (correlation coefficient >0.5) as markers of NF-κB nuclear translocation (30).

Transcriptional profiling by DNA microarray

Total RNA was collected from MDM cultures, as described above. RNA integrity and concentrations were analyzed electrophoretically (Agilent RNA 6000 Nano assay/Agilent 2100 bioanalyzer) (31) and used to generate firstly amplified cDNA and subsequently Cy5-labeled cRNA using the Agilent Low RNA Input Linear Amplification Kit. This was mixed in equal measure with Cy3-labeled reference cRNA derived similarly from a universal human reference RNA mix (Stratagene), and then used to hybridize Agilent 4 × 44K whole human genome cDNA microarrays, according to manufacturer’s instructions (www.agilent.com). Array images were acquired with Aglient’s dual-laser microarray scanner G2565BA (5-μm resolution), and signal data were collected with dedicated Agilent Feature Extraction software (v9.5.1). Log2-transformed red (Cy5) and green (Cy3) data from each channel were first normalized separately, to give equal mean and SD. Preliminary analysis of these normalized data showed that there were no significant differences when comparing any gene on the reference channel between all samples. No further normalization was therefore conducted on the data. Gene expression values were compared by significance analysis of microarray (SAM) using MultiExperiment Viewer v4.0 (32). Significant differences in gene expression across three separate experiments (with different donor cells) were identified using a 1% false detection rate (FDR). SAM tests were performed both with and without pairing of individual experimental/donor samples. Gene lists of interest were annotated using the functional annotation clustering module of the online bioinformatics database, DAVID (http://david.abcc.ncifcrf.gov) (33, 34). In this analysis, gene lists were restricted to those with refseq accession numbers for which contemporary functional annotation is available. Microarray data are available in the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession no. E-MEXP-1904.

Cytokine measurements

The concentration of cytokines in MDM culture supernatants was quantified using a cytometric bead array inflammation kit for TNF-α, IL-1, IL-6, IL-8, IL-10, and IL12p70 (BD Biosciences) using the FACSArray Bioanalyzer System (BD Biosciences), according to manufacturer’s instructions.

Results

HIV-1 infection of MDM

The first objective of this study was to establish a sustainable model of productive HIV-1 infection in macrophages. Day 6 MDM cultures, inoculated overnight with the CCR5-tropic strain Ba-L (MOI 3–5), and subsequently stained for intracellular HIV-1 gag (p24) 7 days later, typically showed 100% p24-positive cells (Fig. 1⇓A). All subsequent experiments were performed at this time point. On occasion, there was incomplete (30–70%) p24-positive staining of cell cultures. Therefore, we performed this assessment of successful HIV-1 infection of MDM in all our experiments and excluded heterogeneously infected (as judged by p24 positivity) cell cultures from this study. Productive HIV-1 infection in this model was demonstrated further by RT-PCR detection of spliced HIV-1 transcript (Fig. 1⇓B) in total RNA samples and by detection of precursor (Pr)55gag protein (Fig. 1⇓C) using immunoblotting of protein lysates from HIV-1 Ba-L-inoculated MDM cultures 7 days after infection. In this model, MDM were not able to support productive HIV-1 infection with the prototypic CXCR4-tropic strain, NL4-3. HIV-1-spliced transcript and Pr55 protein were not detected, and p24 staining was positive in <1% of cells in NL4-3-inoculated cultures. There was no appreciable cytopathic effect of HIV-1 infection in MDM, demonstrated by comparable mitochondrial function in uninfected cells and HIV-1-infected MDM 7 days after infection (Fig. 1⇓D), an observation that is in keeping with the majority of previous studies (31).

FIGURE 1.
FIGURE 1.

A, Intracellular p24 (gag) staining 7 days after inoculation with HIV-1 shows uniform HIV infection of all MDM inoculated with the CCR5-tropic strain Ba-L, compared with sporadic (<1%) positive staining in cell cultures inoculated with the CXCR4-tropic strain NL4-3 and uninfected control MDM. B, Active viral replication in HIV-1 Ba-L-inoculated MDM is demonstrated by RT-PCR of spliced (P659/413MOD) and unspliced (SK38/SK39) products of HIV-1 transcription (B) and by Western immunoblot analysis of concurrent Pr55gag and p24 expression (C). Actin expression is demonstrated in each assay to show equivalent sample loading. D, Comparable mitochondrial function (MTT assay) in Ba-L-infected MDM compared with NL4-3-inoculated and control-uninfected cultures demonstrates that HIV-1 Ba-L infection and replication in MDM are not cytopathic (bars shown mean ± SD of five separate experiments).

