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The Journal of Immunology, 2004, 173: 6864-6872.
Copyright © 2004 by The American Association of Immunologists

RON Receptor Tyrosine Kinase, a Negative Regulator of Inflammation, Inhibits HIV-1 Transcription in Monocytes/Macrophages and Is Decreased in Brain Tissue from Patients with AIDS1

Eileen S. Lee2,3,*,{ddagger}, Parisa Kalantari2,{dagger},{ddagger}, Shigeki Tsutsui§, Alicia Klatt{dagger},{ddagger}, Janet Holden§, Pamela H. Correll*,{dagger},{ddagger}, Christopher Power§ and Andrew J. Henderson4,*,{dagger},{ddagger}

Graduate Programs in * Biochemistry, Microbiology and Molecular Biology, and {dagger} Pathobiology, and {ddagger} Department of Veterinary Science, Pennsylvania State University, University Park, PA 16802; Departments of Clinical Neuroscience, Microbiology, and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada; and § Department of Pathology, St. Paul’s Hospital, Vancouver, British Columbia, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Activation of macrophages and microglia cells after HIV-1 infection and their production of inflammatory mediators contribute to HIV-associated CNS diseases. The mechanisms that initiate and maintain inflammation after HIV-1 infection in the brain have not been well studied. Furthermore, it is not understood why in HIV-associated CNS disease, macrophages and microglia are biased toward inflammation rather than production of mediators that control inflammation. We have focused on the receptor tyrosine kinase RON, a critical negative regulator of macrophage function and inflammation, to determine whether this receptor regulates HIV-1 expression. Overexpressing RON in monocytes/macrophages demonstrates that RON inhibits HIV-1 proviral transcription in part by decreasing the binding activity of NF-{kappa}B to the HIV-1 long terminal repeat. Because macrophages and microglia cells are a critical reservoir for HIV-1 in the CNS, we examined brain tissues for RON expression and detected RON in astrocytes, cortical neurons, and monocytoid cells. RON was detected in all control patients who were HIV seronegative (n = 7), whereas six of nine brain samples obtained from AIDS patients exhibited reduced RON protein. These data suggest that RON initiates signaling pathways that negatively regulate HIV-1 transcription in monocytes/macrophages and that HIV-1 suppresses RON function by decreasing protein levels in the brain to assure efficient replication. Furthermore, HIV-1 infection would compromise the ability of RON to protect against inflammation and consequent CNS damage.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Acquired immunodeficiency syndrome is a multifactorial disease affecting multiple organs, including skin, lung, lymphoid tissue, and brain (1). The widespread effects of AIDS are partly due to a general inflammatory response in which chronic immune activation leads to increased cytokine secretion and enhanced virus replication. These events result in tissue damage and the manifestation of many AIDS-related pathologies (1). A dramatic example of how HIV-1 infection directly and indirectly leads to tissue damage and pathology is HIV-associated dementia (HAD).5 Twenty percent of late-stage AIDS patients not receiving antiretroviral therapy develop HAD, and it has been reported that upon autopsy up to 80% of HIV-1-infected individuals have evidence of neurological lesions (2, 3). The symptoms of HAD range from minor changes in memory and cognitive function to more severe symptoms that include loss of motor skills, seizures, psychiatric disorders, and dementia. Neuropathological features accompanying HAD include infiltration of macrophages, activation of resident microglia and astrocytes, myelin pallor, and subsequent neuronal destruction. CNS pathogenesis associated with AIDS is largely attributed to infection of macrophages and microglia and their production of inflammatory mediators (4, 5, 6, 7, 8). Indeed, the best pathological correlate of HAD is activation macrophages and microglia (9).

Whether through direct or indirect effects of HIV-1 infection, macrophages produce a variety of factors that cause HAD, including cytokines, chemokines, and adhesion molecules (10, 11). Elevated levels of IL-1{beta}, IL-6, and TNF-{alpha} have been detected in brain tissue of AIDS patients, as has increased expression of the TNF-{alpha} receptor on macrophages and microglia (1, 12, 13). In addition to promoting inflammation, these cytokines activate HIV-1 transcription, suggesting an autocrine mechanism for HIV-1 replication (14, 15, 16, 17, 18, 19). Furthermore, dysregulated expression of IFN-{gamma}, IL-4, IL-10, excitatory amino acids, NO, as well as {alpha}- and {beta}-chemokines have all been observed in the CNS of AIDS patients (12, 13). The combined effect of these factors is altered function and survival of microglia cells, endothelium, astrocytes, and neurons, which exacerbates persistent inflammation and tissue destruction (7, 13).

