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(Journal of Leukocyte Biology. 2006;80:1111-1117.)
© 2006 by Society for Leukocyte Biology

Cytomegalovirus blocks intestinal stroma-induced down-regulation of macrophage HIV-1 infection

Akhil Maheshwari*, Lesley E. Smythies{dagger}, Xiaoyun Wu{dagger}, Lea Novak{ddagger}, Ronald Clements§, Devin Eckhoff§, Audrey J. Lazenby{ddagger}, William J. Britt* and Phillip D. Smith{dagger},¶,1

* Departments of Pediatrics,
{dagger} Medicine,
{ddagger} Pathology, and
§ Surgery, University of Alabama at Birmingham, Birmingham, Alabama, USA; and the
VA Medical Center, Birmingham, Alabama, USA

1 Correspondence: Department of Medicine (Gastroenterology), University of Alabama at Birmingham (ZRB 633), 703 19th Street South, Birmingham, AL 35294. E-mail: pdsmith{at}uab.edu

ABSTRACT

Intestinal macrophages, unlike macrophages from other tissues, do not support HIV-1 infection or produce proinflammatory cytokines. In vitro studies suggest this unique, functional phenotype is a result of the exposure of newly recruited blood monocytes to intestinal stromal products. However, in AIDS-related CMV colitis, mucosal macrophages express HIV-1 and proinflammatory cytokines. Therefore, we investigated the mechanism by which CMV confers permissiveness to HIV-1 and cytokine production on intestinal macrophages. We show that intestinal stroma-conditioned media (S-CM) down-regulated monocyte-derived macrophage infection by HIV-1 (pseudotyped with YU2 envelope or vesicular stomatitis virus glycoprotein) and production of TNF-{alpha}, but preinfection of the cells with CMV reversed this down-regulation, enhancing HIV-1 infection, p24 production, and TNF-{alpha} release. The ability of CMV to reverse S-CM down-regulation of macrophage HIV-1 infection was blocked by anti-TNF-{alpha} antibodies and over-ridden by exogenous TNF-{alpha}. Immunohistochemical analysis of monocyte-derived macrophages exposed to CMV and HIV-1 (YU2 pseudotype) revealed that the cells infrequently contained CMV and HIV-1 viral proteins. In addition, analysis of colon tissue sections from HIV-1-infected patients with CMV colitis showed that some macrophage-like cells contained CMV and TNF-{alpha} proteins, others contained HIV-1 and TNF-{alpha} proteins, but cells infrequently contained CMV and HIV-1 proteins. These results indicate that CMV blocks stromal product inhibition of HIV-1 infection in macrophages, and this inhibition is mediated, at least in part, by CMV-induced TNF-{alpha} acting in trans to enhance HIV-1 infection.

Key Words: mucosa • intestinal macrophage • extracellular matrix • TNF-{alpha}

INTRODUCTION

The gastrointestinal mucosa is the largest lymphoid organ and reservoir of macrophages in the body [1 , 2 ]. Macrophages in this organ reside exclusively in the lamina propria, a region of the mucosa characterized by the absence of inflammation, despite the close proximity to immunostimulatory bacteria. To elucidate the role of lamina propria macrophages in the absence of intestinal inflammation, we have shown that lamina propria extracellular matrix (ECM; stroma) products, particularly TGF-ß and IL-8, recruit blood monocytes, the cell from which intestinal macrophages are derived [3 ]. The stromal products also induce a profound loss of monocyte innate response receptors and, via TGF-ß, profound down-regulation of, proinflammatory cytokines, while the cells retain bacteriocidal activity [4 5 6 ]. These findings are consistent with the concept that proinflammatory blood monocytes are recruited to the mucosa by stromal products, which then induce differentiation of the monocytes into noninflammatory intestinal macrophages with retained host defense function [7 ].

In addition to their unique phenotypic and functional profile, intestinal macrophages do not support HIV-1 infection in vitro [8 , 9 ]. In this regard, recent observations indicate that intestinal lamina propria lymphocytes, not macrophages, are the primary mucosal target cell for HIV-1 and SIV in acute infection [10 11 12 13 14 ]. The inability of intestinal macrophages to support HIV-1 replication is likely the consequence of exposure of blood monocytes, newly recruited to the mucosa, to lamina propria stromal products. In this regard, we have reported that incubation of monocyte-derived macrophages with stroma-conditioned media (S-CM) down-regulates the macrophages for HIV-1 RNA/DNA and p24 antigen expression [15 ].