Innate immune cellular activation in HIV-1-infected MDM

The range of innate immune cellular activation pathways uses a complex network of receptors, adaptor molecules, and kinases that appear to converge onto selected intracellular signaling events, detectable by Western blotting. Therefore, we used this strategy to examine degradation of IκBα, and phosphorylation of p38, ERK1/2, and JNK in a time-course study of MDM stimulated with LPS (Fig. 2⇓A). In uninfected MDM, the IκBα signal is diminished at 20 and 60 min, and regenerated by 120 min. Rapid p38 and increased ERK1/2 phosphorylation are also evident, but we did not detect JNK phosphorylation (data not shown). This pattern of signaling events was replicated exactly in control MDM cultures inoculated with HIV-1 NL4-3, a strain that is unable to establish host cell infection. In HIV-1 Ba-L-infected MDM, p38 and ERK1/2 phosphorylation was comparable to controls, but degradation of IκBα was attenuated, albeit with the same time-course profile. These results support the hypothesis that HIV-1 infection of MDM may have a selective inhibitory effect on innate immune signaling pathways downstream of the LPS receptor complex and MyD88 adaptor protein. IκBα binds to the NF-κB p50-RelA heterodimer and inhibits the transcription factor activity of RelA, in large part by promoting its cytoplasmic sequestration. In the classical NF-κB activation pathway, IκBα is phosphorylated by the IκB kinase and is degraded by ubiquitination, allowing for nuclear translocation of the NF-κB complex. Therefore, we performed quantitative confocal immunofluorescence assays of NF-κB RelA nuclear translocation, to assess the functional consequence of attenuated IκBα degradation in HIV-1-infected MDM.

FIGURE 2.
FIGURE 2.

A, Western immunoblot analysis of the time course of intracellular signaling pathways (IκB degradation at 20–60 min, p38 and ERK1/2 phosphorylation) in response to TLR4 stimulation with LPS shows equivalent cellular activation in NL4-3-inoculated and control-uninfected MDM cultures. The p38 and ERK1/2 phosphorylation is comparable in HIV-1 Ba-L-infected MDM, but IκB degradation is modestly attenuated (actin levels are shown to confirm equivalent sample loading). B, Confocal immunofluorescence studies of NF-κB RelA (p65) in HIV-1 Ba-L-infected and uninfected control MDM after 1-h stimulation with LPS (0–100 ng/ml) show equivalent cytoplasmic staining of RelA in unstimulated cells, but greater nuclear translocation in control MDM with escalating dose of LPS (scale bar represents 50 μm). Representative figures are shown of multiple experiments.

RelA exhibits mostly cytoplasmic staining in control and HIV-1 Ba-L-infected MDM before stimulation (Fig. 2⇑B). In response to increasing concentrations of LPS, greater nuclear staining is clearly evident. Quantitative image analysis, by measurement of the ratio of nuclear:cytoplasmic RelA staining and the proportion of cells showing nuclear RelA staining, was then used to compare the dose response to LPS in HIV-1-infected and uninfected MDM cultures (Fig. 3⇓, A–D). We found that nuclear translocation of RelA was attenuated significantly in HIV-1-infected MDM across the LPS dose range (1–100 ng/ml). This effect was evident in 9 of 10 separate experiments with cells from different donors (Fig. 3⇓E). However, testing multiple donors in this way provided a powerful illustration of the natural variance of this response and the effect of HIV-1 infection. The proportion of cells showing RelA nuclear translocation in control (uninfected) MDM cultures stimulated with 10 ng/ml LPS ranged 17–97%, and reduced by a mean value of 28.9% (95% confidence interval of 9.8–46.8% inhibition) in HIV-1-infected MDM. The specificity of our observations was also tested in MDM infected with the dual-tropic HIV-1 strain 89.6 and a synthetic TLR2 stimulus (Pam3CSK4) and showed similarly attenuated NF-κB RelA nuclear translocation (Fig. 3⇓F), suggesting that HIV-1-dependent attenuation of NF-κB activation in response to innate immune stimuli may show broad strain and stimulus specificity.

FIGURE 3.
FIGURE 3.

