Abstract
Despite considerable research, the mechanisms by which HIV disrupts thymic function remain controversial. We have described the phenotypic changes that occur in the thymus of SIV-infected macaques during acute SIV infection. In this study, we analyzed the effects of SIV infection on apoptotic pathways in thymic tissue from newborn macaques infected with SIV. Thymocyte apoptosis was accompanied by a modest increase in surface Fas expression, a profound decrease in the frequency of bcl-2-positive cells, as well as the amount of bcl-2 per cell. With control of viral replication, levels of bcl-2 and Fas returned to baseline together with a return to basal levels of apoptosis. In the thymus, SIV infection resulted in depletion of CD4+CD8+ thymocytes, an increase in apoptosis of thymocytes, and a down-regulation of MHC class I molecules. These changes peaked 14–21 days after infection at or just after peak viremia. This data further suggests disruption of the antiapoptotic pathway regulated by bcl-2 plays a critical role in SIV-induced apoptosis of thymocytes.
Although infection with HIV ultimately results in dysfunction of multiple aspects of immune function, depletion of CD4+ T lymphocytes is the most characteristic abnormality in HIV-infected people. While the mechanisms that contribute to the profound depletion of CD4+ T lymphocytes have not been well defined, multiple processes appear likely to contribute, including both increased peripheral destruction of mature T cells and decreased thymic output.
Evidence documents that the thymus is a major target of HIV and SIV infection and that infection may involve both thymocytes and thymic epithelial cells (1, 2). In vitro studies have shown that HIV-1 can infect thymocytes from uninfected subjects, and in vivo studies in the SCID-hu chimera model have shown that immature thymocytes are infected by HIV-1 and implicate HIV as the direct (3, 4, 5) and indirect (6, 7) cause of thymocyte death. Although information on infection of thymocytes in vivo is limited, a report analyzing thymic tissue obtained from an infant with perinatal HIV-1 infection demonstrated infection in immature thymocytes (1). Pathologic abnormalities in the thymus, such as thymic involution and thymocyte depletion, are common findings at autopsy (8, 9), although the significance of these observations is complicated by the fact that thymic involution and atrophy may occur as a sequel to the debilitating opportunistic infections that accompany AIDS. In addition to HIV infection of thymocytes, data from SCID-hu mice (4) demonstrated the presence of HIV RNA in thymic epithelial cells and degeneration of thymic epithelium. Taken together, these findings suggest that HIV infection of the thymus is likely to thwart efforts to reconstitute immune function in HIV-infected individuals. This is further complicated by the fact that the predominant population of HIV-infected individuals are adults, who, as a consequence of their age, have less residual thymic function, which impacts on the ultimate regenerative capacity of their immune system. This has been further illustrated in studies that have evaluated thymic function in HIV-infected individuals (10) and SIV-infected macaques (11) by measuring TCR excionsal circles. These studies documented a reduced number of TCR exscionsal circles correlated with poor CD4 recovery after antiretroviral therapy and with reduced thymic function as a consequence of HIV or SIV infection.
The exact mechanisms of HIV/SIV-induced thymocyte depletion have not been well characterized, and the relative contributions of direct vs indirect mechanisms of thymocyte depletion remain controversial. Infection of thymocytes by SIV and HIV support direct mechanisms of apoptosis. However, during thymocyte maturation both bcl-2 and Fas-Fas ligand pathways contribute to the regulation of apoptosis during both positive and negative selection (12, 13, 14), and disruption of these pathways as a consequence of retroviral infection may contribute to increased apoptosis. In these studies, we evaluated the role of bcl-2 and Fas in SIV-induced thymocyte depletion.
Materials and Methods
Animal and viral infections
Ten newborn rhesus macaques (Macaca mulatta) were housed in accordance with standards of the Association for Assessment and Accreditation of Laboratory Animal Care. The investigators adhered to the Guide for the Care and Use of Laboratory Animals prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council. The animals were born to dams that were negative for Abs to HIV-1, SIV, type D retrovirus, and simian T cell leukemia virus type 1.
Animals were i.v. inoculated with equal doses (10 ng of SIVp27) of a pathogenic (SIVmac239) molecular clone of SIV. This is ∼20 ng/kg p27, which is equivalent to the dose used in adult animals. In addition, four healthy, age- and sex-matched rhesus monkeys were sacrificed as normal controls. SIVmac239 is a pathogenic molecular clone of SIV (15).
