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Osteopontin Is Increased in HIV-Associated Dementia

  1. Tricia H. Burdo1,
  2. Ronald J. Ellis2 and
  3. Howard S. Fox1,a
  1. 1Molecular and Integrative Neurosciences Department, The Scripps Research Institute, La Jolla, California
  2. 2Department of Neurosciences, University of California, San Diego (UCSD) AntiViral Research Center and HIV Neurobehavioral Research Center, UCSD, San Diego, California
  1. Reprints or correspondence: Dr. Howard S. Fox, University of Nebraska Medical Center, 985800 Nebraska Medical Center, Omaha, Nebraska 68198 (hfox{at}unmc.edu).

  • ↵a Present affiliation: Boston College, Department of Biology, Chestnut Hill, Massachusetts (T.H.B.); University of Nebraska Medical Center, Omaha, Nebraska (H.S.F.).

 
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Abstract

Since the introduction of highly active antiretroviral therapy, survival rates for human immunodeficiency virus (HIV) infection have markedly improved, but less of an effect has been found for HIV-associated neurocognitive disorders. On the basis of our previous findings, we hypothesized that increased production of osteopontin might contribute to the persistence of central nervous system (CNS) dysfunctions. We found increased levels of osteopontin in the brains of humans with HIV encephalitis and monkeys with simian immunodeficiency virus (SIV) encephalitis. In cerebrospinal fluid, osteopontin levels were found to be elevated in HIV-infected individuals, regardless of their neuropsychological status. However, plasma osteopontin levels were significantly increased in individuals with HIV-associated dementia. In addition, a longitudinal study of monkeys revealed that plasma levels of osteopontin increased before the development of SIV-induced neurological and clinical abnormalities. Thus, plasma levels of osteopontin are significantly correlated with HIV-induced CNS dysfunction in the current era of efficacious antiviral treatment, and this finding suggests that the development of interventions to modulate osteopontin production or signaling might be beneficial in the prevention or treatment of HIV-induced CNS disorders.

HIV-positive patients frequently exhibit a wide range of neurological problems due to the neuropathological events that result from HIV-1 infection itself, as opposed to opportunistic infections. Neurological symptoms associated with HIV can include cognitive impairments, motor disturbances, and behavioral changes [1]. HIV-associated neurocognitive abnormalities have been classified into 2 categories: minor cognitive-motor disorder (MCMD), which results in complaints and problems that can compromise everyday functioning; and dementia, which is characterized by more-severe impairments [2]. An update to this nosology has recently been proposed [3]. HIV-associated dementia (HAD) commonly occurs in the late stages of viral infection, when the immune system is compromised, whereas HIV-associated MCMD occurs in the early stages of infection as well.

The neurological complications of HIV infection persist in the current era of highly active antiretroviral therapy (HAART) [4]. Fortunately, after the introduction of HAART, the incidence of HAD decreased from an estimated 20% to <5% among adults with advanced cases of AIDS. However, MCMD develops in 20% of symptomatic HIV-seropositive adults, even those treated with HAART. The condition is associated with shortened survival time and significant effects on factors related to quality of life, such as job-related skills and employment, and its presence is predictive of future development of HIV encephalitis [5, 6]. Thus, despite HAART, clinical neurological dysfunction still occurs.

The etiology of the neuropathogenesis observed in HIV infection is complex. Viral infection of the brain is thought to be an initiating event, but other mechanisms secondary to virus infection are involved in the brain dysfunction. Macrophages, the major target for HIV in the brain, infiltrate the brain; they can produce numerous products potentially harmful to the central nervous system (CNS) and are prime candidates in mediating indirect damage to the CNS initiated by HIV infection [7].

Ongoing research efforts have focused on the identification of dependable correlates for HIV-associated neurocognitive abnormalities. One clue was found in the blood, where an increase in the size of the subset of monocytes that express CD16 in addition to CD69 had been found in individuals with HAD, and supernatants from such cells showed neurotoxic properties [8]. However, with the onset of HAART, this correlation no longer held [9].