Dysregulation of the cytokine network in AIDS patients implies that signaling events that normally control cytokine gene expression are disrupted or altered during HIV-1 infection. Signals that inhibit proinflammatory responses by macrophages have the potential to inhibit HIV-1 transcription, whereas disrupting these signals during the course of HIV-1 infection could contribute to a microenvironment that favors HIV-1 replication and inflammation. However, whether HIV-1 actually targets receptors that upon engagement inhibit inflammatory function and promote activities that prevent tissue damage and resolve inflammation has not been thoroughly examined. The receptor tyrosine kinase, RON, has recently been shown to be a critical regulator of macrophage function and inflammation (20). RON, a member of the MET family of receptor tyrosine kinases, regulates a variety of cellular responses, including proliferation, differentiation, apoptosis, and cell movement (20). The ligand for RON is macrophage-stimulating protein (MSP), a serum protein sharing high homology with the MET ligand, hepatocyte growth factor. Activation of RON by MSP inhibits the production of proinflammatory mediators such as IL-12, TNF-{alpha}, and NO (21, 22, 23) and induces the expression of genes associated with resolving inflammation, including scavenger receptor A, IL-1Ra, and arginase (24). RON activation also promotes CR3-mediated phagocytosis and ICAM-1-dependent adhesion of macrophages (25). Macrophages from RON knockout mice produce elevated levels of NO in response to IFN-{gamma} and LPS and exhibit increased inflammation, tissue damage, and death due to endotoxic shock upon LPS challenge (26). RON knockout mice also have compromised cell-mediated immunity, as demonstrated by an increased susceptibility to Listeria monocytogenes infection (27).

Because HIV-1 transcription is tightly linked to proinflammatory signals, the suppression of this response might be predicted to inhibit virus expression. We directly tested whether the anti-inflammatory signals initiated by RON could inhibit HIV-1 transcription. We demonstrate that RON inhibits HIV-1 transcription in part by targeting NF-{kappa}B activation. Furthermore, we show that RON protein is consistently decreased in brain tissue from AIDS patients, suggesting that chronic HIV-1 infection results in the down-regulation of this receptor tyrosine kinase.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and tissues

The U937 promonocytic cell line was cultured in RPMI 1640 medium supplemented with 10% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.2 M L-glutamine. The 293T human embryonic kidney cell line and the CHME3 microglial cell line were grown in DMEM supplemented with 10% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.2 M L-glutamine. The U87 glioblastoma cell line was cultured in Eagle’s MEM supplemented with 10% bovine calf serum, 100 U/ml penicillin, 100 U/ml streptomycin, 0.2 M L-glutamine, 0.1 mM nonessential amino acids, and 1 mM sodium pyruvate. A stable U937-RON cell line was generated through transduction of U937 cells using a vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped murine stem cell virus (MSCV)-RON retroviral vector that expresses a neomycin resistance gene (28). Transduced pools of cells were maintained for 2 wk after selection with 1 mg/ml G418 (Sigma-Aldrich, St. Louis, MO). In some experiments cells were treated with IL-6, TNF-{alpha}, and IFN-{gamma} (R&D Systems, Minneapolis, MN).

Peripheral blood macrophages were isolated from whole blood obtained from healthy HIV-1-seronegative donors. Mononuclear cells were obtained by differential centrifugation using a Ficoll/Hypaque gradient (Sigma-Aldrich) as previously described (29). The cells were cultured in RPMI 1640 supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.2 M L-glutamine. Monocytes were separated from lymphocytes by an initial adherence to plastic culture flasks overnight. After removing nonadherent cells, monocytes were cultured for 5–7 days to mature into monocyte-derived macrophages (MDM) before infection. Murine resident peritoneal macrophages were obtained from wild-type and RON–/– CD1 mice (26) by peritoneal lavage with 10 ml of RPMI 1640 containing 10% FCS. Cells were plated at ~3.3 x 105 cells/ml overnight, then washed with PBS to eliminate nonadherent cells. Murine macrophages were maintained in RPMI 1640 plus 10% FBS at 37°C in a humidified incubator containing 5% CO2.

The brain tissues used in these experiments have been described in previous reports (30, 31) and were obtained from the AIDS Brain Bank at St. Paul’s Hospital (Vancouver, Canada) and the Neurovirology Laboratory Brain Bank at the University of Calgary. Samples represented frontal white matter that was collected at autopsy from all experimental groups and stored at –80°C. The presence of CNS opportunistic infections was excluded by postmortem histopathologic evaluation of brain tissue sections from adjacent tissue. Control tissues were collected from subjects with a mean age 56 ± 14 yr who were seronegative for HIV; they included three normal brains, two brains from stroke patients, one brain from a patient with endocarditis, and one brain from a patient with anoxic encephalopathy. All HIV-infected patients died of AIDS-related illnesses (mean age, 39 ± 15 year) and had CD4+ T cell counts <200/mm3. The HIV-infected samples were obtained from patients with HIV encephalitis (n = 3), multifocal leukoencephalitis (n = 2), toxoplasma encephalitis (n = 1), microglia nodules (n = 2), and CMV encephalitis (n = 1). Neuropsychological and neurological information was not available for the HIV-infected patients from whom tissues were collected (29, 30). Fetal brain tissues were provided by Drs. B. Wigdahl (Drexel University, Philadelphia, PA) and R. Frisque (Pennsylvania State University, University Park, PA). Astrocytes were provided by Dr. D. Volsky (Columbia University, New York, NY).

