INTRODUCTION
Human immunodeficiency virus (HIV) infects cells of the immune system, such as CD4
+ T cells and macrophages (MΦs). HIV can enter the central nervous system (CNS) early in the course of infection (
1,
2), and viral replication in brain macrophages (Br-MΦs) can lead to HIV-associated neurocognitive disorders (HAND) in 20 to 30% of infected individuals in the later stages of HIV infection (
3). Clinical determination of HAND is associated with the presence of multinucleated giant cells (MNGCs) at the time of autopsy due to productive infection of Br-MΦs and microglial cells, a hallmark of HIV-induced encephalitis (HIVE) (
4–14). Administration of combination antiretroviral therapy (cART) can greatly reduce the incidence of fulminant HIV encephalopathy and improve cognitive function; however, a milder form of HAND, minor cognitive motor disorder (MCMD), has become more commonplace and is associated with persistence of virus in the CNS and a worse prognosis than that in fully suppressed patients (
6,
15,
16).
While studies of HIV-infected patients with neurological complications of AIDS (neuroAIDS) are no longer feasible due to the efficacy of antiviral drug treatment, an examination of the older literature on the pathology of HIVE/neuroAIDS revealed that lymphocytes were frequently observed in the brains of these patients (
5,
8–14). Studies of HIV-infected dementia patients are complicated by the fact that this clinical syndrome encompasses both the direct effects of virus replication in the CNS, as in HIV encephalitis, as well as opportunistic infections of the brain (cytomegalovirus [CMV], polyomavirus, and
Cryptococcus spp., to name a few) and lymphoma. In a majority of cases, opportunistic infections in the brain were identified, in addition to microglial nodules, MNGCs, and lymphoproliferative lesions of the neuroparenchyma (
8,
13). Progressive diffuse leukoencephalopathy (PDL) and multifocal giant cell encephalitis (MGCE) was observed in most cases examined. MGCE was characterized by perivascular and parenchymal infiltrates of both macrophages and lymphocytes (
5,
13). In these patients, a spectrum of neurological abnormalities existed, along with varied expression of neurological symptoms. Severe dementia was observed in patients with perivascular and parenchymal macrophages, as well as MNGCs, while milder cases of dementia were noted in the patients with scattered perivascular lymphocytes and macrophages (
5). The authors also noted that even in severe cases of dementia, histopathological findings were nonexistent, leading them to note that histopathology is not uniform in cases of HIV-induced dementia. The pathophysiology differs from patient to patient; thus, it becomes important to have various animal models that can cover all complexities of the disease.
Simian immunodeficiency virus (SIV)-infected nonhuman primates are widely used as a model for AIDS pathogenesis. Infection of these animals with neurotropic SIV can result in SIV-induced encephalitis (SIVE)/neuroAIDS), with neuropathologic findings reminiscent of HIVE in humans, including the presence of MNGCs. SIV infection of rhesus macaques (RMs) allows for sampling of the cerebrospinal fluid (CSF) and brain tissue throughout all stages of disease progression under controlled conditions. Current models that dominate studies evaluating SIVE include the use of immunomodulation in order to induce rapid progression to neuroAIDS. One model uses pigtailed macaques coinoculated with a neurovirulent clone virus, SIVmac17E-Fr (17E-Fr), and an immunosuppressive, uncloned virus, SIVsmB670 (
17). This dual infection model results in the peripheral depletion of CD4
+ T cells by B670, which appears to allow for efficient replication of 17E-Fr in Br-MΦs. The advantage of this model is high reproducibility of SIVE in animals (90%); however, animals rapidly progress to neuroAIDS within 3 to 6 months postinfection. A second model uses immunomodulation to induce neuroAIDS. RMs are administered anti-CD8 antibodies prior to inoculation with SIV (SIVmac251 or SIVmac239), and just as with the pigtailed macaques, animals rapidly progress within 3 to 6 months postinfection (
18,
19). A third model uses anti-CD4 antibodies to deplete CD4
+ T cells in RMs prior to infection with SIVmac251. This results in a rapid progression to neuroAIDS within 3 months postinfection due to productive infection in microglia (
20,
21).