In contrast to the profound inability of normal intestinal macrophages to support HIV-1 infection or produce proinflammatory cytokines, mucosal macrophages in AIDS patients with CMV colitis are associated with HIV-1 expression [16 ] and the production of proinflammatory cytokines, including TNF-{alpha} [17 18 19 ]. The inflammatory lesion in CMV colitis shares features with CMV lung and retinal disease, including the presence of viral inclusion cells and accumulation of mononuclear inflammatory cells and tissue necrosis, suggesting a common pathogenesis of involving the migration of blood mononuclear cells latently infected with CMV [20 21 22 23 ] to tissue sites. To understand the mechanism by which CMV infection confers permissiveness to HIV-1 and proinflammatory cytokine production in intestinal macrophages, we investigated the effect of CMV infection on HIV-1 replication and TNF-{alpha} production by moncytes induced by intestinal stromal products to differentiate into cells with an intestinal macrophage phenotypic and functional profile.

MATERIALS AND METHODS

Blood monocytes
PBMC from healthy HIV-1/CMV seronegative donors were isolated by Ficoll Hypaque sedimentation, enumerated by an automated cell counter (Beckman Coulter, Fullerton, CA), and then plated in serum-free RPMI in 48-well plates at a concentration of 1 x 106 monocytes/well [24 ]. After 1 h, the nonadherent lymphocytes were removed, and the media were replaced with RPMI containing 10% human AB serum, recombinant human M-CSF (rhM-CSF; 10 ng/mL, R&D Systems, Minneapolis, MN), 1% penicillin-streptomycin, and 50 µg/mL gentamicin.

Viruses
CMV, including a laboratory strain (AD169) [25 ] and a recent clinical isolate (TR; a kind gift of Dr. Jay Nelson, University of Oregon, Portland, OR), [26 ] were propagated in human telomerase RT-immortalized human foreskin fibroblasts using standard protocol [27 ]. Virus titers were determined by a quantitative immunofluorescence assay for CMV immediate-early protein-1 described previously [28 ]. All experiments with CMV included UV-inactivated virus [29 ] as control. Briefly, 3 x 106 pfu of virus were exposed to 150 mJ UV irradiation in a cross-linking chamber (Bio-Rad, Hercules, CA), followed by addition of sodium pyruvate to a final concentration of 5 mM to neutralize any free oxygen radicals produced during UV inactivation.

To study the effect of CMV infection on HIV-1 replication, a reporter HIV-1 with enhanced GFP (eGFP) was constructed using a protocol described previously [30 ]. Briefly, the eGFP reporter virus was derived from HIV-1 pNL4-3, gp120 envelope (Env) gene was deleted, and the eGFP gene was inserted between the Env and nef genes. To rescue nef gene expression, the internal ribosome entry site element was inserted between the eGFP and nef genes, which were expressed in bicistronic mRNA. To generate the YU2 Env and vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped virus, eGFP reporter virus proviral DNA was cotransfected with the YU2 Env or VSV-G expression plasmids (pMD-G) into 293T cells. After 72 h, the transfection cell culture supernatant was harvested, and the titer of the eGFP reporter virus was determined by eGFP expression in JC53BL cells derived from CXCR4+ HeLa cells.

Intestinal S-CM
Lamina propria stroma was generated from sections of normal human jejunum obtained from otherwise healthy subjects undergoing gastrojejunostomy for obesity as described previously [6 ]. Briefly, cell-depleted lamina propria stroma (1 g wet weight stromal tissue/mL) was cultured in RPMI without serum for 24 h. The harvested culture supernatant was filter-sterilized (0.2 µm syringe filter, Corning Inc., Corning, NY), and endotoxin and protease levels were measured by commercially available ELISA assays. The protein content of endotoxin-free and protease-free S-CM was determined, and the S-CM was stored frozen at –80°C.