Quantitative image analysis of confocal nuclear and RelA staining is presented as percentage of RelA-positive nuclei (A, C, and E) and nuclear:cytoplasmic (N:C) ratios (B, D, and F) in MDM stimulated with a dose range of LPS (A–D) or Pam3CSK4 (F). Data points represent image analysis from five separate high power fields, with best-fit lines across the stimulus dose range. Data from A and B and C and D show two paired examples of response to LPS stimulation, and F shows example of response to Pam3CSK4, from multiple experiments. In each of these experiments, HIV-1 Ba-L- or HIV-1 89.6-infected MDM showed attenuated RelA nuclear translocation dose responses to innate immune stimulation (p < 0.01, ANOVA). E, Shows summary data from 10 separate experiments of RelA nuclear translocation in HIV-1-infected and control MDM stimulated with 10 ng/ml LPS. Data points represent image analysis from five separate high power fields, and lines indicate paired HIV-1-infected and control MDM. Analysis of paired responses shows less RelA nuclear translocation in HIV-1-infected MDM in 9 of 10 experiments (p < 0.01, Wilcoxon signed rank test).

Innate immune responses by macrophages are known to be augmented by IFN-γ from T cells in the classical Th1-type paradigm for adaptive immune responses. Therefore, we also investigated the effect of IFN-γ priming on activation of the NF-κB pathway in HIV-1 Ba-L-infected MDM. We found that RelA nuclear translocation in HIV-1-infected MDM was enhanced by prestimulating MDM (24 h earlier) with 10 ng/ml IFN-γ and equivalent to control (HIV-1-uninfected) MDM (Fig. 4⇓, A and B). In view of this finding, we considered the possibility that some variability in HIV-1-mediated inhibition of NF-κB activation may be due to the presence of IFN-γ, potentially as a result of minor T cell contamination of MDM cultures. We therefore analyzed all cell culture supernatants from the experiments performed in this study for IFN-γ, but found none detectable by ELISA at a sensitivity of 10 pg/ml (data not shown). We also considered the mechanism by which IFN-γ-attenuated NF-κB activation in response to LPS is corrected in HIV-1-infected cells. This may be due to priming of the innate immune activation pathway directly or by an effect of IFN-γ on HIV-1. Comparison of LPS-induced nuclear translocation of NF-κB RelA in uninfected MDM with and without IFN-γ prestimulation showed no significant differences (Fig. 4⇓, C and D), suggesting that there is no direct effect independent of HIV-1. However, in keeping with previous reports (35), we found that addition of IFN-γ to HIV-1 Ba-L-infected MDM cultures potently inhibited HIV-1 replication (Fig. 4⇓, E and F), supporting the hypothesis that IFN-γ priming of NF-κB activation in this model is mediated through an inhibitory effect on HIV-1. Further detailed study of this mechanism is not addressed in this work.

FIGURE 4.
FIGURE 4.

Quantitative image analysis of confocal nuclear and RelA staining, presented as percentage of RelA-positive nuclei (N:C) ratios (A and C) and nuclear:cytoplasmic (B and D) in MDM stimulated with a dose range of LPS. MDM pretreated with 10 ng/ml IFN-γ 24 h before stimulation with a dose range of LPS show equivalent RelA nuclear translocation responses in HIV-1-infected and control-uninfected MDM (A and B). Similar IFN-γ pretreatment does not enhance RelA nuclear translocation in uninfected MDM (C and D). Data points are derived from image analysis from five separate high power fields, with best-fit lines across the stimulus dose range, and are representative of three separate experiments. The inhibitory effect of IFN-γ on HIV-1 replication in MDM is shown by significant (p < 0.01, ANOVA) reduction of p24-positive cells in MDM cultures stimulated with 10 ng/ml IFN-γ 24 h after inoculation (E and F). Bars represent mean ± SD of three separate experiments (E), and images (F) show representative p24 staining of IFN-γ-treated and untreated MDM cultures infected with MOI 5.