Quantitation of viral load
Peripheral blood was collected for viral isolation before inoculation and at days 3, 7, 14, 21, 35, and 50 after inoculation. Quantitative viral cultures were performed as described previously (16).
Briefly, serial 3-fold dilutions were performed in duplicate beginning with 106 PBMC. PBMC dilutions were cocultured with 105 CEMX174 cells in a volume of 1 ml. Cultures were split 1:2 twice weekly until day 21, when the cultures were assayed for virus production by enzyme immunoassay for SIV p27 (Coulter, Miami, FL). Results are expressed as the number of SIV+ cells/106 PBMC.
Virion-associated SIV RNA in plasma was quantified by using a real-time RT-PCR assay on an Applied Biosystems Prism 7700 sequence detection system (Foster City, CA) as described previously (17, 18). Results shown are averages of duplicate determinations. Analyses of viral RNA levels were performed by Drs. Jeffrey Lifson and Michael Piatak (Science Applications International Corp.-Frederick, Frederick, MD).
Tissue collection and processing
Two animals from each group were sacrificed at 3, 7, 14, 21, and 50 days postinfection by i.v. injection of sodium pentobarbital. Thymus and other tissues were collected in 10% neutral buffered formalin, embedded in paraffin, sectioned at 6 μm, and stained with hematoxylin and eosin by routine histologic techniques. Adjacent sections were subjected to in situ hybridization and immunohistochemistry. Adjacent blocks of fresh tissue were collected for flow cytometry and snap-frozen in optimum cutting temperature compound (OCT, Miles, Elkhart, IN) by immersion in 2-methylbutane cooled in dry ice for immunohistochemistry.
Flow cytometric analysis of thymocyte progenitors
Thymic tissue was obtained at the time of euthanasia, minced into small fragments, and then digested into a single-cell suspension by incubation in PBS with 0.5 mg/ml of collagenase (Sigma, St. Louis, MO) and 2 U/ml DNase1 (Sigma) at 37°C for 60 min with frequent agitation. The cell suspension was then washed once in PBS with 2% normal mouse serum and filtered through a 70-μm nylon mesh. Abs used for immunophenotyping of rhesus thymocytes included anti-CD3 (6G12) (kindly provided by J. Wong, Massachusetts General Hospital) (19), anti-CD4 (OKT4) (Ortho Diagnostics, Raritan, NJ), anti-CD8 (Leu-2a) (Becton Dickinson, San Diego, CA), anti-MHC class I (w632) (Dako, Carpinteria, CA), anti-HLA-A, B, C (PharMingen, San Diego, CA), anti-Fas (Immunotech, Miami, FL), anti-Fas ligand (PharMingen), and anti-CD34 (Qbend-10) (Immunotech). Cells were stained in the presence of staining media (PBS with 2% mouse serum). After Ab staining, the cells were fixed with fresh 2% paraformaldehyde. Three- and four-color flow cytometry analysis of the cells was performed using a FACScan with CellQuest software (Becton Dickinson).
For intracellular cytokine staining, previously cryopreserved thymus cells were quick thawed and washed three times in PBS (Life Technologies, Grand Island, NY). Cells were surface stained with anti-CD4 FITC (custom conjugate), anti-CD4 PE (custom conjugate), and anti-CD8 peridinin chlorophyll protein (Becton Dickinson) for 30 min at 4°C in the dark. Cells were washed once with stain media (2% normal mouse serum (Sigma) in PBS). One hundred microliters of reagent A from the Fix and Perm cell permeabilization kit (Caltag, South San Francisco, CA) was added to each tube. Cells were incubated for 15 min at room temperature in the dark. Cells were washed once. One hundred microliters of reagent B from the Fix and Perm cell permeabilization kit (Caltag) was added to each tube. The appropriate intracellular cytokine Ab conjugates were also added (rat IgG2a PE, IL-10 PE, IL-2 PE, IL-4 PE, IL-7 PE, IL-12 PE, IFN-γ PE, and TNF-α PE, all from PharMingen). Cells were incubated for 15 min at room temperature in the dark. Samples were washed once in stain media as before. Cells were resuspended in 250 μl of 2% paraformaldehyde and then analyzed using a FACScalibur (Becton Dickinson).