Other research has focused on the cerebrospinal fluid (CSF), and findings have been reported for diverse metabolites and proteins [10–15]. However, these correlations were demonstrated mainly in the era before HAART was available. In the current era of treatment, the association between CSF markers and neurologic status is altered [16–18], and this lack of biomarkers has hindered work on HIV-induced CNS disorders, which continue to afflict infected individuals.

Infection of macaques with simian immunodeficiency virus (SIV) remains the best model of HIV disease in humans. Progressive lymphoid changes, opportunistic infections, a wasting syndrome, and central nervous system disease characterize the syndrome in both humans and monkeys [19]. When we used the SIV model, our previous gene-array expression analysis of the brains of rhesus monkeys with SIV encephalitis (hereafter, “SIVE+ monkeys”) [20] revealed a significant increase in the level of osteopontin, an extracellular protein involved in differentiation and immune cell activation, as well as cell attachment and migration [21]. We subsequently showed that osteopontin increased monocyte retention and protected monocytes from apoptosis, and we proposed that these mechanisms are an underlying cause of macrophage accumulation during HIV-1 infection [22].

We then hypothesized that increased expression of osteopontin may correlate with the development of neurological disease associated with HIV-1 infection. To test this hypothesis, we examined the expression of osteopontin in both plasma and cerebral spinal fluid in humans, as well as in SIV-infected monkeys. Our results clearly demonstrate a correlation between plasma levels of osteopontin and the severity of neurologic disease.

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Methods

Rhesus macaques. Rhesus monkeys were infected with derivatives of SIVmac251 [23, 24]. Animals were categorized as having either SIV encephalitis (7 animals for RNA analysis and 6 for ELISA analysis) or SIV infection without encephalitis. The latter group was subdivided into the following categories: animals with simian AIDS without encephalitis (4 animals for ELISA analysis) and SIV-positive animals without encephalitis and without simian AIDS (12 animals for RNA analysis, and 4 for ELISA analysis). In addition, for quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR) analysis, we used RNA from uninfected controls animals (n = 9).

RNA isolation. RNA was purified from frontal lobe samples by use of TRIzol reagent (Invitrogen), and it was then further purified utilizing the RNeasy Mini Kit (Qiagen). RNA was quantified by 260 nm of UV light absorption.

Reverse transcription. RNA was incubated with random primers and SuperScript II reverse transcriptase (Invitrogen), followed by heat inactivation and treatment with ribonuclease H (New England BioLabs).

Quantitative real-time RT-PCR. Specific RNA transcripts were quantified through the use of real-time RT-PCR performed with dual-labeled (FAM-TAMRA) hydrolysis probes; reactions were optimized and validated by dilutional analysis. For osteopontin, the primers were 5′-AGA-A-G-T-T-C-C-G-C-A-G-A-C-C-T-GAC-3′ and 5′-GCT-T-T-C-C-A-C-A-T-G-T-G-A-G-G-TGA-3′, and the probe was 5′-CAG-T-A-C-C-C-T-G-A-T-G-C-T-A-C-A-G-A-C-G-AGG-3′. For TATA box-binding protein, the primers were 5′-AAA-G-A-C-C-A-T-T-G-C-A-C-T-T-C-GTG-3′ and 5′-GGT-T-C-G-T-G-G-C-T-C-T-C-T-T-A-TCC-3′, and the probe was 5′-TCC-C-A-A-G-C-G-G-T-T-T-G-C-T-G-CAG-3′.

Primers and probes were obtained from Eurogentec. Real-time RT-PCR reactions were performed in duplicate by using Platinum qPCR Supermix-UDG (Invitrogen) in a Stratagene MX3000 machine. To compute the relative amounts of osteopontin mRNA in the samples, the average cycle threshold (Ct) of the primary signal of TATA box-binding protein (as a housekeeping gene) was subtracted from that for the osteopontin to obtain the change in Ct (dCt), and relative units were calculated as 2−dCt.