Innate immune transcriptional responses in HIV-1-infected MDM

Innate immune stimulation of MDM is known to invoke wide-ranging transcriptional responses (25), mediated by NF-κB and other transcription factors. In view of the apparent attenuation of NF-κB activation in HIV-1-infected MDM, we tested the hypothesis that resultant transcriptional responses would also be affected. Therefore, genome-wide transcriptional profiling was performed in unstimulated cells, and after 3- and 24-h stimulation with LPS (10 ng/ml), to make a comprehensive assessment of the effects of HIV-1 on this response. Significant gene expression changes were identified across three separate (different donor) experiments by SAM (1% FDR) comparison of transcriptional profiles from unstimulated MDM with those from 3- and 24-h stimulated cells, for HIV-1-infected and uninfected cultures separately. Gene expression changes were clearly greater at 3 h (∼400 genes up-regulated and 100 genes down-regulated) when compared with 24 h (∼40 genes up-regulated and 15 genes down-regulated). The overall profile of significant changes in gene expression levels, as assessed by frequency and magnitude compared with unstimulated cells, was comparable in HIV-1-infected and uninfected MDM (Fig. 5⇓, A and B, and supplementary figure S1).5 The transcriptional responses to LPS were then interrogated qualitatively by aligning all significantly affected genes in an expression matrix showing mean fold change in 3- or 24-h LPS-stimulated cells compared with corresponding HIV-1-infected or uninfected unstimulated MDM (Fig. 5⇓C). Functional annotation of these genes was performed by identifying statistically overrepresented gene ontology clusters using the online DAVID bioinformatics database (33, 34) and indicating the alignment of individual genes within the expression matrix. This analysis shows LPS induced up-regulation of a wide repertoire of genes that cluster together within functionally related immune response ontology groups, which demonstrate highly significant enrichment in comparison with the whole human genome (Fig. 5⇓C, supplementary figure S2, and supplementary Table I).5 By contrast, LPS induced down-regulated genes cluster within ontological groups that are not directly related to immune responses and not significantly enriched. Genome-wide transcriptional responses of HIV-1-infected and uninfected MDM after LPS stimulation for 3 or 24 h appear to be similar in this analysis also (Figs. 5⇓C and 6⇓, A and B).

FIGURE 5.
FIGURE 5.

Analysis of mean fold changes (three separate experiments) in expression of all significantly (SAM, 1% FDR) up-regulated (>1) or down-regulated (<1) genes at 3 and 24 h after LPS stimulation (10 ng/ml) compared with unstimulated cells in HIV-1 Ba-L-infected and uninfected MDM cultures shows comparable overall profile (A and B). Significantly up-regulated (>0) and down-regulated (<0) genes in the transcriptional response to LPS (at 3 and 24 h) from HIV-1-infected and uninfected MDM are aligned for comparison (C). Gene ontology term associations for each gene (row) are shown for significantly (p < 0.05) overrepresented functionally related gene clusters, using the online DAVID functional annotation clustering analysis tool. Gene ontology terms are arranged in order of statistical significance (p values 10−14–10−2). This expression matrix and analysis are restricted to genes with refseq accession numbers, for which contemporary functional annotation is available.

FIGURE 6.
FIGURE 6.

The proportion of genes up-regulated by LPS (derived from three separate experiments) associated with each significantly overrepresented gene cluster (A) and the fold enrichment of each gene cluster compared with the whole human genome (B) is comparable in HIV-1-infected and uninfected MDM.

To assess the effect of HIV-1 infection on LPS responses directly, the expression levels of the cumulative set of 581 LPS-responsive (up and down-regulated) genes were compared in HIV-1-infected and uninfected MDM at 3 and 24 h after LPS stimulation (Fig. 7⇓). Statistically significant differences in gene expression were identified in 85 of the LPS-responsive genes by paired testing of HIV-1-infected and uninfected MDM from individual donors. The major effect was attenuation of a proportion of up-regulated LPS-responsive genes in HIV-1 Ba-L-infected MDM compared with controls (Fig. 7⇓, A and B), and the extent of attenuation varied between 20 and 70% of control values, corresponding to 1.2- to 3-fold reduction in expression levels. Alignment of these genes with the gene ontology groups identified by the functional annotation clustering analysis of LPS-responsive genes showed that LPS responses attenuated by HIV-1 included a range of proinflammatory cytokines and immune-related genes (Fig. 7⇓, A and B). These differences were not evident when comparing HIV-1-infected and uninfected MDM as two groups without pairing individual donor samples, suggesting that differences in gene expression attributable to HIV-1 infection were smaller than the variability between different donors.

FIGURE 7.
FIGURE 7.

Differences in the transcriptional response to LPS between HIV-infected and uninfected MDM are tested by analysis of LPS-responsive genes in paired HIV-infected and uninfected samples from three separate donors for statistically significant differences (SAM, 1% FDR) at 3-h (A) and 24-h (B) LPS stimulation. The fold difference in HIV-infected MDM compared with uninfected cells, in each of three separate experiments, is shown together with the gene symbol and gene ontology term associations.