For the detection of apoptosis using TdT, isolated thymocytes were stained (1 × 106 cells/tube) for the presence of surface CD4 and CD8 before their permeabilization/fixation with 0.1% Tween 20 in 2% paraformaldehyde (37°C for 30 min, followed by 30 min at room temperature) for the subsequent assessment of apoptosis employing Oncor’s ApopTag kit (Oncor, Gaithersburg, MD). Each tube was given 76 μl equilibration buffer and gently vortexed, and then ∼3 ml of PBS was added. The tubes were spun at 1500 rpm for 5 min at 25°C as is the case for all centrifugations in the protocol.
The liquid was aspirated and each tube’s content resuspended in 38 μl reaction buffer and 16 μl TdT enzyme. The tubes were capped, vortexed, and placed in a 37°C heat block (covered with foil) for 30 min. Then 3 ml/tube of the stop/wash buffer were added upon removal from the heat block. The tubes were centrifuged, the liquid aspirated off, and the cells were resuspended in 56 μl blocking solution and 49 μl antidigoxigen FITC Ab and then incubated at room temperature for 30 min. The excess Ab was removed by two 0.1% Triton X-100/PBS washes, and the cells were resuspended in 300 μl/tube of 2% paraformaldehyde for flow analysis using a FACScalibur (Becton Dickinson) the next day. Analysis was performed through a “live” gate established on forward scatter (FSC)3/side scatter (SSC) plots to exclude debris and monocytic macrophages.
For detection of apoptosis using Phi Phi Lux (OncoImmunne, Gaithersburg, MD), 2 × 106 thymocytes/tube were aliquoted into 1.5-ml conical microcentrifuge tubes. Microfuge tubes were spun at 1200 rpm for 7 min at 4°C to allow for removal of all media by vacuum aspiration. Then 150 μl of the 10 μM Phi Phi Lux G1D2 caspase substrate and 10 μl FBS were then added to each tube. The cells were gently resuspended. The open tubes were incubated in a 5% CO2 incubator at 37°C for 60 min in the dark.
Cells were washed with 500 μl/tube of OncoImmune and resuspended in 500 μl/106 cells of fresh flow cytometry media and placed on ice for FACS analysis. For additional surface staining, the cells were resuspended in 50 μl flow cytometry media with the appropriate amount of Ab. The cells were incubated on ice for 15 min, washed, spun, and resuspended in 500 μl flow cytometry media for FACS analysis using a FACScalibur (Becton Dickinson). Analysis was performed through a live gate established on FSC/SSC plots to exclude debris and larger monocytic macrophages.
Localization of SIV-infected cells
Localization of infected cells was performed by immunohistochemistry for viral Ags and in situ hybridization for viral DNA and RNA. In situ hybridization was performed on formalin-fixed paraffin-embedded sections. The DNA probe used was labeled with digoxigenin-11-dUTP by random priming (Boehringer Mannheim, Indianapolis, IN) as previously described (2, 16). Sections were examined microscopically and scored semiquantitatively on a scale as follows. The absence of positive cells was given a score of negative; 1–5 positive cells per section were given a score of 1+; 5–15 positive cells per 10× field were given a score of 2+; 15–30 positive cells per 10× field were given a score of 3+; and >30 positive cells per 10× field were given a score of 4+.
Adjacent snap-frozen blocks of tissue were used in immunohistochemical procedures to localize virus as previously described (2, 20, 21). Briefly, tissue sections cut at 6 μm were fixed in 2% paraformaldehyde and immunostained using an avidin-biotin-HRP complex technique with diaminobenzidine as the chromogen. The primary Ab used was Senv71.1 (a gift from C. Colignon and C. Thiriart, SmithKline Beacham, Belgium), which recognizes SIVgp120.
Immunophenotype of infected cells
To examine the expression of bcl-2 in relation to the presence of SIV-infected cells, we performed double labels for SIVgp120 (Senv71.1) and bCL-2 (Becton Dickinson). Briefly, immunohistochemistry for SIVgp120 was performed as described previously (2) using Vector red (Vector Laboratories, Burlingame, CA) followed by monoclonal anti-bCL-2 directly conjugated to FITC. The slides were then examined by immunofluorescence and confocal microscopy taking advantage of the intense fluorescence of Vector red. Controls consisted of thymus from uninfected age-matched controls and substitution of isotype-matched Ig for primary Ab.