Experimental protocol
To determine the effect of CMV on HIV-1 production by S-CM-treated macrophages, adherent monocyte-derived macrophages (hereafter referred to as macrophages) in 48-well plates (106 cells/well) were inoculated with CMV [multiplicity of infection (MOI)=1] in two sequential exposures at 48 and 72 h of culture. In each exposure, CMV was added to the macrophage cultures for 2 h, after which, the cells were washed three times and refreshed with RPMI plus AB serum and rhM-CSF, 10 ng/mL (R&D Systems). Parallel cultures of macrophages were mock-infected with medium or UV-inactivated CMV (MOI=1). CMV-exposed cells were cultured for 2 days and then exposed to varying concentrations of S-CM (5–500 µg/mL protein) for 2 or 24 h, washed, and inoculated with VSV-G or YU2 Env-pseudotyped, GFP-labeled HIV-1 (MOI=1) overnight. After 48 h, the experiment was terminated, and green fluorescent cells were enumerated by counting in a blinded protocol. Culture supernatants were assayed for HIV-1 p24 by ELISA.

To determine the effect of CMV on TNF-{alpha} production by S-CM-treated macrophages, macrophages were infected with CMV and treated with S-CM as above. However, instead of adding HIV-1, the cultures were inoculated with LPS, 1 µg/mL, for 1 h (without removing the S-CM). Subsequently, cells were washed, and S-CM was refreshed. The culture supernatants were harvested at 24 h and assayed for TNF-{alpha} by ELISA (R&D Sytstems). The frequency of CMV-infected cells in both experimental protocols was determined by immunofluorescence as described below.

Enumeration of CMV- and HIV-1-infected cells
CMV-infected macrophages were enumerated by quantitative immunofluorescence using one or more of the following antibodies: p63-27 (specific for IE-1, UL123, the major immediate early gene product-1), 65-8 (specific for pp65, UL83, a major tegument protein), or 36-17 (specific for pp150, UL32, a late protein in the tegument of the mature virion). The binding of the primary antibody was detected with Cy3-conjugated polyclonal goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA), diluted 1:300 in PBS with 1% FCS PCR serum (45 min at 37°C). Control cells were stained with mouse IgG of the same isotype (BD Biosciences, San Diego, CA). GFP-expressing, HIV-1-infected cells were enumerated by quantitative imunofluorescence. Cell nuclei then were stained with 4',6-diamidino-2-phenylindole (DAPI) and mounted for confocal microscopy.

Immunohistochemistry
Colon tissue sections from two AIDS patients with CMV colitis and normal colonic mucosa from a seronegative subject (negative control) were analyzed for CMV and HIV-1 antigens and TNF-{alpha} protein by immunofluorescence. Briefly, tissue sections were deparaffinized with xylene, hydrated in a series of graded ethanols, and rinsed in several changes of distilled water. Antigen retrieval was achieved by immersion of the sections in 10 mM sodium citrate buffer, pH 6.0, heated to 95°C for 5 min, and allowed to cool for 20 min. Sections next were treated with a serum-free protein-blocking solution (Dako Corp., Carpinteria, CA) and stained with biotinylated mouse anti-p24 antibodies (Beckman Coulter, Hialeah, FL), followed by FITC-conjugated avidin (Santa Cruz Biotechnology, CA), and sequentially, with goat anti-TNF-{alpha} (Santa Cruz Biotechnology), followed by donkey anti-goat Cy3 antibody (Jackson ImmunoResearch Laboratories); mouse anti-CMV pp150 followed by Cy3-conjugated polyclonal goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) and sequentially, with FITC-conjugated mouse anti-TNF-{alpha} (R&D Systems) or FITC-conjugated goat anti-p24 antibodies (Fitzgerald Industries International, Concord, MA); and appropriate controls. In this way, sections were dual-stained for CMV and TNF-{alpha}, HIV-1 and TNF-{alpha}, or CMV and HIV-1. All sections were stained for 2 h at room temperature, then counterstained with DAPI (15 min), and analyzed by confocal microscopy.