Plasmids and transient transfections

293T cells were transiently transfected using CaPO4 (32). The pCI-RON construct was generated by subcloning the XhoI and NotI 4.5-kb RON fragment from the pvuless-hRON (provided by Dr. R. Breathnach, Institut de Biologie, Nantes, France) into the XhoI-NotI sites of pCI-neo vector (Promega, Madison, WI). An XhoI-EcoRI hRON fragment was subcloned into the XhoI and EcoRI sites of MSCV2.2 for MSCV-RON. Long terminal repeat (LTR) reporter constructs LTR-Luc, –158 LTR-Luc, and NF-{kappa}B-Luc and pSV2-LUC were previously described (33). DNA for transfections was prepared using plasmid purification systems from Marligen Biosciences (Ijamsville, MD) following protocols provided by the manufacturer. MFG-GFP was used to control for transfection efficiency and was assayed 12 h post-transfection by fluorescent microscopy or flow cytometry. RON overexpression did not influence the transfection efficiencies of the different cell lines (data not shown).

HIV-1 Infections

Replication-deficient virus was generated by transfecting 293T cells with 15 µg of pNL43-Luc+Env– DNA (34), 3 µg of VSV-G-Env DNA, and 3 µg of Tat by CaPO4 transfection. HXB.2 or HXBnPLAP (35) (HIV-PLAP; obtained from the AIDS Research Reference Reagent Program, National Institutes of Health) replication competent virus was generated similarly by cotransfecting proviral cDNA, and Rev expression constructs. Transfection efficiency was assessed by luciferase (Luc) activity or p24 levels. We consistently generated titers of 1.0 x 106 infectious particles/ml. One milliliter of undiluted viral stocks was added to 1.0 x 106 cells for 24 h, and the spent medium was replaced. Cells were harvested 48 h postinfection and assayed for viral transcription by Luc assays. Cells (1.0 x 106) were lysed in 1x Reporter Lysis Buffer (Promega), and supernatants were collected. Twenty microliters of cell extract was added to 100 µl of Luc substrate (Promega), and activity was measured using a luminometer. In addition, virus replication was assessed by p24 ELISA (PerkinElmer, Wellesley, MA).

Nuclear extract preparations and EMSAs

Nuclear extracts were prepared as described previously (36) by lysing 2.0 x 106 cells with 10% Nonidet P-40 in buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 0.5 mM PMSF). The extracts were recovered in 50 µl of buffer C (20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF). Fifty nanograms of annealed –161 to –94 HIV-1 LTR binding site DNA (5'-GATCGCCCGAGAGCGCATCCGGAGTACTTCAAGAACTGCTGACATCGA-3' and 5'-GATCGCGGAAAGTCCCTTGTAGCAAGCTCGATGTCAGCAGTTCTTGAAG-3') or C/EBP binding site DNA (5'-GATCGCCTAGCATTTCATCACACGT-3' and 5'-GATCACGTGTGATGAAATGCTAGGC-3') was end-filled with [{alpha}-32P]dCTP using bacterial Klenow fragment (Promega). The DNA probe was used at a specific activity of 108–109 cpm/µg and incubated with 5 µg of nuclear extract samples in a reaction mixture of a final volume 20 µl containing 3 µg of dI-dC (Amersham Biosciences, Arlington Heights, IL), 0.25 M HEPES (pH 7.5), 0.6 M KCl, 50 mM MgCl2, 1 mM EDTA, 7.5 mM DTT, and 9% glycerol for 20 min at 25°C. A 50-fold excess of unlabeled NF-{kappa}B, C/EBP, or Sp1 binding site DNA was used as the specific and nonspecific competitors. Anti-NF-{kappa}B p65 Ab (0.5 µg; Santa Cruz Biotechnology, Santa Cruz, CA) was used to supershift complexes. The samples were run on a 6% polyacrylamide gel and visualized by autoradiography.

RNA extraction and RT-PCR

Total cellular RNA was prepared by lysing cells with 4 M guanidinium thiocyanate, 25 mM sodium citrate (pH 7), 0.5% sarkosyl, and 0.1 M 2-ME, followed by phenol-chloroform extraction (37). cDNA was prepared from 2 µg of cellular RNA using murine leukemia virus reverse transcriptase and random primers. To amplify the cDNA, a 30-cycle PCR of 94°C for 1 min, 55°C for 2 min, and 72°C for 3 min was performed with 0.2 µg of cDNA using various primers. RON cDNA was amplified using the upstream primer 5'-TAGCAGTGCAACCCCTCTTT-3' and the downstream primer 5'-GTAAAGCCAGCAGCTCCATC-3'. {beta}-Actin cDNA was amplified using the upstream primer 5'-CCTAAGGCCAACCGTGAAAAG-3' and the downstream primer 5'-TCTTCATGGTGCTAG GAGCCA-3'.