The release of inflammatory cytokines represents one of the major functional outcomes of macrophage innate immune responses. It was therefore of significant interest that expression levels of IL-1, IL-6, IL-8, and IL-12 were included among the LPS transcriptional responses attenuated by HIV-1 infection. To establish whether these transcriptional response differences were translated to significant differences in protein levels, we quantified inflammatory cytokine concentrations in culture supernatants collected from the same transcriptional profiling experiments in unstimulated and 3- and 24-h LPS-stimulated MDM with and without 7 days HIV-1 infection (Fig. 8⇓). In addition to the cytokines highlighted by the transcriptional profiling differences, TNF-α and IL-10 levels were measured as well-established components of MDM cytokine responses to LPS that were not found to have attenuated gene expression levels in HIV-1-infected MDM. Significant concentrations of IL-8, but none of the other cytokines tested, were detectable in unstimulated MDM culture supernatants and were unchanged by HIV-1 infection. Concentrations of TNF-α, IL-6, IL-8, and IL-10 clearly increased following LPS stimulation, but were equivalent in HIV-1-infected and control samples. Similarly, no significant difference was evident in levels of IL-1β, which increased just above the detection threshold in response to LPS. IL-12p70 was not detected in any samples, an observation in keeping with previous analyses of this MDM model (36, 37). We undertook additional experiments to measure the same cytokines in culture supernatants after 6-h stimulation with LPS. These experiments also showed no significant difference attributable to HIV-1 infection (data not shown).

FIGURE 8.
FIGURE 8.

The concentrations of selected cytokines (A–F) in cell culture supernatants of HIV-1-infected and control-uninfected MDM at baseline and after stimulation with LPS for 3 and 24 h are comparable. Data represent mean ± SD of three separate experiments. Shaded area represents data values below the detection threshold of the standard curves used in this assay.

Discussion

In the present study, we aimed to address the hypothesis that HIV-1 infection of macrophages inhibits innate immune cellular activation and may therefore have significant effects on downstream immunological and host-defense responses, and hence contribute to immunodeficiency.

An ex vivo model was established using replication-competent HIV-1 for uniform infection of MDM cultures. This model avoids some of the limitations of previous reports using rHIV-1 proteins, expression vector systems with individual HIV-1 components, myeloid leukemia cell lines used as models for macrophages, and primary cells from HIV-1-infected patients that generally are infected with HIV-1 at low frequency (1). We principally used the long-established laboratory CCR5-tropic HIV-1 Ba-L strain and found that we were able to generate reproducible productive infection without cytopathic effect. This strain was favored over primary CCR5-tropic HIV-1 strains because although such primary strains are readily accessible, they are not easily propagated to adequate high titer virus required for the experimental scale used in this study. HIV-1 Ba-L is not available as a molecular clone, but is propagated in PBMC and therefore prone to genetic drift. Like some other laboratories (38), we alternately passage Ba-L in MDM to retain its macrophage tropism and exert some selection pressure against significant genetic mutations or deletions. Selected accessory protein genes (nef, vpu, vpr), which have been previously reported to interact with cellular innate immune signaling pathways, were sequenced from infected MDM samples to ensure that they were expressed and did not show marked differences with published Ba-L sequences. It is also important to note that in ex vivo models, MDM display heterogenous biological properties as a result of alternative differentiation protocols (36). We used M-CSF-differentiated MDM that are known to support HIV-1 replication better than GM-CSF-differentiated MDM (39, 40, 41), but typically show less inflammatory capacity (36). This feature is illustrated by the reported deficiency of IL-12 cytokine secretion by LPS-stimulated M-CSF-differentiated MDM that is also evident in our results (37).

Innate immune responses to LPS were studied because this is the best-characterized and most potent minimal stimulus for MDM, which is known to signal mainly through TLR4. The effect of HIV-1 infection on TLR signaling pathways, genome-wide transcriptional responses, and inflammatory cytokine secretion was investigated. Our results show that established HIV-1 infection in MDM has a selective inhibitory effect on the classical NF-κB activation pathway, reflected in diminished IκBα degradation and NF-κB RelA nuclear translocation. This effect is also evident in MDM infected with an alternative HIV-1 strain 89.6 and alternative innate immune stimulation with Pam3CSK4, a TLR2 ligand. By contrast, the MAPK pathway involving phosphorylation of p38 is not affected. These findings are consistent with existing data in other model systems that show inhibitory interactions between the HIV-1 accessory proteins (1). However, inhibition of NF-κB activation is not complete. The inhibitory effect exhibits considerable experimental/donor variability and is rescued by prior exposure to IFN-γ, possibly reflecting the strong inhibition of viral replication induced by this cytokine. The attenuation of innate immunity may therefore be aggravated by the widespread deficiency of Th1 responses that is the hallmark of progressive HIV-1 immunodeficiency. Importantly, we did not detect any endogenously produced IFN-γ that may have been released by contaminating T cell populations and confounded our results.