In situ hybridization
Mucosal cells containing HIV-1 transcripts were detected by in situ hybridization using our protocol described previously [16 , 17 ]. Briefly, deparaffinized colonic tissue sections from two AIDS patients with CMV colitis were permeabilized with proteinase K (100 µg/mL), acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine buffer (pH 8.0), and rehydrated in graded ethanols. After prehybridization in 50% formamide-1x Denhardt’s solution-2x saline sodium citrate (SSC)-50 mM Tris-l mM EDTA-0.5% mg/ml tRNA for 3 h at 40°C, the sections were incubated with a hybridization mixture containing the 35S-labeled cDNA probe (5x105 cpm/slide) encoding human TNF-{alpha} [17 ] labeled with 35S-uridine triphosphate (Promega Biotec, Madison, WI) overnight at 48°C. Control probe (sense with the same sequence but reverse orientation) was included in each hybridization. The slides were washed sequentially in 2x SSC; 2x SSC, 1 mM EDTA, 5 mM DTT, 0.1 Triton X at 56°C, and the same again, except with 0.1x SSC; and then treated with 40 µg/ml RNase-10 U/ml RNase T (Sigma Chemical Co., St. Louis, MO) at 37°C for 40 min, washed in 2x SSC, and dehydrated in graded ethanols. Slides were prepared for autoradiography with NT-4 emulsion (Kodak, Rochester, NY), developed after 3 days, and stained with H&E.

Statistical methods
Effects of different treatments were tested by one-way ANOVA or Kruskall-Wallis H test, using SigmaStat 3.1.1 software (Systat Software, Point Richmond, CA). Individual comparisons were made by t-test/Mann-Whitney U-test where appropriate. All data are expressed as mean ± SEM unless stated otherwise. A P value of <0.05 was considered significant.

RESULTS

CMV blocks S-CM down-regulation of macrophage HIV-1 infection
Intestinal macrophages are normally down-regulated for HIV-1 infection and proinflammatory cytokine production, likely through the inhibitory activity of lamina propria stromal products [6 , 15 ]. In contrast, the gastrointestinal mucosa in HIV-1-infected persons with CMV mucosal disease is characterized by local macrophage HIV-1 replication and cytokine production [16 17 18 ]. Therefore, we determined whether CMV infection of macrophages blocks the ability of S-CM to down-regulate macrophage HIV-1 infection and TNF-{alpha} production. We first established that 45.3 ±1.7% of the macrophages exposed to AD169 and 38.7 ±1.8% exposed to TR expressed CMV gene products (n=3), indicating CMV infection (data not shown). Next, mock- and CMV-infected cultures were treated with an optimal concentration of S-CM [15 ] and then inoculated with GFP-labeled HIV-1, pseudotyped with YU2 Env or VSV-G, and the number of HIV-1-infected macrophages was determined by quantitative immunofluorescence. As shown in Figure 1 , macrophages supported HIV-1 infection by YU2- and VSV-G-pseudotyped viruses. Pretreatment of the macrophages with S-CM caused a marked reduction in the number of macrophages that became infected with HIV-1, but when the cells were infected with CMV (AD169; upper panels) prior to S-CM treatment, the proportion of HIV-1-infected cells was increased above that of macrophages infected with HIV-1 without S-CM pretreatment (P<0.01 for pair-wise differences in and between each subgroup). Infection with CMV TR (lower panels), a recent clinical isolate, also reversed S-CM down-regulation of HIV-1 infection (P<0.001). Moreover, the addition of ganglicovir, an acyclic nucleoside analog (10 µM), to the cultures prior to CMV inoculation prevented CMV-related up-regulation of HIV-1 infection (data not shown). Although the pseudotyped HIV-1 used in these experiments resulted in only single-round infection, the amount of p24 released into the culture supernatants corroborated the infection results for VSV-G (Fig. 2 )- and YU2 (data not shown)-pseudotyped viruses. Importantly, the ability of CMV to block S-CM down-regulation of HIV-1 permissiveness was dependent on CMV gene expression, as the results with UV-irradiated virus were similar to mock-infection.


Figure 1
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  		Figure 1. CMV TR blocks intestinal stromal product down-regulation of macrophage HIV-1 infection. Macrophages were infected with pseudotyped HIV-1; pretreated with intestine-derived S-CM (500 µg/ml) and then infected with pseudotyped HIV-1; or infected with CMV AD169 or TR, then treated with S-CM, and then inoculated with pseudotyped HIV-1. Values are the mean ± SEM percent macrophages infected with HIV-1 in five high power fields (original, x200) in duplicate or triplicate wells from a representative experiment (n=3).