Immunoblotting

Cells (5 x 106) were lysed in 100 µl of phosphatase inhibitor lysis buffer (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA (pH 8.0), 2 mM sodium vanadate, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 1% Nonidet P-40, 1 mM PMSF, and 1 mM pepstatin). An equal volume of whole cell lysate was added to 2x SDS loading buffer (100 mM Tris-HCl (pH 6.8), 4% SDS, 0.2% bromophenol blue, 20% glycerol, and 200 mM DTT) and run on a 6% SDS-polyacrylamide gel. After transfer of proteins to the nitrocellulose membrane, rabbit-anti-RON polyclonal Ab (Santa Cruz Biotechnology) was used to probe for RON, or rabbit-anti-I{kappa}B{alpha} polyclonal Ab (Santa Cruz Biotechnology) was used to detect I{kappa}B{alpha}. HRP-conjugated, goat anti-rabbit IgG (Sigma-Aldrich) was used as the secondary Ab. Proteins were detected using the ECL Plus Western blotting detection system (Amersham Biosciences) and exposure to film. Filters were stripped 45 min at 55°C using 100 mM {beta}-ME, 62.5 mM Tris-HCl (pH 6.7), and 2% SDS and were reprobed with mouse anti-{beta}-actin (Sigma-Aldrich), which was detected with HRP-conjugated, goat anti-mouse secondary Ab. For detection of nuclear p65 by immunoblots, 20 µg of nuclear extracts were separated by 6% SDS-PAGE and transferred to nitrocellulose as described above. Primary staining was performed with rabbit anti-p65 polyclonal Ab (Santa Cruz Biotechnology), and the secondary Ab was HRP-conjugated, goat anti-rabbit IgG (Sigma-Aldrich).

Immunohistochemistry

Human brain tissues were embedded in paraffin and sectioned into 5-µm samples. After removal of paraffin and hydration with decreasing concentrations of ethanol, sections were boiled in 0.01 M citrate buffer in preparation for staining. To block endogenous peroxidases, samples were incubated in 0.3% H2O2. Sections were blocked with 10% normal goat serum/0.5% Triton X-100 before adding primary Abs diluted in PBS supplemented with 10% normal goat serum. Primary Abs included rabbit-anti-RON polyclonal Ab and anti-CD45 (DakoCytomation, Carpinteria, CA), whereas controls were isotype-matched IgG. Primary Abs were detected with biotinylated goat anti-rabbit or biotinylated goat anti-mouse Abs, followed by avidin-biotin-peroxidase complexes. Peroxidase activity was detected by diaminobenzidine or alkaline phosphatase (Vector Laboratories, Burlingame, CA), as previously reported (31). Hematoxylin was used to counterstain sections to assist in identifying cells expressing RON.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
RON receptor tyrosine kinase inhibits HIV-1 transcription

RON has been shown to play a key regulatory role during macrophage activation. Because HIV-1 transcription in macrophages is associated with inflammation, we posited that RON would suppress HIV-1 transcription. To investigate the ability of RON to influence HIV-1 LTR activity, 293T cells were transiently transfected with an LTR-Luc reporter construct (33) in the absence or the presence of RON. LTR activity was assessed by measuring Luc activity. As shown in Fig. 1A, the presence of RON inhibited LTR activity by 3-fold compared with LTR-Luc transfected in the absence of RON. This response is specific to the HIV-1 LTR, because SV40 early promoter (pSV2-Luc) activity was comparable in the absence or the presence of RON (Fig. 1). These data suggest that the RON receptor tyrosine kinase inhibits HIV-1 transcription. It should be noted that we observed constitutive signaling when RON was overexpressed, and addition of MSP had no significant effect on our results (data not shown). This is consistent with previous studies showing that overexpression of RON leads to constitutive activity of the receptor (21, 38). In addition, the phenotype of the RON knockout mice, which is a deficiency in resolving inflammation, is more severe than the modest phenotype observed in mice lacking MSP, suggesting that RON has additional uncharacterized ligands or ligand-independent activity (26).



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  		FIGURE 1. Decreased HIV-1 transcription in the presence of RON. A, 293T cells were transfected with 1 µg of LTR-Luc or pSV2-Luc and 1 µg of pCI empty vector or pCI-RON. Cells were harvested 48 h post-transfection and assayed for Luc activity. B, Primary MDM transduced with MSCV or MSCV-RON were infected with HIV-LUC (1 x 106 infectious particles/1 x 106 cells), and 24 h postinfection cells were harvested to measure Luc activity. The data in A and B are presented as the percentage of Luc, with Luc activity in the cells lacking RON being set at 100%. C, U937 cells transduced with MSCV (MOCK) or MSCV-RON were infected with HIV-Luc. Twenty-four hours postinfection, cells were cultured in 1% serum medium for 6 h, then treated with 10 ng/ml IL-6 and 10 ng/ml TNF for 12 h before performing Luc assays. Data are presented as Luc activity, and the values shown in the figure are for unstimulated U937-MOCK and U937-RON cells. {blacksquare}, Data obtained with mock-transfected or transduced cells; {square}, data for cells overexpressing RON. For transfections, MFG-GFP was used to control for transfection efficiency (see Materials and Methods), and RON overexpression did not influence the transfection efficiencies of the different cell lines (data not shown). Each data point represents three independent transfections, and error bars show the SD of these replicates. Each set of data is representative of at least three experiments.