Immunohistochemical analysis. Formalin-fixed, paraffin-embedded brain sections from rhesus monkeys were deparaffinized with xylene and hydrated in graded alcohols. Immunohistochemical staining was performed in accordance with a basic indirect protocol that used a citrate antigen retrieval method. The primary antibody used was directed toward osteopontin (Panomics; 1:2), detected with PicturePlus universal secondary antibody-horseradish peroxidase polymer reagent (Zymed), developed with NovaRed chromogen (Vector Laboratories), and, finally, counterstained with hematoxylin (Sigma-Aldrich). Control slides included omission of primary antibodies and irrelevant antibodies. Image capture was performed using a Spot RT Color CCD camera and Spot RT software (version 3.4.2 for MacOS; Spot Diagnostic Instruments) with the use of a Leica Diaplan microscope (Leica).

Immunofluorescence. Before immunofluorescence experiments were performed, slides of formalin-fixed, paraffin-embedded brain sections from rhesus monkeys were exposed to a dual black light-natural sunshine light for 18 h to minimize autofluorescence [25]. To examine whether osteopontin was expressed in infiltrating or perivascular macrophages or in activated microglia, we used an antibody directed toward CD163 (Novocastra; 1:100) [26] in conjunction with osteopontin. These sections were pretreated as for the immunohistochemical analysis. Sections were blocked with 10% normal goat serum (NGS) in CAS block (0.5% casein and 0.05% thimerosol in PBS) for 30 min and were incubated with primary antibodies overnight. For the osteopontin, the secondary antibody was biotinylated anti-rabbit IgG (Vector; at 1:100 for 30 min), followed by streptavidin AlexaFluor 488 conjugate (Molecular Probes; at 1:500 for 30 min in the dark). For CD163, the secondary antibody was anti-mouse AlexaFluor 594 (Molecular Probes; 1:500). For the detection of DNA and thus nuclei with the use of 4′,6-diamidino-2-phenylindole (DAPI), sections were overlaid with 25 µL of Vectashield mounting media containing 1.5 µg/µL DAPI (Vector) and observed by fluorescence microscopy (Axiovert 200 inverted microscope [Zeiss] for osteopontin [green], DAPI [blue], and CD163 [red]). Green, red, and blue images were merged by using AxioVision software (Zeiss).

Human plasma and CSF samples. EDTA-anticoagulated plasma, and CSF centrifuged free of cells were obtained from 17 HIV-negative individuals and from 3 groups of HIV-infected individuals, who were categorized on the basis of their neurocognitive diagnosis: 33 individuals were classifed as being neuropsychologically normal (similar to a Memorial Sloan-Kettering classification [MSKC] of 0), 37 were classified as having MCMD (similar to a MSKC of 1), and 25 were classified as having HAD (similar to a MSKC of 2, 3, or 4). All plasma and CSF samples were obtained from the 4 National NeuroAIDS Tissue Consortium (NNTC) sites. The NNTC uses a series of 14 neuropyschological tests to determine neurocognitive diagnosis [27, 28]. Plasma and CSF samples from HIV-negative individuals were obtained from investigators at a single NNTC site, whereas samples from HIV-positive individuals were provided by all 4 sites. Viral loads were converted from the number of copies per milliliter to the log10 number of copies of RNA per milliliter. For plasma viral loads, any values that were <400 copies/mL or were undetectable were reported as <2.6 log10 copies of RNA/mL. For viral loads in CSF samples, any values reported as <50 copies/mL or as undetectable were reported as <1.7 log10 copies of RNA/mL.

Collection of rhesus monkey plasma. Rhesus macaques had blood samples serially collected while they were anesthetized with ketamine. For plasma isolation, blood was placed in EDTA-treated tubes and centrifuged for separation from cells and erythrocytes.

Cytokine measurement. The levels of osteopontin in the plasma and CSF samples obtained from humans and monkeys, as well as the chemokine ligand 2 (CCL2) and interleukin (IL)-6 levels in monkey plasma samples, were measured by use of a human osteopontin ELISA (Immuno-Biological Laboratories, IBL-America), human CCL2 ELISA (R & D Systems), or human IL-6 ELISA (R & D Systems), respectively. Samples were analyzed in duplicate in accordance with the manufacturer's instructions.