 

Figure 2
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  		Figure 2. CMV blocks intestinal stromal product down-regulation of macrophage HIV-1 p24 production. Macrophages were infected with HIV-1, pseudotyped with VSV-G; pretreated with S-CM (500 µg/ml) and then infected with pseudotyped HIV-1; or infected with CMV AD169 CMV, treated with S-CM, and then infected with pseudotyped HIV-1, after which 48-h culture supernatants were harvested and analyzed for p24. Values represent the mean ± SEM p24 levels released by the monocyte-derived macrophages in triplicate wells from one of two experiments.

 
CMV and HIV-1 infection of macrophages
In cells infected with CMV and HIV-1, the immediate-early gene region 2 of CMV transactivates the HIV-1 promoter [31 ], causing increased HIV-1 production [32 , 33 ]. Therefore, we evaluated macrophages exposed to CMV and HIV-1 for dual infection. As shown in Figure 3 , GFP-expressing macrophages, indicating HIV-1 infection, infrequently contained red fluorescent CMV gene products, indicating dual-infected cells. This low proportion of cells infected with both viruses is consistent with the concept that in our system, CMV enhances HIV-1 expression through a mechanism other than direct molecular transactivation, such as through CMV-induced TNF-{alpha} production, a cytokine capable of stimulating HIV-1 production [34 ].


Figure 3
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  		Figure 3. CMV and HIV-1 infection in macrophages. Cultures of macrophages infected with AD169 or TR CMV were exposed to YU2- or VSV-G-pseudotyped, GFP-labeled HIV-1, and the cells were analyzed by fluorescence microscopy for infection as described in Materials and Methods. Photomicrographs show (A) nuclear staining with DAPI (blue), (B) CMV TR-infected macrophages identified by nuclear and cytoplasmic immunoreactivity for pp150 (red), (C) GFP-expressing, HIV-1-infected macrophages (green), and (D) cells infected with CMV and HIV-1 identified by overlay (orange; original, x500).

 
CMV infection increases macrophage TNF-{alpha} production
To investigate the possible role of TNF-{alpha} in the CMV-induced up-regulation of HIV-1 expression, mock-infected and CMV-infected macrophages were analyzed for spontaneous and LPS-induced TNF-{alpha} production. CMV infection enhanced basal and LPS-stimulated TNF-{alpha} production by the macrophages, consistent with previous findings [17 , 34 ], and stromal products present in S-CM suppressed TNF-{alpha} production markedly in mock CMV-infected macrophages in a dose-dependent manner (Fig. 4 ). However, prior infection of the macrophages with CMV AD169 reversed the S-CM down-regulation of inducible TNF-{alpha} production (P<0.05 for pair-wise comparisons between mock and CMV-infected cells). CMV TR also reversed stromal down-regulation of TNF-{alpha} production (data not shown).


Figure 4
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  		Figure 4. CMV infection increases macrophage TNF-{alpha} production. Macrophages were infected with CMV (AD169) and then treated with S-CM (5–500 µg/mL) for 2 h followed by LPS (1 µg/mL) stimulation (1 h). Culture supernatants were harvested after 24 h and assayed for TNF-{alpha}. Each experiment was performed using macrophages from separate donors; S-CM was derived from the intestinal tissue from four donors. Values are the mean ± SEM TNF-{alpha} levels from triplicate wells from a representative experiment (n=6). Differences in TNF-{alpha} production by mock-infected and CMV-infected macrophages were significant (P<0.05).

 
Because CMV reversed S-CM down-regulation of inducible TNF-{alpha} production, we next investigated whether CMV also inhibited S-CM down-regulation of HIV-1-induced TNF-{alpha} production. Although HIV-1 infection of the macrophages was associated with modest TNF-{alpha} production (158±6 pg/mL), S-CM suppressed HIV-1-induced TNF-{alpha} production (74±12 pg/mL, P<0.01), and this suppression was reversed when the macrophages were preinfected with CMV (153±7 pg/mL). Thus, CMV infection also blocked the ability of S-CM to inhibit HIV-1-induced TNF-{alpha} production.