 
Human monocytic cell lines or primary macrophages that express RON are not readily available because this protein is found predominantly on tissue resident macrophages (20, 39, 40, 41, 42). Therefore, to study the effects of RON on HIV-1 expression in relevant cell types, primary monocyte-derived macrophages were transduced with an MSCV-RON retroviral vector and subsequently infected with a replication defective HIV-1 in which a Luc reporter gene (HIV-Luc) was inserted into Nef (34); Luc serves as a reporter for proviral transcription. The expression of RON in these cells was confirmed by RT-PCR and immunoblotting (Fig. 2). Overexpression of RON in primary macrophages inhibited HIV-Luc proviral transcription by >4-fold compared with macrophages expressing empty MSCV vector (Fig. 1B). To more readily study the biochemical mechanism by which RON inhibits HIV-1, we transduced U937 cells with an MSCV-RON retroviral vector to establish a U937 cell line stably expressing RON (U937-RON). RON expression was confirmed by RT-PCR and immunoblotting (Fig. 2). U937-RON cells and U937 cells transduced with the empty vector (U937-MSCV) were infected with HIV-Luc and assayed for Luc activity. Overexpression of RON in U937 cells inhibited HIV-Luc transcription compared with U937-MSCV controls and dramatically diminished cytokine-induced activation of HIV-1 transcription in monocytic cells (Fig. 1C). The observed decrease in HIV-1 transcription is not due to a RON-mediated block in virus entry, because no difference was seen in proviral integration as determined by PCR and Southern blot in the U937-MSCV and U937-RON cell lines (data not shown).



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  		FIGURE 2. RON expression in brain tissue and cell lines. A, mRNA was extracted from cell lines, primary MDM, and 17-wk fetal human brain tissue, and RT-PCR was performed for RON expression. B, Whole cell extracts were prepared from fetal human brain, primary astrocytes, transduced U937 cells, and transduced primary MDM and analyzed for RON protein expression by immunoblotting with anti-RON Ab.

 
To determine whether RON could alter HIV-1 replication, U937 and U937-RON cells were infected with HXB2 virus and assayed for virus replication over 3 wk by p24 ELISA. Three weeks postinfection, a 2- to 4-fold decrease in HIV-1 replication was observed in cells expressing RON compared with cells not expressing RON (Fig. 3A). HIV-1 replication was similarly inhibited in MDM that were transduced with RON (Fig. 3B). Taken together, our data suggest that RON inhibits HIV-1 transcription and replication.



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  		FIGURE 3. Ectopic RON expression inhibits HIV-1 replication. A, U937-mock (solid line) and U937-RON (dashed line) cells were infected with HXB2 virus (1 x 106 infectious particles/1 x 106 cells). Various times postinfection, supernatants were collected and assayed for viral replication by p24 ELISA. Each data point represents three independent infections, and error bars show the SD of these replicates. These data are representative of four independent experiments. B, MDM transduced with MSCV or MSV-RON were infected with HIV-PLAP virus (1 x 106 infectious particles/1 x 106 cells), and p24 was measured 8 days postinfection. These data represent at least four independent infections.

 
The above experiments depend on overexpressing RON and may exaggerate the ability of RON to negatively regulate HIV-1 transcription. However, as mentioned, human cells that express RON are not readily accessible. Therefore, to determine whether endogenously expressed RON was capable of regulating HIV-1 transcription, we infected peritoneal macrophages obtained from RON wild-type and RON-deficient mice (26) with VSV-G pseudotyped HIV-LUC. Although HIV-1 does not replicate in mouse cells, the murine system recapitulates aspects of HIV-1 Tat-independent transcriptional regulation observed in the human cells (43, 44, 45). As shown in Fig. 4 the RON-deficient cells supported HIV-1 transcription more efficiently than the wild-type controls. These data suggest that physiological levels of RON are sufficient to influence HIV-1 transcription.



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  		FIGURE 4. HIV-1 transcription is enhanced in RON–/– macrophages. Peritoneal macrophages from wild-type and RON–/– CD1 mice were transduced with HIV-LUC virus pseudotyped with the VSV-G envelope. Luc activity was measured 48 h postinfection as an indication of proviral transcription. Comparable levels of provirus were detected in the various macrophages by PCR of genomic DNA for HIV-1 proviral sequences (data not shown). Each data point represents three independent infections, and error bars show the SD of these replicates. These data are representative of three independent experiments.

 
RON targets NF-{kappa}B to inhibit HIV-1 transcription

Signaling through RON potentially alters the activity of cellular transcription factors necessary for HIV-1 transcription. It has been demonstrated that RON signaling inhibits NF-{kappa}B activity (21), which is an important transcriptional activator for HIV-1 (46, 47, 48). To determine whether RON is targeting NF-{kappa}B activity to block HIV-1 transcription, 293T cells were cotransfected with an NF-{kappa}B-Luc reporter and a RON expression construct or empty vector control. A 7-fold decrease in Luc activity was observed in 293T cells cotransfected with NF-{kappa}B-Luc and RON compared with cells cotransfected with NF-{kappa}B-Luc and empty vector (Fig. 5A). RON had no effect on the activity of an NFAT reporter control (data not shown).