We have recently reported on this SIVE/neuroAIDS model using uncloned SIV isolated after 4 serial passages of nonneurovirulent SIVsmE543-3 (E543-3) through RMs and subsequently generated a neurovirulent molecular clone virus, SIVsmm804E-CL757 (CL757) (
22). Infection with this clone virus leads to SIVE in 50% of infected RMs approximately 1 year postinfection. In the current study, we examined the brains of RMs infected with CL757 and other nonneurovirulent strains of SIV from sooty mangabey monkeys (SIVsmm) to identify which cellular subsets in the brain are targeted by the virus. We show that in macaques that conventionally progress to neuroAIDS, both brain memory CD4
+ cells (Br-mCD4s) and Br-MΦs harbor replication-competent SIV DNA. We also show that Br-mCD4s harboring SIV DNA infiltrate the neuroparenchyma and localize to the site of viral replication. Unexpectedly, Br-mCD4s harbored SIV DNA in animals that showed no pathological or clinical signs of neuroAIDS. This finding indicates an unanticipated role for Br-mCD4s as a potential additional viral reservoir.
DISCUSSION
Shortly after infection, HIV establishes itself in the CNS where it persists throughout the course of disease and in untreated patients eventually results in encephalitis and neuroAIDS. Although the mechanism of entry into the CNS remains unknown, a hallmark of HIVE/neuroAIDS is the infection and replication of HIV in macrophages and the resulting formation of multinucleated giant cells (MNGCs). Studies of neuroAIDS in HIV-infected individuals are difficult due to the limitations of direct sampling of the CNS; therefore, animal models that closely resemble the disease conditions in humans are of utmost importance. We used one such model of neuroAIDS that results in a conventional (rather than rapid) disease progression and results in SIVE/neuroAIDS in about half of the inoculated macaques to study the cellular targets in the brain at terminal disease endpoints. Using isolated CNS mononuclear cells and tissue from a panel of chronically infected RMs, we demonstrate that the development of SIVE in CP RMs leads to the formation of lymphoid-rich lesions that are associated with glial nodules and MNGCs at the site of chronic viral replication and inflammation in the neuroparenchyma. In RMs without SIVE, viral DNA was not found in Br-MΦs; however, replication-competent viral DNA was discovered in the Br-mCD4s. The finding of virus in the Br-mCD4s of RMs with or without SIVE was unexpected since most studies have focused on the role of MΦs in this disease process. Indeed, MΦs appear to be the sole cell type harboring SIV in the brains of RP RMs with SIVE and are clearly strongly associated with the pathology of SIVE. Infection of macrophages was only observed in association with pathological evidence of encephalitis linking this cell type to the pathogenesis of inflammation in the CNS in neuroAIDS. Our current model can aid in the elucidation of the pathophysiology of neuroAIDS using CP RMs and indicates an unanticipated role for mCD4s as an additional potential viral reservoir in the CNS.
Recruitment of T cells to the CNS has been previously reported in cases of CNS disease, although these studies have focused predominately on brain CD8
+ T cells isolated from RMs that rapidly progressed to SIVE (
25,
26). Lymphocyte infiltration of the CNS is thought to arise due to hematopoietic lymphocytes surveying the CNS via the meninges and choroid plexus and encountering their cognate antigen presented by an antigen-presenting cell (APC)/Br-MΦs, which leads to two subsequent events. First, cells are trafficked in through high-endothelial vessels and localize to the site of virus infection (
27,
28); second, mCD4s clonally expand in attempt to control and clear infection (
29,
30). Past examinations of HIV-infected patients with neuroAIDS, prior to the discovery of antiretroviral drug treatment, showed that along with the development of MNGCs, lymphocytes were present in the neuroparenchyma of these patients (
5,
8–14). These past studies show that histopathology is not uniform in cases of AIDS-induced dementia. The pathophysiology of brain sections differed from patient to patient, even in those with severe cases of dementia; thus, it becomes important to have various animal models that can cover all complexities of the disease.