CMV blocks S-CM down-regulation of macrophage HIV-1 infection through TNF-{alpha}
Because CMV infection of macrophages reverses stromal down-regulation of HIV-1 infection and TNF-{alpha} production, we investigated whether TNF-{alpha} mediates CMV blockade of S-CM down-regulation of macrophage HIV-1 infection. Macrophages were cultured for 4 days and then incubated for 24 h in the presence of anti-TNF-{alpha} antibodies (20 µg/mL) or rhTNF-{alpha} (0.1–10 ng/mL, concentrations comparable with the levels of TNF-{alpha} released by CMV-infected macrophages), after which the cells were treated with S-CM and then exposed to HIV-1. As shown in Figure 5 , anti-TNF-{alpha} antibodies inhibited CMV reversal of S-CM down-regulation of HIV-1 infection, and rhTNF-{alpha} increased macrophage HIV-1 infection (inset). These findings suggest that TNF-{alpha} mediates, at least in part, CMV blockade of stromal product inhibition of HIV-1 infection of macrophages.


Figure 5
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  		Figure 5. TNF-{alpha} mediates S-CM-induced inhibition of macrophage HIV-1 infection. Macrophage cultures were inoculated with VSV-G-pseudotyped, GFP-expressing HIV-1; treated with S-CM 500 µg/mL and then inoculated with HIV-1; infected with CMV prior to S-CM treatment and then inoculated with HIV-1; or infected with CMV and then incubated with anti-TNF-{alpha} antibodies prior to S-CM treatment and then inoculated with HIV-1. Values represent the mean ± SEM percent macrophages infected with HIV-1 in five high power fields (original, x200) in triplicate wells from a representative experiment (n=2). (Inset) Cultures of macrophages were inoculated with HIV-1 (VSV-G pseudotype); treated with S-CM and then inoculated with HIV-1; or treated with rhTNF-{alpha} prior to S-CM treatment and then inoculated with HIV-1.

 
CMV-infected colonic macrophages in AIDS patients with CMV colitis express TNF-{alpha}
To further explore the role of TNF-{alpha} in CMV-induced HIV-1 expression, we analyzed tissue specimens from HIV-1-infected patients with CMV colitis for TNF-{alpha} production. As shown in Figure 6A , mononuclear cells with macrophage-like morphology displayed colocalization of TNF-{alpha} protein and CMV pp150 (top panels), TNF-{alpha} protein and HIV-1 p24 (middle panels), and infrequently, CMV pp150 and HIV-1 p24 (bottom panels). (B) The HIV-1-infected cells expressed multiple copies of HIV-1 mRNA. Thus, in AIDS patients with CMV colitis, CMV- and HIV-1-infected mucosal cells may express TNF-{alpha}, but mucosal cells are infected infrequently by both viruses. The infrequency of dual infection is consistent with the concept that TNF-{alpha}, induced by CMV and possibly HIV-1, plays a more important role in local HIV-1 replication than direct CMV transactivation of the HIV-1 promoter in dual-infected cells.


Figure 6
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  		Figure 6. Colocalization of CMV and TNF-{alpha} in mucosal mononuclear cells. (A) Sections of colon tissue from a patient with HIV-1 infection and CMV colitis were examined by immunofluorescence for TNF-{alpha} (green) and CMV pp150 (red) proteins (top panels), TNF-{alpha} (red) and HIV-1 p24 (green) proteins (middle panels), and CMV pp150 (red) and HIV-1 p24 (green; bottom panels; original, x200). Image overlay confirmed the infrequent colocalization of CMV and TNF-{alpha} proteins (orange). In control tissue, HIV-1, CMV, and TNF-{alpha} mRNA and/or proteins were not identified. (B) A macrophage-like colonic mucosal cell from the same patient displays multiple copies of HIV-1 mRNA.