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  		FIGURE 5. Inhibition of NF-{kappa}B activity upon RON overexpression. A, 293T cells were transfected with 1 µg of NF-{kappa}B-Luc and 1 µg of pCI empty vector or pCI-RON DNA. Cells were harvested 48 h post-transfection and assayed for Luc activity as an indication of transcription. MFG-GFP was used to control for transfection efficiency as described in Materials and Methods. Error bars are the SD for three independent transfections. These data are representative of results from three experiments. B–E, U937-mock (MOCK) or U937-RON (RON) extracts were untreated or treated with 10 ng/ml IL-6, 10 ng/ml TNF-{alpha}, and 100 U/ml IFN-{gamma} (cytokines) for 6 h before preparing nuclear or whole cell extracts. B, EMSA with 4 µg of nuclear extracts using a radiolabeled probe spanning –161 to –94 of the HIV-1 LTR. A 50-fold excess of cold NF-{kappa}B and Sp1 binding site oligonucleotides were used as specific and nonspecific competitors, respectively. Supershifts were performed using 1 µg of anti-p65 Ab. Isotype control Ab did not alter the complex (data not shown). C, Immunoblots for detection of nuclear p65. Twenty micograms of nuclear extracts were probed with anti-p65 polyclonal Ab. D, Whole cell extracts from the indicated cells were probed for I{kappa}B{alpha}, stripped, and reprobed for {beta}-actin. E, EMSA with 4 µg of U937-MSCV or U937-RON nuclear extracts treated with cytokines for 6 h using a radiolabeled C/EBP binding site. A 50-fold excess of cold C/EBP (COMP) and Sp1 binding site oligonucleotides (NS) were used as competitors.

 
To investigate whether RON affects NF-{kappa}B binding activity, EMSAs were performed using a probe derived from the HIV-1 LTR spanning nucleotides –161 to –94, which contains sites for multiple factors, including NF-{kappa}B. U937-RON and U937-MSCV cells were stimulated with IL-6, TNF-{alpha}, and IFN-{gamma} to strongly induce NF-{kappa}B. A decrease in binding activity was observed in extracts from U937-RON cells compared with U937-MSCV cells (Fig. 5B). This complex could be competed away with cold NF-{kappa}B binding site oligonucleotide, but not with nonspecific binding site competitor and could be supershifted with an anti-p65 Ab. Consistent with this decrease in binding activity, nuclear extracts from RON-expressing cells had decreased p65 after induction with IL-6, TNF-{alpha}, and IFN-{gamma} compared with MSCV-transduced controls, as determined by immunoblotting (Fig. 5C). Furthermore, I{kappa}B-{alpha}, a negative regulator of NF-{kappa}B nuclear translocation, was more resistant to degradation after cytokine activation in RON-expressing cells (Fig. 5D). These data indicate that RON signaling decreases NF-{kappa}B activation.

Because the HIV-1 LTR contains binding sites for multiple host factors, we were interested as to whether other transcription factors might be affected by RON. Besides NF-{kappa}B, another important factor for HIV-1 replication in monocytes/macrophages is C/EBP{beta} (33, 49, 50, 51). However, EMSA using labeled C/EBP binding site oligonucleotides showed equivalent complex formation in both U937-RON and U937-MSCV cells (Fig. 5E), suggesting that C/EBP factors are not downstream of RON signaling in monocytic cells.

RON is expressed in human brain

Given the pivotal role of macrophages and microglia in supporting HIV-1 replication in the brain and the development of HAD, we examined the expression of RON in the human brain. RON mRNA has previously been shown to be expressed in the mouse hippocampus and hypothalamus and in dorsal root ganglia (52, 53), however, the expression of RON in human brains has not been examined. We used RT-PCR, immunoblots, and immunohistochemistry to examine RON expression in human tissues. Seventeen-week gestation human fetal brain tissue was examined for RON mRNA by RT-PCR. As shown in Fig. 2, RON mRNA was detected in brain samples, whereas monocytic cell lines and primary MDM did not exhibit RON expression. This is consistent with previous observations that RON is preferentially expressed on tissue-resident macrophages (20, 39, 40, 41, 42). Furthermore, RON protein was detected by immunoblots in samples prepared from human brain tissue as well as primary astrocytes isolated from in vitro cultures (Fig. 2B). To define which cells express RON, we examined tissue sections from white matter and cortex, which showed abundant RON immunoreactivity detected by immunohistochemistry (Fig. 6A). RON was present on ~50% of cells that were immunopositive for CD45 (Fig. 6A), indicating that RON is expressed in a subset of monocytoid cells within the brain. In addition, RON immunoreactivity was present on cortical neurons (data not shown). Fewer RON-immunopositive cells were observed in white matter sections from HIV patients (Fig. 6B) compared with controls. MSP immunoreactivity was also observed in white matter of both control and HIV patients (Fig. 6C), largely in CD45-immunopositive cells (Fig. 6C, inset). Taken together, these data suggest that RON is expressed in monocyte-derived cells where it potentially regulates inflammatory responses.