Statistical analysis. For the osteopontin ELISA data in plasma and CSF samples from humans, outlier analysis and test of fit to normality were performed using Statgraphics Centurion (build 15.02; StatPoint). Outliers were defined as data points that lay >3.0 times outside the interquartile range and that had a median absolute deviation (MAD) z score >3.5. Outliers were identified by using standard scores and Grubb's test, and the result was verified by Dixon's test, where applicable. The fit to normality was tested by using the Shapiro-Wilk test and standardized skew. With the use of raw values, 4 outliers were identified, and 4 of 8 samples did not fit the assumption of normality. The log10 transformation was tested, and a better fit to normality was obtained in which only 1 sample did not fit (1 CSF sample from the neuropsychologically normal group) and only 2 log10 values met our outlier criterion (1 large value in plasma samples from the HAD group and 1 small value in CSF samples from the HAD group).

For other statistical analyses, we used Prism software (version 4.0b; GraphPad) and Delta Graph software (version 5.7.3; Red Rock Software).

Regulatory approval. All animal studies were performed with the approval of the Scripps Institutional Animal Care and Use Committee, and the studies followed Scripps and National Institutes of Health guidelines. Human studies were performed after obtaining written informed consent from the patients. Samples were collected under the guidelines and approval of regulatory committees at the institution where the samples were procured, and the studies reported were performed under the guidelines of the Scripps Human Subjects Committee.

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Results

Identification of osteopontin upregulation during SIV encephalitis. To validate prior DNA array results indicating upregulation of osteopontin in SIVE+ monkeys [20], we performed quantitative real-time RT-PCR analysis on RNA isolated from the frontal lobes of uninfected control animals, SIV-infected animals without encephalitis, and SIV-infected animals that spontaneously developed SIV encephalitis. We indeed found that osteopontin mRNA is significantly upregulated in the brains of SIVE+ animals (figure 1).

Figure 1
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Figure 1

Osteopontin (OPN) mRNA expression in the frontal lobe of uninfected animals and simian immunodeficiency virus (SIV)-infected animals with and without encephalitis (SIVE+ and SIVE− animals). Quantitative real-time polymerase chain reaction analysis was used to examine OPN mRNA expression in the frontal lobe of uninfected control monkeys (n = 9), SIVE− animals (n = 12), and SIVE+ animals (n = 7). OPN mRNA is significantly upregulated in the brains of SIVE+ monkeys, compared with expression in uninfected control animals and SIVE− animals.

To examine the cellular source of osteopontin in the brain, we next performed immunohistochemical analysis, and we found that osteopontin was indeed detectable in the brains of SIVE+ animals, especially in perivascular regions (figure 2A), but it was not detectable in uninfected animals or in SIV-infected monkeys without encephalitis (data not shown). Osteopontin was also expressed in the brains of human patients who experienced HAD (figure 2B). Double-label immunofluorescence was performed to determine the cell types expressing osteopontin in the brains of SIVE+ monkeys (figure 2C–E). Colocalization of CD163 (a scavenger receptor expressed on macrophages) and osteopontin was seen (figure 2E, arrows), indicating that these cells were expressing osteopontin during encephalitis. Osteopontin was thus expressed in the brains of monkeys and humans with encephalitis, and it is produced by macrophages, which are key cells in the neuropathogenesis of HIV.

Figure 2
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Figure 2

Osteopontin expression in the brains of monkeys with simian immunodeficiency virus encephalitis (SIVE+) and the brains of humans with HIV encephalitis (HIVE+). A and B, Immunohistochemical analysis revealed the presence of osteopontin (*) in the brains of SIVE+ monkeys (A) and the brains of HIVE+ humans (B). CD163+ perivascular cells express osteopontin in the brains of SIVE+ monkeys. C–E, Immunofluorescence was performed on prepared sections of brain from SIVE+ monkeys. C, Osteopontin is shown in green and 4′,6-diamidino-2-phenylindole (DAPI) in blue. D, CD163 is shown in red with DAPI in blue. E, Merged image; the arrows point to coexpression of CD163 and osteopontin (yellow). All images were taken through a 63× magnification lens.