Confocal microscopy was performed using a Leica TCS SP laser scanning microscope (Leica Microsystems, Exton, PA) fitted with a ×100 Leica objective (PL APO, 1.4NA) and using the Leica image software. Images were collected at 512 × 512 pixel resolution. The stained cells were optically sectioned in the z-axis, and the images in the different channels (photomultiplier tubes) were collected simultaneously. The step size in the z-axis was varied from 0.2 to 0.5 μm to obtain 30–50 optical sections per imaged field.
Results
Animal and viral infections
To examine the early effects of SIV on the neonatal thymus, 10 newborn macaques were injected i.v. with 10 ng of SIV p27. This is approximately equivalent to 20 ng/kg of SIV p27, which is an equivalent dose to what we have previously used in juvenile and adult animals (2). Two animals from each group were sacrificed at 3, 7, 14, 21, and 50 postinfection, and thymus was collected for flow cytometry, histopathology, immunohistochemistry, and in situ hybridization. Peripheral blood was collected at these same time points and used to evaluate cell-associated viral load and lymphocyte subset analysis in all animals alive at that time. Four additional, healthy age-matched animals were sacrificed as normal controls.
SIV isolation and quantitation
Virus was recovered from all animals by 3 days postinfection. Viral loads rose quickly and peaked at 7–14 days postinfection, (Fig. 1⇓). Cell-associated viral loads in neonates were as high as 4 × 104 virus-positive cells per 106 PBMC, which is as high or higher than what is typically observed in juvenile or adult animals infected with the same viral stocks, but the difference did not reach statistical significance (data not shown).
Viral loads in rhesus neonates infected with SIVmac239 determined by limiting dilution culture of peripheral blood mononuclear cells (A) or quantitation of plasma viral RNA (B).
Viral localization and histopathology of the thymus
Coincident with peak viral loads at 7 days postinfection, virus was detected in the thymic medulla by immunohistochemistry for SIVgp120 and in situ hybridization for SIV nucleic acid (Table I⇓ and Fig. 2⇓). Most of the infected cells were present in the thymic medulla with rare positive cells in the cortex consistent with previous observations (2, 22, 23).
Thymus from a neonatal macaque infected at birth with SIVmac239. Hematoxylin- and eosin-stained section of thymus at 14 days postinfection is shown in A, and in situ hybridization for SIV on an adjacent section of the same thymus is shown in B. The SIV-positive cells in B are in the thymic medulla near a cystic Hassall’s corpuscle. A higher magnification of the infected cells is shown in the inset in B.
Thymic morphology and localization of SIV in neonatal macaques
Histopathologic alterations of the thymus were fairly mild and similar to those described previously in the older macaques infected with SIV (2). At 7 and 14 days after infection, the thymus showed varying degrees of dysinvolution with thinning of the thymic cortex followed by an apparent increase in the thickness of the cortex at 21 (Fig. 3⇓) and 50 days (data not shown) postinfection. The apparent thinning of the cortex and subsequent rebound in cortical thickness occurred coincident with alterations in thymocyte apoptosis (see below).
Hematoxylin- and eosin-stained sections of thymus from a normal neonatal macaque at 21 days of age (A), as well from neonates infected at birth, 7 days (B), and 21 days (C) postinfection. All photomicrographs are reproduced at the same magnification.
Alterations in the phenotype of thymocytes
Alterations in thymocyte subpopulations was evident at 3 days postinfection, where we observed a decrease in the relative frequency of CD4+CD8+ double-positive cells. This subset continued to decline with a nadir at 21 days postinfection followed by a return to baseline levels by 50 days postinfection (Fig. 4⇓A). This relative decrease in double-positive cells was accompanied by a relative increase in CD4−CD8+ single-positive cells (from 2% at baseline to 7% by day 21), and a relative increase in CD4−CD8− cells (from 1% at baseline to 15% by day 21) (Fig. 4⇓B). Using three- and four-color flow cytometry, it was evident that a significant number of the CD4− CD8− cells were in fact CD34+ progenitors (data not shown). Consistent with our previous observations in juvenile macaques infected with SIV (2), we observed a rebound phenomenon in thymic T progenitors during the course of acute SIV infection. The most profound alterations observed both by histology and flow cytometry were evident by 14–21 days postinfection. Thymic atrophy and involution was followed by profound increases in CD34+ progenitors at 14 and 21 days postinfection and restoration of normal thymic architecture as well as thymocyte phenotype. By day 50 postinfection, the frequency of CD34+ T progenitors in the thymus had also decreased to levels observed before SIV infection (Fig. 4⇓A). The phenotypic changes described above were not detected in normal age-matched controls (data not shown), demonstrating that these fluctuations were a consequence of SIV infection.