The inhibition of the NF-κB signal was associated with attenuation of the transcriptional response to LPS. However, this decrease was restricted, both in terms of the number of LPS-responsive genes affected (85 of 581 gene expression changes) and the magnitude of the effect. Indeed, this effect was smaller than the interdonor variability observed, because the differences could only be detected using paired sample analysis (with/without HIV-1 infection in cell cultures from same experiment/donor). The differences in transcription observed for three key inflammatory cytokines, IL-1β, IL-6, and IL-8, could not be detected at protein level, suggesting that either the magnitude of transcriptional differences detected is not biologically significant or the existence of compensatory posttranslational regulatory mechanisms, which are acting to preserve the core innate immune response. Furthermore, genome-wide bioinformatic analysis shows that the functional clusters of genes in the innate immune transcriptional response to LPS are substantially the same in HIV-1-infected and uninfected MDM, suggesting that from a systems perspective, LPS responses are preserved in our model of HIV-1-infected MDM. The classical NF-κB activation pathway is thought to be a key signaling pathway for innate immune activation of macrophages and can regulate inducible expression of wide-ranging genes involved in immune responses and cell cycle regulation, including apoptosis (23). The effects of HIV-1 infection on LPS-induced gene expression in this model were therefore unexpectedly modest. Our observations suggest that sufficient redundancy may exist in innate immune signaling to compensate for relative inhibition of the NF-κB pathway in macrophages. This hypothesis is supported by the finding that other signal transduction pathways, such as the MAPK pathway involving p38 phosphorylation (42), that contribute to innate immune responses are not affected by HIV-1 infection in our model.

Our results contrast with reports of deficient TNF-α responses to LPS in the myeloid leukemia HIV-1-infected U1 cell line compared with the uninfected parental U937 cell line (13, 14). This difference is most likely to reflect differences in the biology of leukemic cell lines and MDM (43), or heterogeneity between U937 subclones. Indeed, the variability observed in our extended experiments using cells from 10 different donors highlights the limitations of cell lines to model host-pathogen interactions in a physiologically relevant fashion. The results presented in this study are not incompatible with many reports that show impaired innate immune responses in ex vivo MDM or alveolar macrophages from HIV-infected patients. These cells are likely to harbor only low-frequency HIV-1 infection (1, 10, 11, 12). Therefore, their phenotype is unlikely to be due directly to HIV-1 infection, but to result from the in vivo effects of HIV-1 on other components of the immune system. For example, the effect of IFN-γ in our experiments shows clearly the close interrelationship between adaptive and innate immunity. T cell depletion in progressive HIV-1 infection is therefore very likely to compromise innate immune activation of macrophages.

In conclusion, our study shows preservation of innate immune transcriptional responses by HIV-1-infected macrophages in genome-wide functional clustering analysis, but also supports the hypothesis that HIV-1 infection of macrophages impairs both the major pathway of innate immune cellular activation and a subset of subsequent transcriptional responses, and leaves open the extent to which the ability of HIV-1 to manipulate macrophage immune responses may contribute to immunodeficiency and viral persistence. Further studies are in progress to investigate the ability of the HIV-1-infected macrophages to respond effectively to the wider range of minimal innate immune stimuli, as well as bacterial, fungal, and viral copathogens.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • 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 a Wellcome Trust fellowship (WT077161) to M.N.

  • ↵2 Address correspondence and reprint requests to Dr. Mahdad Noursadeghi, Infection and Immunity, University College London, Windeyer Building, 46 Cleveland Street, London W1T 4JF, U.K. E-mail address: m.noursadeghi{at}ucl.ac.uk

  • ↵3 Current address: Institute for Virology, University Hospital, Ulm, Germany.

  • ↵4 Abbreviations used in this paper: MDM, monocyte-derived macrophage; AF, Alexa Fluor; DAPI, 4′,6-diamidino-2-phenylindole; FDR, false detection rate; HS, human serum; MOI, multiplicity of infection; NHS, normal HS; SAM, significance analysis of microarray; Pam3CSK4, N-palmitoyl-S-[2,3-bis(palmitoyloxy)- (2RS)-propyl]-Cys-Ser-Lys4.

  • ↵5 The online version of this article contains supplementary material.

  • Received August 19, 2008.
  • Accepted October 27, 2008.

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