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  		FIGURE 6. RON and MSP protein expression in human brain. A, RON immunoreactivity in white matter glia (arrows) from an HIV-seronegative patient. The inset shows double-label immunoreactivity displaying CD45 (brown) and RON (blue) immunoreactivity on microglia (inset, arrow). B, RON immunoreactivity in white matter glia (arrow) from an HIV encephalitis patient. C, MSP immunoreactivity was detected in white glia (arrowhead), which were largely CD45-immunopositive (inset).

 
To determine whether RON inhibited HIV-1 transcription in the context of cells derived from the CNS, we cotransfected RON with HIV-LUC cDNA into brain-derived cell lines: CHME3, a transformed human microglia line (54), and U-87MG, a glioblastoma (55). RON inhibited HIV-1 transcription by >5-fold in both these cell types (Fig. 7), suggesting that the signals downstream of RON that suppress HIV-1 expression are operative in CNS-derived cell types.



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  		FIGURE 7. RON inhibits HIV-1 transcription in CNS-derived cell lines. CHME3 and U-87MG were cotransfected with HIV-LUC cDNA and pCI-RON (RON) or pCI (MOCK). Twelve hours post-transfection, cells were assayed for Luc activity. {blacksquare}, Cells that lack RON; {square}, cells overexpressing RON. Data are presented as the percentage of Luc, with Luc activity in the cells lacking RON being set at 100%. Error bars are the SD among three independent transfections. These data represent a single experiment that was performed four times. MFG-GFP was used to control for transfection efficiency, as described in Materials and Methods.

 
RON expression is decreased in HIV-associated CNS inflammatory diseases

RON negatively regulates inflammation; therefore, we would predict that RON expression might be altered during chronic inflammation induced by HIV-1 infection of the brain. We obtained adult human brain tissues from uninfected control individuals or AIDS patients (30, 31) and examined them by immunoblotting for RON protein. Neuropathological evaluation of the samples from AIDS patients revealed that three patients had HIV encephalitis, and the remaining cases exhibited other neuropathological features (30, 31). RON was detected in all seven control patients who were HIV seronegative, whereas six of the nine samples obtained from AIDS patients, including all three that exhibited encephalitis, had reduced RON expression. A subset of these samples is shown in Fig. 8. These data suggest that HIV-1 may directly or indirectly target RON to assure a microenvironment that is favorable for virus replication.



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  		FIGURE 8. RON is decreased in AIDS patients with CNS disease. Brain tissue extracts were prepared from AIDS patients, including two patients with HIV encephalitis (HIVE) or an uninfected patient (control), and RON protein was detected by immunoblotting. Filters were stripped and reprobed for {beta}-actin as a loading control. Each tissue sample was examined for RON expression by immunoblotting at least three times.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
HIV-1 infection of macrophages is associated with activation and elevated expression of inflammatory cytokines, which potentially feed back in an autocrine fashion to further induce HIV-1 transcription as well as contribute to the tissue destruction and dysregulated growth observed in AIDS-associated diseases of the brain, skin, lymph nodes, and lung. The mechanisms that contribute to the inappropriate macrophage function are not clear, although it seems likely that HIV-1 specifically targets receptors and signaling events that negatively regulate inflammation. Critical regulators of inflammation include Fc{gamma}R, phosphatidylserine receptor, and the Tyro 3 family of receptors (56, 57, 58, 59). HIV-1 has been associated with a decrease in the expression of Fc{gamma}Rs (60) and inhibition of Fc-mediated phagocytosis (61). Our data suggest that RON receptor tyrosine kinase, which has also been shown to negatively regulate inflammation and macrophage function (20), inhibits HIV-1 expression and may be a target of HIV-1 infection.

RON signaling has been demonstrated to inhibit the transcription of iNOS, TNF-{alpha}, and IL-12 (21, 22, 23, 62) and, based on our findings, HIV-1. RON activation has been previously shown to inhibit NF-{kappa}B activity (21), and our data are consistent with these findings. Because NF-{kappa}B is critical for efficient HIV-1 transcription in monocytes/macrophages (46, 47, 63), inhibition of NF-{kappa}B would be expected to compromise HIV-1 LTR activity and proviral transcription. RON does not lead to a general decrease in transcription factor activity, because C/EBP binding activity remained unchanged regardless of RON expression. However, physical and functional interactions between NF-{kappa}B and other factors might be important for the transactivation of the HIV-1 LTR (64, 65).