Flow cytometric analysis of thymocyte subsets during acute neonatal SIV infection. Thymus was obtained at the time of euthanasia, and a single-cell suspension was stained with Abs to CD4, CD8, and CD34. The frequency of CD34+ and CD4+CD8+ thymocyte subsets is shown in A, and CD4+, CD8+, and CD4−CD8− thymocytes in B, during the course of acute SIV infection. CD4+CD8+ double-positive thymocytes demonstrate a relative decrease in frequency during peak viral replication. Similarly, a reciprocal increase in CD4−CD8− thymocytes is observed.
Down-regulation of MHC class I expression on immature thymocytes
We analyzed the expression of MHC-class I on double-positive and single-positive thymocytes in normal and SIV-infected neonates using two Abs that cross-react with rhesus epitopes. As previously described by others (7), we detected a lower level of MHC class I expression on CD4+CD8+ thymocytes compared with CD4+ and CD8+ thymocytes in normal thymic tissue (Fig. 5⇓). In tissue derived from age-matched SIV-infected (day 21 postinfection) neonates, the level of MHC class I molecules on double-positive cells was similar to uninfected controls. In contrast, in SIV-infected animals the difference in surface MHC class I expression was most evident in CD4+ single-positive cells where thymocytes from SIV-infected animals expressed levels of class I MHC in a range comparable to CD4+CD8+ double-positive thymocytes. To control for differences that may result from instrument settings, samples from naive and SIV-infected neonates were stained and analyzed on the same day using the same instrument settings. As alterations in the microenvironment have been incriminated in inducing change in MHC class I expression (7), we also examined cytokine levels in thymus from SIV-infected neonates. Additional unstimulated samples were stained to detect intracellular levels of IL-2, IL-4, IL-7, IL-10, IL-12, IFN-γ, and TNF-α. No significant differences were detected in thymus from SIV-infected neonates as compared with uninfected animals (data not shown). As we and others have previously published (2, 22, 23), the number of thymocytes infected with SIV is <5% of cells, whereas MHC class I down-regulation affects >70% of all thymocytes. Thus, down-regulation of MHC-class I in the thymus is likely an indirect effect of SIV infection, in contrast to the direct effects reported due to infection of cells with either HIV or SIV (24).
Class I MHC expression thymocytes from normal neonates and SIV-infected neonates as evaluated by flow cytometry. Significantly decreased expression of MHC class I is seen in SIV-infected animals in all subsets examined.
Evaluation of thymocyte apoptosis as a result of SIV infection
SIV infection contributes to an increase in apoptosis during acute SIV infection (2). As shown in Figs. 6⇓A and 7, we were able to detect an increase in apoptotic cells 3 days postinfection using TdT staining and detection by flow cytometry. The frequency of TdT+ cells peaked at 14 days postinfection, coincident with the peak viremia and thymic involution (Figs. 1⇑ and 7⇓). In addition, the TdT+ cells were more frequent in CD4+ and CD4+CD8+ cells than CD8+ cells (data not shown). This population bias suggests an association with SIV replication.
Flow cytometric assessment of apoptosis in the thymus during acute SIV infection. Thymus was obtained at the indicated time points after SIV infection at birth. Single-cell suspensions were evaluated for apoptosis using TdT (A) as well as a caspase substrate (Phi Phi Lux) (B). Analysis was performed through a live gate established on FSC/SSC plots to exclude debris.
The kinetics of apoptosis in the thymus during acute SIV infection. The frequency of apoptotic cells was established using TdT staining and analysis by flow cytometry. Analysis was performed through a live gate established on FSC/SSC to exclude debris.
