Osteopontin enhances HIV replication and is increased in the brain and cerebrospinal fluid of HIV-infected individuals

Abstract

Despite effective and widely available suppressive anti-HIV therapy, the prevalence of mild neurocognitive dysfunction continues to increase. HIV-associated neurocognitive disorder (HAND) is a multifactorial disease with sustained central nervous system inflammation and immune activation as prominent features. Inflammatory macrophages, HIV-infected and uninfected, play a central role in the development of HIV dementia. There is a critical need to identify biomarkers and to better understand the molecular mechanisms leading to cognitive dysfunction in HAND. In this regard, we identified through a subtractive hybridization strategy osteopontin (OPN, SPP1, gene) an inflammatory marker, as an upregulated gene in HIV-infected primary human monocyte-derived macrophages. Knockdown of OPN in primary macrophages resulted in a threefold decrease in HIV-1 replication. Ectopic expression of OPN in the TZM-bl cell line significantly enhanced HIV infectivity and replication. A significant increase in the degradation of the NF-κB inhibitor, IκBα and an increase in the nuclear-to-cytoplasmic ratio of NF-κB were found in HIV-infected cells expressing OPN compared to controls. Moreover, mutation of the NF-κB binding domain in the HIV-LTR abrogated enhanced promoter activity stimulated by OPN. Interestingly, compared to cerebrospinal fluid from normal and multiple sclerosis controls, OPN levels were significantly higher in HIV-infected individuals both with and without neurocognitive disorder. OPN levels were highest in HIV-infected individuals with moderate to severe cognitive impairment. Moreover, OPN was significantly elevated in brain tissue from HIV-infected individuals with cognitive disorder versus those without impairment. Collectively, these data suggest that OPN stimulates HIV-1 replication and that high levels of OPN are present in the CNS compartment of HIV-infected individuals, reflecting ongoing inflammatory processes at this site despite anti-HIV therapy.

Fig. 1

The role of osteopontin in HIV replication. a Quantitative PCR for SPP1 and 18S rRNA message was performed on cDNA prepared from HIV-1 SF162, and BaL and infected macrophages and controls. A representative of two independent experiments with mean and standard deviation with three different donors is shown. b Western blot analysis for OPN and actin 48 h after transfection of macrophages with anti-OPN siRNAs (SPP1, 3, 4, 6). c Scatter plots and graph: quantification of GFP⁺ cells by flow cytometry. A representative of two independent experiments with mean and standard deviation with three different donors is shown. d Western blot analysis for OPN on uninfected TZM-bl cells or cells infected with HIV and recombinant adenovirus expressing OPN or red fluorescent protein (RFP). e Increased replication and syncytia formation in TZM-OPN-expressing cells infected with HIVR3Nef+GFP by fluorescent microscopy and f quantified by flow cytometry (top graph). The mean fluorescent intensity (MFI) of GFP expression in HIV-infected cells or HIV-infected cells over-expressing OPN is shown in the middle graph. Activation of the HIV-LTR-beta-galactosidase promoter is shown in the bottom graph. Means and standard deviations are shown

Fig. 1

The role of osteopontin in HIV replication. a Quantitative PCR for SPP1 and 18S rRNA message was performed on cDNA prepared from HIV-1 SF162, and BaL and infected macrophages and controls. A representative of two independent experiments with mean and standard deviation with three different donors is shown. b Western blot analysis for OPN and actin 48 h after transfection of macrophages with anti-OPN siRNAs (SPP1, 3, 4, 6). c Scatter plots and graph: quantification of GFP⁺ cells by flow cytometry. A representative of two independent experiments with mean and standard deviation with three different donors is shown. d Western blot analysis for OPN on uninfected TZM-bl cells or cells infected with HIV and recombinant adenovirus expressing OPN or red fluorescent protein (RFP). e Increased replication and syncytia formation in TZM-OPN-expressing cells infected with HIVR3Nef+GFP by fluorescent microscopy and f quantified by flow cytometry (top graph). The mean fluorescent intensity (MFI) of GFP expression in HIV-infected cells or HIV-infected cells over-expressing OPN is shown in the middle graph. Activation of the HIV-LTR-beta-galactosidase promoter is shown in the bottom graph. Means and standard deviations are shown

Fig. 2

Single-cell analyses reveal that the NF-κB inhibitor IκBα is decreased in HIV-infected cells expressing OPN. TZM-bl cells infected with HIVR3Nef+GFP (green) were fixed and immunostained for IκBα (red), nuclei with DRAQ5 (blue). a TZM-bl cells only; b TZM-bl infected with Ad-OPN; (c) HIV-infected TZM-bl; d HIV-infected TZM-bl expressing OPN. The relative normalized pixel intensity of IκBα is shown (top right graph). All pictures were taken on a LSM 510 Meta at the same pinhole and intensity settings. The means and standard deviations of a representative of two independent experiments are shown. Western blot analysis for IκBα is shown in the middle panel and quantification shown in the bottom graph: HIV-infected TZM-bl cells only (HIV) or co-infected with Ad-RFP (HIVRFP) or Ad-OPN (HIVOPN); HIV-uninfected cells and exposed to Ad-RFP (RFP) or Ad-OPN (OPN). A representative of two independent experiments is shown

Fig. 3

NF-κB activity is increased in HIV-infected cells expressing OPN. TZM-bl cells infected with HIVR3Nef+GFP were fixed and immunostained for NFκB (red) and nuclei with DRAQ5 (blue). a TZM-bl cells only; b HIV-infected TZM-bl; c HIV-infected TZM-bl expressing OPN; d TZM-bl infected with Ad-OPN; (I) the relative normalized nuclear:cytoplasmic intensity of NFκB is shown. All pictures were taken on a LSM510 Meta at the same pinhole and intensity settings. (II) TZM-bl cells were co-transfected with HIV-LTR-CAT reporter constructs encoding an intact (pHIV-CAT) or mutated NFκB (pDkB-HIV-CAT) binding domain and control (pRC) or OPN-expressing plasmid (OPN). CAT protein was measured by ELISA. The means and standard deviations of a representative of two independent experiments are shown

Fig. 4

OPN is increased in the CSF and brain of HIV-infected persons. a Samples were grouped by MSK with MSK 0 (n=21), representing normal cognition and MSK 0.5 (n=35), MSK 1 (n=33) and MSK 2 (n=13) representing increasing severity of cognitive impairment; population means are indicated by hash mark. Inflammatory (n=30) and non-inflammatory (n=27) CSF samples were obtained from HIV-uninfected individuals (Table 1). Data was analyzed with one-way ANOVA, Dunn’s multiple comparison post test: *p<0.05, **p<0.01, ***p<0.001. b Osteopontin is increased in brain tissue of HIV-infected persons with cognitive disorder. Cell lysates prepared from frozen brain tissue from HIV-infected patients with stable neuropsychological diagnoses from the NNTC (Table 2, supplemental data) were analyzed by Western blot for OPN and β3-tubulin and quantified (c). HAD HIV-associated dementia, MCMD minor cognitive motor disorder, Sub subsyndromic, Normal neurocognitively normal, Cont control TZM-bl lysate. The Westerns were also probed for the HIV Nef protein. A representative of three independent experiments is shown

Fig. 5

No biologically significant associations between CSF OPN levels and plasma or CSF HIV viral load, CD4 T-cell count or age. Correlation analyses were determined assuming sampling from a Gaussian distribution. Pearson r and R² are indicated