We examined tissue and mononuclear cells isolated from the CNS from a panel of 15 RMs all chronically infected with either CL757 (
n = 5) or other relevant SIVsmm strains (
n = 10). Three CL757-infected RMs developed SIVE, as determined by high CSF viral loads (VLs) and MNGCs, which are indicators of viral replication in Br-MΦs. Inflammatory responses initiated from infected MΦs have been previously shown to recruit adaptive immune cells from the periphery to the target tissue, with these infiltrating cells consisting mainly of T cells, B cells, and MΦs (
31,
32). We observed a significant increase in the number of mononuclear cells in the brains of animals with SIVE and identified classical adaptive immune cells, CD4
+ and CD8
+ T cells, B cells, and MΦs, within this population. There were significant increases in the percentages of CD4
+ T cells, B cells, and MΦs, all of which correlated with an increase in the CSF viral load, while the population of CD8
+ T cells remained constant regardless of the SIVE status of the animal. Of the CD4
+ and CD8
+ T cells isolated from the CNS, they were all of a memory phenotype, which confirmed previously reported findings that lymphocytes of the CNS are mainly of the memory subset (
33). Br-MΦ populations have previously been shown to expand proportionally with the formation of MNGCs due to the proliferation of infected MΦs (
34). Using an immunofluorescence assay (IFA) coupled with both DNA and RNAscope, we see that RMs with SIVE harbor SIV DNA that is transcriptionally active in Br-MΦs within lymphoid-rich lesions. We have previously shown that CL757 is highly adapted to replicate in the CNS compared to its parental clone, SIVsmmE543-3 (E543-3), which is generally excluded from replicating in the CNS (
22). Our study shows that while trafficking does occur between the CNS and the periphery, Br-mCD4s appear to harbor replication-competent virus in the CNS in the absence of SIVE. This consistent with CD4s trafficking from the periphery to the CNS, which may allow for the CNS to serve as a reservoir for SIV in Br-mCD4s. Since this study did not examine target cells in the brain during primary infection or following the initiation of antiretroviral therapy (ART), we can only speculate as to the initial targets for virus in the CNS and the persistent reservoir. Further studies will be required to determine whether mCD4s constitute a significant reservoir in fully cART-suppressed macaques with critical implications for eliminating the viral reservoir to effect a cure.
In summary, variations in neurological disease outcomes are common in HIV-infected individuals. Studies of neuroAIDS in humans are complicated by limitations on samples collected and the widespread use of antiviral drug therapy, which has greatly reduced the incidence of fulminant HIVE/neuroAIDS. CL757-infection of RMs leads to the development of SIVE/neuroAIDS at a conventional pace closely resembling the tempo of disease in HIV-infected patients. This model shows infiltration and/or proliferation of classical adaptive immune cells to the CNS of RMs with SIVE, and of these, a high percentage are MΦs and mCD4s. Both cell types are carriers of replication-competent SIV DNA and, along with B cells, localize to the site of viral replication in the neuroparenchyma. While SIV-infected RMs without SIVE do not harbor virus in Br-MΦs, mCD4s harbored replication-competent SIV DNA in such animals, implicating mCD4s as a potential long-term viral reservoir in the brain.
MATERIALS AND METHODS
Ethics statement and animal studies.
This study was carried out in strict accordance with the recommendations described in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, the Office of Animal Welfare, and the U.S. Department of Agriculture. Colony-bred rhesus macaques of Indian origin were obtained from the Morgan Island, SC, rhesus monkey breeding colony. All animal work was approved by the NIAID Division of Intramural Research Animal Care and Use Committee (IACUC) in Bethesda, MD (animal study protocol LMM-6). The animal facility is accredited by the American Association for Accreditation of Laboratory Animal Care. All procedures were carried out under ketamine anesthesia by trained personnel under the supervision of veterinary staff, and all efforts were made to ameliorate the welfare of the animals and to minimize animal suffering in accordance with the recommendations of the Weatherall report on the use of nonhuman primates. Animals were housed in adjoining individual primate cages, allowing for social interaction, under controlled conditions of humidity, temperature, and light (12-h light/12-h dark cycles). Food and water were available ad libitum. Animals were monitored twice daily (pre- and postchallenge) and fed commercial monkey chow, treats, and fruit twice daily by trained personnel. Early endpoint criteria, as specified by the IACUC-approved score parameters, were used to determine when animals should be humanely euthanized.
Study animals and sample processing.
This study consisted of 14 chronically infected SIV-infected RMs (3 RMs chronically infected with variants of SIVsmmE543-3, 5 RMs with variants of SIVsmmE660, 1 RM with SIVmac239, and 5 RMs with SIVsmm804E-CL757;
Table 1). Viral clones designated with “SS” were distinguished by the introduction of a double mutation, P37S-R89S, in the Gag protein that conferred resistance to TRIM-5
TFP restriction, and viral clones designated with “CT” encoded an Env protein in which the entire cytoplasmic was truncated (
35–38). Although all study animals were evaluated at terminal disease endpoints, neuroAIDS was only observed in those animals inoculated with CL757. Peripheral blood was collected from all animals terminally, and peripheral blood mononuclear cells (PBMCs) were isolated via standard density gradient centrifugation using LSM solution (MP Biomedicals, Santa Ana, CA) and then analyzed from frozen samples attained at necropsy. Brain tissue from each animal was processed fresh, and mononuclear cells were isolated from frozen samples and analyzed.
Isolation of brain mononuclear cells.