We performed additional assays using a substrate (Phi Phi Lux) for mitochondrial caspases associated with early events in apoptosis (25). As demonstrated in Fig. 6⇑B, thymus from SIV-infected macaques demonstrates a marked increase in the activity of mitochondrial caspases, indicative of apoptosis. This increase in activity is biased toward CD4+ cells, although it is increased in all subsets. This confirms the observations made using TdT to detect apoptosis. In addition, this observation is supportive of indirect mechanisms associated with the induction of apoptosis as the frequency of apoptotic cells exceeds the relative number of SIV-infected cells. Furthermore, as previously described (2), most infected cells are in the thymic medulla, whereas the majority of changes we observed are in immature thymocytes that would be located in the thymic cortex.
Alterations in Fas, Fas ligand, and bcl-2 in the thymus of SIV-infected neonates
Thymocyte depletion during acute SIV infection is accompanied by an increase in apoptosis as determined by TdT incorporation of labeled nucleotides as well as increased mitochondrial caspases activity. The frequency of apoptotic cells increased dramatically shortly after peak viral loads were observed in both blood and tissues including the thymus. We examined the two major pathways associated with apoptosis, namely Fas and bcl-2. The surface levels of Fas on thymocytes from normal neonates is generally low (1–3% of all cells) and does not demonstrate any specific phenotypic distribution (data not shown). After SIV infection, the level of surface Fas changes modestly, increasing to a maximum of 8% of thymocytes on day 14 postinfection (Fig. 8⇓A). Although the overall alterations in surface Fas expression were modulated in all thymocytes, when gating on single-positive cells more significant fluctuations were detected. The level of Fas on CD4+ single-positive thymocytes increased four to five times when compared with either CD4+CD8+ thymocytes or CD8+ single-positive cells (data not shown). In normal age-matched neonates, the level of Fas expression was generally equivalent in CD4+ and CD8+ single-positive thymocytes. This data demonstrates that up-regulation of Fas on CD4+ thymocytes may contribute to an increase in apoptosis of this subset. This was accompanied by increases in surface expression of Fas ligand predominantly on CD4+CD8+ and CD8+ single-positive thymocytes, particularly at 14 and 21 days postinfection (Fig. 8⇓A), demonstrating modest fluctuations in both Fas and Fas ligand in acute SIV infection.
Detection of bcl-2, Fas, and Fas ligand expression using flow cytometry during acute SIV infection. Fas and Fas ligand surface expression are shown in A. Bcl-2 was quantitated using both the frequency of bcl-2-positive cells as well as the quantity of bcl-2 per cells determined using mean channel fluorescence (MCF) as shown in B.
Using immunohistochemical detection of bcl-2 and confocal microscopy, we were able to detect bcl-2 in thymocytes in both uninfected and SIV-infected thymocytes (Fig. 9⇓). However, it was difficult to quantitate bcl-2 levels in various thymocyte subsets using this technique. Therefore, we analyzed bcl-2 expression using flow cytometry where levels could be quantitated in various subsets using mean channel fluorescence.
Localization of SIV and bcl-2 expression in the thymus. To examine the relationship between SIV infection and bcl-2 expression, we performed double labels for SIV and bcl-2 in the thymus. The images were captured by confocal microscopy with individual channels on the left and the larger merged image on the right. SIVgp120 was detected by immunohistochemistry using Vector red as the substrate. This chromogen is visible in the differential interference contrast (DIC) image as a dark precipitate and also fluoresces brightly in the red channel (SIV) when excited with the 568 nm line of the laser. Bcl-2 was detected using an Ab directly conjugated to FITC and appears in the green channel. In the larger merged image, SIV-infected cells appear red, bcl-2-positive cells appear green, and the two SIV-infected, bcl-2-positive cells appear yellow.
Uninfected neonatal thymocytes express large amounts of bcl-2, consistent with the observations of others. However, during the course of SIV infection the number of bcl-2 positive cells rapidly decreases, as well as the amount of bcl-2 per cell as detected by flow cytometry (Fig. 8⇑B). The lowest levels of bcl-2 occur coincident with the largest amount of apoptosis, detected by TdT and Phi Phi Lux staining. By day 21, the levels of virus have reached a set point, and this is associated with the resumption of normal thymic function. Specifically, by day 21, the number of apoptotic cells is drastically reduced, and the number of bcl-2 cells begins to approach baseline levels. The level of bcl-2 on a per cell basis recovers only by day 50 postinfection. Based on these observations, it would appear that dysregulation of the bcl-2 pathway is the predominant mechanism associated with the apoptosis that accompanies retroviral infection of the thymus.