 
DISCUSSION

We have shown previously that intestinal macrophages, which are derived from blood monocytes [3 ], are unable to support HIV-1 infection [8 , 9 ] or produce proinflammatory cytokines [5 , 6 ]. We also have shown that intestinal ECM products present in S-CM down-regulate blood monocyte-derived macrophages for HIV-1 infection [15 ] and proinflammatory function [6 ], inducing in these cells the functional profile of intestinal macrophages. Here, we show that CMV infection blocks S-CM down-regulation of macrophages for HIV-1 infection in vitro, reflected in increases in the number of HIV-1-infected cells and p24 production. CMV infection of the macrophages also reversed S-CM down-regulation of TNF-{alpha} release. CMV and HIV-1 infection of the macrophages induced TNF-{alpha}, a known stimulator of HIV-1 replication [34 ], but few macrophages in vitro and in the mucosa of HIV-1-infected persons with CMV were infected with both viruses. It is important that anti-TNF-{alpha} antibodies neutralized the ability of CMV to reverse S-CM down-regulation of macrophage HIV-1 infection. Together, these findings support the notion that CMV-induced TNF-{alpha} mediates CMV reversal of S-CM down-regulatory activity. Although CMV encodes a chemokine receptor, which can act as a cofactor for HIV-1 entry [35 ], the ability of CMV to reverse S-CM down-regulation of HIV-1 pseudotyped with VSV-G, the entry of which is coreceptor-independent, suggests that CMV up-regulation of macrophage HIV-1 infection in our system is independent of enhanced HIV-1 entry.

The ability of CMV infection to over-ride the down-regulatory effect of intestinal stromal products on HIV-1 replication in macrophages suggests that the intestinal mucosa may be a site of increased macrophage HIV-1 expression during CMV mucosal disease. Indeed, our findings show that macrophage-like cells in the mucosa of HIV-1-infected subjects with CMV colitis contain HIV-1 mRNA and p24. In addition, the findings that CMV- and HIV-1-infected mucosal cells contain TNF-{alpha} proteins suggest that virus-induced TNF-{alpha} may play a key role in HIV-1 replication in mucosal macrophages in patients with CMV mucosal disease [16 17 18 ]. Our finding that CMV-induced TNF-{alpha} reversed the nonpermissiveness of S-CM-treated macrophages strengthens this speculation. However, other mechanisms, such as superantigen-enhanced replication [36 ], could also be involved in CMV-induced HIV-1 replication in the gastrointestinal mucosa.

Macrophages play a fundamental role in HIV-1 disease at all stages of infection [37 ]. Although blood monocytes may serve as a reservoir for HIV-1 during antiretroviral therapy [38 , 39 ], blood monocytes are refractory to HIV-1 infection in vitro until they differentiate into macrophages [40 41 42 43 ]. Differentiation-associated up-regulation of CCR5 was originally thought to account, at least in part, for the HIV-1 permissiveness of macrophages and the lack of permissiveness of blood monocytes, which usually do not express CCR5 [41 , 44 ]. However, recent studies [43 , 45 , 46 ] suggest that post-entry restrictions in viral replication in monocytes more likely contribute to the inability of blood monocytes to support HIV-1 infection. That intestinal macrophages do not support HIV-1 infection contrasts sharply with the paradigm that differentiated macrophages are permissive to HIV-1 [4 5 6 7 ]. In this regard, intestinal macrophages not only restrict replication of HIV-1 [8 , 9 ], but they also restrict infection and gene expression by lentiviral vectors pseudotyped with VSV-G-encoding indicator genes driven by different promoters, including the HIV-1 long terminal repeat, CMV, and ubiquitin promoters [7 ]. Intestinal macrophages also are resistant to gene expression following infection with respiratory syncytial virus, a single-stranded RNA virus, and adeno-associated virus-2, a single-stranded DNA virus [7 ], suggesting that multiple restrictions in viral replication by intestinal macrophages limit the infection of these cells by an array of harmful viruses. Dissecting the mechanism(s) by which CMV infection causes intestinal macrophages to lose their inherent down-regulation for HIV-1 infection and cytokine production will provide new insight into the role of macrophages in the immunobiology of HIV-1 and CMV mucosal disease.

ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health (DK-74033, DK-47322, DK-54495, HD-41361, DE-16005), the Crohn’s and Colitis Foundation of America, the Research Service of the Veterans Administration, and the Children’s Center for Research and Innovation at the Children’s Hospital of Alabama.

Received March 30, 2006; revised June 9, 2006; accepted June 12, 2006.

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