Osteopontin levels in plasma, but not in CSF, correlate with neurological status in HIV-infected individuals. Although plasma samples are easier to obtain clinically, CSF samples provide a window into the CNS that is accessible by clinical testing. To examine whether osteopontin levels in plasma or CSF may help indicate the presence of HIV-induced CNS disease, we investigated the osteopontin levels in the plasma and CSF of individuals with HIV infection and uninfected individuals. Examination of osteopontin levels in the plasma and CSF was performed for 4 specific groups of individuals: HIV-negative individuals, HIV-positive individuals with normal neuropsychological test results, HIV-positive individuals with MCMD, and HIV-positive patients with HAD. All samples were collected from patients who were alive, and all HIV-positive individuals were neuropsychologically tested and classified into these categories on the basis of validated, reproducible criteria [27, 28]. Other than the variation in neuropsychological classification, the groups were fairly well matched for demographic and clinical characteristics (table 1). The HIV-positive subjects were mostly middle-aged men who were moderately immunosuppressed and were receiving combination antiretroviral therapy, although approximately half of them had detectable plasma viral loads. Neither plasma nor CSF viral loads were significantly different between the three groups of HIV-infected subjects (i.e., the neuropsychologically normal, MCMD, and HAD groups).

Figure 3
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Figure 3

Relationship of plasma and cerebrospinal fluid (CSF) osteopontin levels and neurological status. Osteopontin (OPN) was measured in samples of plasma (A) and CSF (B) from HIV-negative individuals (Neg) and the following 3 groups of HIV+ individuals: those who were neuropsychologically normal (NPN), those with minor cognitive-motor disorder (MCMD), and those who had HIV-associated dementia (HAD). The values in the groups are shown as box and whisker plots, indicating the median, the 25th and 75th percentiles, and the largest and smallest values. The groups were significantly different by analysis of variance; brackets and asterisks, significant difference by post hoc testing, as described in Results.

Figure 4
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Figure 4

Relationship between plasma osteopontin (OPN), chemokine ligand 2 (CCL2), and interleukin (IL)-6 levels and development of simian immunodeficiency virus (SIV) encephalitis. Cytokine plasma levels were examined in a longitudinal study after SIV infection. Graphs are plotted as plasma levels of OPN versus weeks prior to necropsy. A, OPN levels in animals that received a pathological diagnosis of SIV encephalitis (SIVE+). B, OPN levels in animals without encephalitis (SIVE−), a group that included animals that were infected with SIV and developed simian AIDS, but not encephalitis (closed symbols), and animals that were infected with SIV, but were killed while healthy (open symbols). C, Plasma CCL2 levels in SIVE+ animals. D, Plasma CCL2 levels in SIVE− animals. E, Plasma IL-6 levels in SIVE+ animals. F, Plasma IL-6 levels in SIVE− animals.

Table 1
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Table 1

Demographic and clinical characteristics of the study population.

The analysis of plasma osteopontin levels revealed a significant difference between the groups (figure 3A) (P < .001, by analysis of variance [ANOVA]). More specifically, the levels were increased in both groups with deficits. The plasma osteopontin levels in the MCMD group were significantly different from those in the HIV-negative group (P < .01, by Tukey's multiple comparison test), whereas osteopontin levels in the HAD group were significantly different from those found in the HIV-negative and the HIV-positive neuropsychologically normal groups (P < .001 and P < .05, respectively, by Tukey's multiple comparison test). These results demonstrate that osteopontin levels correlate with neurological status. The osteopontin levels in the plasma were lowest in the HIV-negative individuals and the highest in the patients with HAD. In the CSF samples, osteopontin levels were again significantly different between the groups (figure 3B) (P < .001, by ANOVA). However, the differences between the groups were limited to all of the HIV-positive groups (the neuropsychologically normal, MCMD, and HAD groups) having levels significantly higher than those of the HIV-negative group (P < .01, P < .001, and P < .01, respectively). Thus, the presence of HIV infection correlated with increased osteopontin levels in CSF, regardless of neuropsychological status. For this reason, CSF osteopontin levels, although elevated by HIV infection, are not a good marker of neurological disease status, whereas plasma osteopontin levels reveal a significant correlation.