All animals were saline perfused before necropsy to remove blood from the tissues. Whole brain was sliced in half along the sagittal plane, and one half was preserved in neutral-buffered 10% formalin solution (Sigma-Aldrich) for subsequent histopathology immunohistochemistry and DNAscope/RNAscope. The remaining half of the brain was used to isolate mononuclear cells using a protocol adapted from a study by Cardona et al. (
39). Briefly, tissue was diced and then enzymatically digested for 1 h in digestion solution composed of Hanks’ balanced salt solution (HBSS) without Ca
2+ and Mg
2+ (Sigma-Aldrich), supplemented with 0.05% collagenase D (catalog no. 110-888-82-001; Sigma), 0.5% Dispase II (catalog no. D4693-1G; Sigma), 0.025 U/ml DNase I (catalog no. D4527-10KU; Sigma), and 0.025 U/ml
Nα-
p-tosyl-
l-lysine chloromethyl ketone [TLCK] (catalog no. T7254-100 mg; Sigma) at 37°C with agitation (
39). Homogenized tissue was centrifuged at 300 ×
g for 10 min and the cell pellet washed with 1× HBSS twice to remove any residual enzymes. The washed pellet was resuspended at a 1:1 ratio in 100% Percoll (diluted with 1× HBSS). A Percoll gradient was formed using 10 ml of 70% Percoll and 1× HBSS in the bottom of a 50-ml conical tube and then overlaid with 15 ml homogenized tissue mixed with equal parts of undiluted Percoll solution. Ten milliliters of 30% Percoll solution was slowly layered on top, followed by 15 ml of 1× HBSS, and the gradient was centrifuged for 40 min at 500 ×
g. Cells were harvested from the 30 to 50% interphase and washed with 1× HBSS 3 times to remove residual Percoll, and the cells were suspended in 10 ml of 1× HBSS. Cells were cryopreserved in freezing medium (90% fetal bovine serum and 10% DMSO) in liquid nitrogen.
Immunophenotyping.
Polychromatic cell sorting was performed on stained mononuclear cells isolated from fresh rhesus macaque brain tissue utilizing a FACSAria II flow cytometer (BD Biosciences, Franklin Lakes, NJ) equipped with the FACSDiva software (BD Biosciences). Cells were first stained with a LIVE/DEAD fixable aqua dead cell dye (Life Technologies, Carlsbad, CA). For live-cell sorting, the following panel of monoclonal antibodies (MAbs) was used: CD14 fluorescein isothiocyanate (FITC; clone M5E2), BD CD123 phycoerythrin (PE; clone 6H6; Pharmingen), CD28 ECD (clone CD28.2; BioLegend), CD95 PECy5 (clone DX2; Beckman Coulter), CD11c BV605 (clone 3.9; BioLegend), HLA-DR APC-H7 (clone L243; BioLegend), CD4 BV650 (clone OKT4; BD), CD11b BV785 (clone ICRF44; BioLegend), CD45 V450 (clone D058-1283; BioLegend), CD206 APC (clone 19.2; BD Horizon), CD3 Alexa700 (clone SP34-2; BD Pharmingen), and CD20 PE-Cy7 (clone L27; BD Pharmingen). The results were analyzed using the FlowJo software v9.9 (TreeStar, Ashland, OR). A threshold cutoff of 200 cells of the parent population was used for all cell subset analyses.
Quantitative PCR for SIV DNA.
From isolated brain mononuclear cells, we sorted 30,000 live memory CD4+ T cells (live, CD45+ CD3+ CD4+ CD28+ CD95+) and 30,000 live MΦs (live, CD45+ CD4+ CD20− CD11b+ HLA-DR+) cell populations using a BD FACSAria II flow cytometer and then lysed the sorted cells with 25 μl of a 1:100 dilution of proteinase K (Roche, Indianapolis, IN) in 10 mM Tris buffer. We performed quantitative PCR with 5 μl of cell lysates per reaction using the following reaction conditions: 95°C for 5 min and 40 cycles of 95°C for 15 s, followed by 60°C for 1 min. We used TaqMan gene expression master mix (Life Technologies) with the following primers and probes against SIVsmE543: For, GGC AGG AAA ATC CCT AGC AG; Rev, GCC CTT ACT GCC TTC ACT CA; and probe, AGT CCC TGT TCR GGC GCC AA. A StepOnePlus PCR machine and software (Applied Biosystems) were used to amplify genes of interest.
Immunohistochemistry for lymphocytes.