RON signaling requires two critical tyrosine residues, 1353 and 1360, in the C-terminal domain that serve as multifunctional docking sites for SH2-containing signaling proteins, including Grb2, phospholipase C-{gamma}, Src homology domain 2-containing phosphatase 1, SHIP, and p85, the regulatory subunit of PI3K (66, 67). RON activates PI3K signaling, and inhibition of NO production is PI3K-dependent (22, 25). Previous studies have indicated a role for PI3K in negatively regulating HIV-1 transcription in T cells (68), although whether PI3K inhibits HIV-1 transcription in macrophages has not been determined. In addition, there are two tyrosine residues, 1238 and 1239, in the catalytic domain of RON that are crucial for kinase activity of the receptor (69), but whether these residues are required for suppression of HIV-1 transcription has not been investigated. Other signaling pathways downstream of RON include the MAPK pathway and STAT3 activation (20), both of which have been reported to regulate HIV-1 replication (70, 71, 72, 73).

Gene targeting studies have indicated that RON has a critical role in mouse development and normal immune function (26, 74). Macrophages from RON-deficient mice do not properly resolve inflammation, leading to elevated levels of NO in response to IFN-{gamma} and LPS, increased tissue damage, and death due to endotoxic shock (26). Similarly, HIV-1-infected macrophages in the brain are chronically activated and produce elevated levels of inflammatory cytokines, resulting in destruction of the blood-brain barrier and surrounding brain tissue (12, 13). This chronic activation of brain macrophages could be in part attributed to a breakdown in pathways required for immune homeostasis in the brain microenvironment. We propose that in the brain RON is a protective barrier against inflammation, and that HIV-1 infection, either directly or indirectly, alters RON function, contributing to an inflammatory microenvironment that favors HIV-1 replication. RON and MSP are expressed in multiple cell types in the CNS (this study and data not shown) (75, 76), and MSP has been suggested to act as neurotrophic factor for subsets of sensory and sympathetic neurons during development (53, 75, 76), although we have not seen altered MSP expression in brains from HIV patients (data not shown). However, the consistent decrease in RON protein in AIDS patients suggests that chronic HIV-1 infection alters RON protein levels.

It is not clear whether HIV-1 infection directly or indirectly down-regulates RON expression, although in regions exhibiting encephalitis, usually representing high levels of HIV replication, RON immunoreactivity was markedly reduced. We are currently examining mechanisms by which HIV-1 may directly alter RON expression and function. HIV-1-encoded proteins such as Nef and Tat have a myriad of activities that influence gene expression, signal transduction, receptor turnover, and cell growth and differentiation. Nef interacts with a variety of signal transduction proteins and has been shown to down-regulate surface expression of CD4, CD28, MHC class I molecules, and Fc{gamma}Rs (77, 78, 79). Tat also has been demonstrated to have several activities, including regulating transcription and cell growth and differentiation (80). Furthermore, cytokine production and inflammatory mediators initiated by HIV-1 infection in the brain (12, 13) could influence RON expression and function through an autocrine/paracrine mechanism (20, 81). Finally, HIV-1-induced inflammation may lead to the death of cells that express RON, including neurons and astrocytes, as well as the infiltration and/or expansion of RON-negative lymphocytes and macrophages within the brain, resulting in an overall decrease in RON protein in the brain. For example, although our preliminary immunostaining suggest a reduction in the percentage of cells expressing both RON and CD45 in HIV-infected brain samples, these tissues typically had more CD45+ cells than control brain samples (S. Tsutsui and C. Power, unpublished observation).

In summary, we have demonstrated that the tyrosine kinase receptor RON, which normally controls inflammatory activities of macrophages, inhibits HIV-1 transcription. Furthermore, we present evidence that RON protein is decreased in the brain in a subset of AIDS patients. These findings imply that HIV-1 infection compromises immune function in part by disrupting normal signals that actively suppress inflammation and provide unique insights into the progression of diseases such as HAD.


   Acknowledgments
 
We thank Lisa Finkelstein (National Human Genome Research Institute/National Institutes of Health) for assistance with generating the RON expression constructs, and Margaret Kensinger and Sarah Shelby for technical support. We also thank Dr. Brian Wigdahl (Drexel University, Philadelphia, PA) for cells from primary glia cultures, and Dr. David Volsky (Columbia University, New York, NY) for purified astrocytes. In addition, the General Clinical Research Center at Pennsylvania State University assisted with blood collection.


   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 National Institutes of Health Grant AI46261 (to A.J.H.) and Tobacco Formula Funds (to A.J.H. and P.H.C.). Back

2 E.S.L. and P.K. contributed equally to this work. Back

3 Current address: Department of Molecular and Cell Biology, Immunology Division, University of California, Berkeley, CA 94720-3200. Back

4 Address correspondence and reprint requests to Dr. Andrew J. Henderson, Department of Veterinary Science, 115 Henning Building, Pennsylvania State University, University Park, PA 16802-3500. E-mail address: ajh6{at}psu.edu Back

5 Abbreviations used in this paper: HAD, HIV-associated dementia; LTR, long terminal repeat; Luc, luciferase; MDM, monocyte-derived macrophages; MSP, macrophage-stimulating protein; VSV-G, vesicular stomatitis-glycoprotein; MSCV, murine stem cell virus. Back

Received for publication March 31, 2004. Accepted for publication October 1, 2004.


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