Longitudinal study of SIV-infected rhesus monkeys revealed that plasma osteopontin levels are predictive of the onset of symptoms. After this demonstration that plasma osteopontin levels in HIV-infected subjects were further increased in HIV-positive patients with the severe clinical neurological diagnosis of HAD, we assayed plasma osteopontin levels over the course of disease in an experimental model system, the SIV-infected rhesus monkey, to assess whether the increase in plasma osteopontin levels may be useful in predicting the development of CNS disease. We thus serially analyzed levels of osteopontin in the plasma of SIVE+ monkeys (figure 4A) and SIVE− monkeys (figure 4B); the latter group included monkeys that developed simian AIDS (figure 4B, closed symbols) and monkeys that did not (figure 4B, open symbols). The mean increase (±SD) in plasma osteopontin levels in animals that developed encephalitis (34.8 ± 19.1 ng/mL per week) was significantly greater than that noted in animals with simian AIDS that did not develop encephalitis (6.2 ± 4.4 ng/mL per week) and the levels observed in SIV-infected animals that did not develop simian AIDS (1.3 ± 3.2 ng/mL per week) (P = .004, by ANOVA; P < .05 and P < .01, respectively, by post hoc Tukey's tests). Plasma osteopontin levels thus correlated with the development of AIDS with encephalitis, as opposed to correlating with the development of AIDS without encephalitis or correlating with SIV infection itself. Furthermore, because the animals did not receive therapy, they were killed soon after showing symptoms of disease. In most of the animals, the increase in osteopontin level occurred 1–3 weeks before the onset of clinical disease.

To assess whether the plasma levels of other potentially inflammatory cytokines were also increased, we assayed the levels of CCL2 and IL-6 in the same animals (figure 4C–F). CCL2 levels varied inconsistently during the course of disease in SIVE+ animals (figure 4C), and the level of IL-6 was highly increased in only 2 of 6 SIVE+ animals at necropsy (figure 4E). CCL2 and IL-6 levels remained stable in all SIVE− animals (figure 4D and 4F). Thus, the increase in plasma osteopontin levels did not reflect a general increase in proinflammatory markers, and osteopontin was the best biomarker in the plasma to predict the onset of CNS disease.

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Discussion

Our study is a unique combination and cross-validation of work in humans and the rhesus macaque animal model. We have demonstrated that osteopontin mRNA, as well as protein, was upregulated in the brains of SIVE+ rhesus monkeys. In HIV-infected humans, CSF osteopontin levels were found to be increased relative to those observed in uninfected individuals, but it was the levels of osteopontin in the plasma (not the levels in the CSF) that correlated with neurocognitive diagnosis. Among HIV-infected individuals, plasma osteopontin levels were significantly increased in those with HAD, compared with those without neuropsychological abnormalities. Furthermore, the plasma osteopontin levels in individuals with MCMD and HAD were increased relative to those in HIV-negative individuals. Finally, a longitudinal study of SIV-infected rhesus monkeys revealed that osteopontin in plasma is a dependable correlate of SIV encephalitis. It is also predictive, because osteopontin levels were increased before clinically apparent neurological disease in SIVE+ rhesus monkeys.

From the clinical point of view, one goal is to be able to use osteopontin in conjunction with other biomarkers in plasma to diagnose and/or predict clinical symptomatology associated with HIV-associated neurocognitive disorders. To advance this goal further, a longitudinal study of the plasma of HIV-infected individuals needs to be performed in conjunction with assessment of other molecules and neuropsychological evaluation. Interestingly, it was recently demonstrated that plasma osteopontin levels were elevated before increases in disease activity in patients with relapsing-remitting multiple sclerosis [29]. In a similar manner, plasma osteopontin levels in individuals without dementia could be monitored, in conjunction with other markers, to investigate osteopontin levels indicative of a progression toward HAD.