Brain tissue was collected at the time of necropsy and preserved in 10% neutral-buffered formalin solution (HT501128; Sigma). Tissue sections were then embedded in paraffin and made into unstained slides cut to 5-μm thickness by American Histolabs, Inc. (Gaithersburg, MD). For CD3, CD4, CD20, and joint CD63/CD168 (for myeloid lineage cells) staining, slides were heated at 60°C for 1 h and deparaffinized in xylene washes and 50% xylene–50% ethanol wash, and then they were rinsed twice in 100% ethanol. Tissues were then slowly rehydrated with a series of ethanol and double-distilled water (ddH2O) washes, added to boiling Target Retrieval solution (catalog no. 32200; ACDBio) for 30 min at 98°C, and then washed in 1× Tris-buffered saline with Tween 20 (TBST) buffer. For CD8 staining, tissues were deparaffinized, rehydrated, and heated in Trilogy pretreatment solution (Calle Marque, Rocklin, CA, USA), using a pressure cooker at 110 to 120°C for 15 min, and then hot washed with Trilogy solution, followed by a second hot wash with 1× TBST. All slides were then treated with 0.03% hydrogen peroxide reagent for 30 min and then incubated at room temperature for 2 h with 10% goat normal serum (Vector Laboratories, Burlingame, CA). Slides were incubated with primary antibodies overnight at 4°C with dilutions as follows: rabbit monoclonal anti-CD3, 1:200 (clone SP7; Thermo Scientific); rabbit monoclonal anti-CD4, 1:200 (clone EPR6855; Abcam); rabbit monoclonal anti-CD8, 1:50 (clone SP16; Invitrogen); mouse monoclonal anti-CD20, 1:200 (clone L26; Dako); mouse monoclonal anti-CD68, 1:400 (clone KP1; BioCare Medical); and mouse monoclonal anti-CD163, 1:400 (clone 10D6; Leica Biosystems). Secondary antibodies (1:1,000, goat anti-mouse horseradish peroxidase [HRP], ab205719; and goat anti-rabbit HRP, ab205718; Abcam) were incubated at room temperature, and hybridization signal was detected using 3,3′-diaminobenzidine tetrahydrochloride (DAB) substrate. Slides were counterstained with chloramphenicol acetyltransferase (CAT) hematoxylin and visualized and photographed with a Zeiss Axio Imager Z1 microscope.
SIV DNAscope/RNAscope with immunofluorescence.
To immunophenotype the cells containing viral DNA, we combined SIV-specific DNAscope with immunofluorescence-targeting cell markers using antibodies for T cells and myeloid lineage cells as listed above. We performed DNAscope following the protocol described above using RNAscope Probe-SIVmac239-sense (catalog no. 314071; American Cell Diagnostics [ACD]), and after the last amplification step, viral DNA was developed using the Tyramide Signal Amplification (TSA) Plus Cy3.5 kit (PerkinElmer). Next, slides were directly incubated overnight at 4°C with antibodies to phenotype T cells and myeloid lineage cells, as stated above. Slides were washed and incubated with secondary antibodies donkey anti-rabbit IgG-Alexa 488 and donkey anti-mouse Alexa 647 (all from Molecular Probes/Thermo Fisher Scientific) for 1 h at room temperature. To decrease autofluorescence, the tissues were incubated with Sudan Black solution (0.1% in 80% ethanol [ENG Scientific, Inc.] plus 1× TBS) for 30 min at room temperature, washed, counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (ready to use [RTU]; ACD) for 10 min, washed in TBS, and coverslipped using the ProLong Gold reagent (Invitrogen). Tiled images were collected on a Leica DMI6000 wide-field microscope equipped with a 20×/0.4 numerical aperture (NA) objective, an EL6000 light source, appropriate filter cubes, and a DFC360FX monochrome camera. The remaining images were taken with 2 by 2 binning, and mosaic tiled images were stitched automatically using the Leica Application Suite X (LAS X) software. IFA images were visualized and photographed using a Zeiss Axio imager Z1 microscope affixed with ApoTome. RNAscope was performed as described above for DNAscope except using RNAscope Probe-V-SIVmac239-vif-env-nef-tar probe (catalog no. 416131; ACD).
Statistical analysis.
Statistical analyses of cell correlations and averages were performed using Prism 285 (v7.0; GraphPad Software, Inc.). The Mann-Whitney t test (unpaired, nonparametric) was used to determine if there were significant differences between average populations. Analyses between the size of the mononuclear cell population and the CSF VL were calculated using the Spearman correlation.
ACKNOWLEDGMENTS
We thank Richard Herbert, Heather Cronise, and Joanna Swerczek of the NIHAC for excellent animal care.
Funding was mainly provided by the Intramural Program of the National Institute of Allergy and Infectious Diseases. This project has also been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract HHSN261200800001E.
The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.