Somewhat surprisingly, although HIV infection itself led to increased CSF osteopontin levels, it was the plasma levels that correlated with neurocognitive status. The increased plasma osteopontin levels could potentially provide a mechanism for CNS disease in patients with HIV infection. The combination of HIV-induced increases in expression of osteopontin in the CNS, along with the development of increased plasma levels in a subset of HIV-infected individuals, may drive the development of neurological disease. Mechanistically, our previous work indicated that the potential role of osteopontin in the plasma would be to protect monocytes from apoptosis, whereas, in the brain, osteopontin could decrease the number of macrophages returning to the circulation [22], yielding increased numbers of macrophages in the brain, which is the best pathological correlate of HAD [30]. A relevant analogy has recently been reported in a mouse model of obesity. The disease, induced by a high-fat diet, is characterized by elevated levels of plasma osteopontin and increased osteopontin-containing macrophages in adipose tissue. The use of osteopontin knockout mice revealed a central role for osteopontin in this model [31]. Similarly, we found that, in CNS disease induced by HIV and SIV, there are increased plasma osteopontin levels and increased numbers of brain macrophages, which express osteopontin. However, we note that, in addition to differences in absolute levels, osteopontin is functionally regulated by glycosylation, phosphorylation, and proteolytic cleavage [32–35], and changes in these processes may also alter the quantification of osteopontin by ELISA.

In the present study, we have demonstrated correlations between plasma osteopontin levels and neurological status in HIV-infected individuals, and we intend to conduct future studies to examine whether there are any causal relationships between increased osteopontin levels and CNS disease. The potential contribution of osteopontin to HIV neuropathogenesis, as opposed to osteopontin serving as a marker of macrophage activation, awaits further investigation. Interestingly, both peroxisomes proliferator-activator receptor (PPAR)-α and PPAR-γ agonists have been shown to reduce osteopontin production by macrophages [36, 37]. We note that, although we have identified macrophages as a source of osteopontin in the brain, there may be multiple cellular sources of the osteopontin circulating in the plasma. Nonetheless, both fibrates, which are PPAR-α agonists, and thiazolidinediones, which are PPAR-γ agonists, have been used in HIV-infected individuals to treat HAART-induced lipodystrophy. Although no data have been reported for thiazolidinedione treatment, the use of benfibrate treatment for patients with type 2 diabetes led to reduced plasma levels of osteopontin [36]. It would be of interest to assess whether similar reductions can occur in HIV-infected individuals and to determine the influence of such treatment on CNS disease.

In conclusion, we have shown that osteopontin levels are increased in the CSF of HIV-infected individuals and that osteopontin levels in plasma exhibit a stair-step increase across diagnostic categories of HIV-associated neurocognitive disorders. These findings are consistent with a pathophysiological model in which osteopontin amplifies brain macrophage accumulation, which is the pathological substrate of HAD. Furthermore, longitudinal analysis of osteopontin levels in SIV-infected monkeys suggested the value of osteopontin as a plasma biomarker for neurological disease associated with AIDS. Thus, plasma osteopontin levels may be used, along with other criteria, to help predict the course of neurological disease in patients with AIDS. Means for decreasing osteopontin production or its signaling can be attempted therapeutically to prevent or ameliorate this disorder.

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Acknowledgments

We thank Claudia Flynn, Jason Lee, Ryan Ojakian, Debbie Watry, and Michelle Zandonatti for technical assistance; Caroline Lanigan for the outlier analysis; and J. Lindsay Whitton for use of the fluorescence microscopy system.

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Footnotes

  • Potential conflicts of interest: none reported.

  • Financial support: National Institutes of Health (NIH; grants NS045534 to H.S.F. and MH073490 to H.S.F.); National Institute of Mental Health (NIMH) Core Support Program for Mental Health/AIDS Research (grants MH62261 [the TSRI SNAPS] to H.S.F. and MH62512 [HIV Neurobehavioral Research Center, University of California, San Diego] to R.J.E.); National Research Service Award (postdoctoral fellowship F32 NS048830 to T.H.B.). The National NeuroAIDS Tissue Consortium is supported by the NIMH and the National Institute of Neurological Disorders and Stroke (contract NIH-N01MH32002).

  • Received October 11, 2007.
  • Accepted December 27, 2007.
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  1. J Infect Dis. (2008) 198 (5): 715-722. doi: 10.1086/590504
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