Discussion
This study demonstrates that in acute SIV infection of neonates, thymocyte infection is accompanied by profound thymocyte depletion and increased thymocyte apoptosis. We and others have previously published that HIV and SIV infect the thymus, and this is accompanied by thymocyte depletion and thymic atrophy (1, 4). During acute SIV infection, this appears to be somewhat reversible (2). The alterations in the relative frequency of thymocyte subsets in the neonates is consistent with previous observations (2). These fluctuations most likely include events associated with depletion of CD4+CD8+ thymocytes, but also reflect an influx of thymocyte precursors that are CD4−CD8−, thus reflecting an increase in the double-negative subset. In addition, we cannot exclude the role of the infiltrating CD8+ cells (including CTL and NK cells) as a consequence of acute viral infection, contributing to the relative increase in CD8+ single-positive cells. Apoptosis in the thymus during acute SIV infection has been shown to be accompanied by the subsequent increases in the levels of cellular proliferation particularly in the thymic cortex, but also in the medulla (2). The contribution of proliferation of thymocyte subsets cannot be excluded as contributing to these changes observed in the relative frequency of thymocyte subsets. In this study, we demonstrate that thymic atrophy and thymocyte destruction occur as a consequence of increased apoptosis.
As programmed cell death is an integral feature of normal thymocyte development, we examined the frequency of apoptotic cells in normal and SIV-infected neonatal thymus. To further determine the potential pathways associated with alterations in the number of apoptotic thymocytes, we determined the levels of Fas, Fas ligand, and bcl-2 in both normal and SIV-infected neonatal tissue.
Destruction of the thymus has been demonstrated to contribute to a failure to regenerate new T cells during HIV or SIV infection and thus impact directly on the course of disease. Understanding how thymopoiesis is disrupted is the first step to design interventive strategies that may aid in sparing the thymus during retroviral infection. Consistent with previous observations (2), we detected a rapid increase in the frequency of apoptotic thymocytes during neonatal SIV infection. In general, this occurred shortly after peak viral loads were detected. A greater number of apoptotic cells were detected using flow cytometry, and this was coincident with thymic atrophy detected by histology. The two major mechanisms of apoptosis are associated with either up-regulation of Fas expression, which allows Fas ligand to induce a signal for apoptosis, or bcl-2, which protects cells from apoptosis-inducing signals. We examined the levels of both Fas and bcl-2 expression in the thymus of SIV-infected neonates and were able to demonstrate that the levels of bcl-2 are substantially decreased just before and during the period of peak apoptosis. Both the frequency of bcl-2-positive cells and the level of bcl-2 on a per cell basis were found to be significantly decreased. This mechanism is observed in normal T cell ontogeny, where cells that are not positively selected in the thymus down-regulate bcl-2 expression (12). In concert with this, bcl-2 is up-regulated or induced to re-express in cells that are destined to undergo positive selection (12, 13). However, these fluctuations are much more pronounced in acute SIV infection and are observed in the majority of thymocytes. Again, consistent with the fact that productively SIV-infected cells are detectable at a low frequency using in situ hybridization and immunohistochemistry (1, 2), an indirect mechanism must be implicated as being associated with this down-regulation of bcl-2 expression. This would imply that the presence of SIV in only a small number of cells within the thymus is adequate to disrupt the microenvironment, and this in turn results in decreased bcl-2 expression and thus increased apoptosis. The accompanying increase in cellular proliferation associated with thymocyte apoptosis may contribute to some of the alterations observed in bcl-2 expression, although the decrease in bcl-2 observed was evident to some extent in all thymocyte subsets, including double-positive and single-positive cells. Of potential relevance is our previous observation of infection of cells of monocyte-macrophage lineage in the thymus of SIV-infected macaques (2). These cells contribute to establishing the thymic microenvironment, and so retroviral infection of these cells may further disrupt this environment. It should be further noted that bcl-2 is down-regulated in both CD4+CD8+ double-positive and CD4+ or CD8+ single-positive thymocytes. However, it is predicted that the consequence of bcl-2 down-regulation will be the most overt in CD4+CD8+ double-positive cells, as bcl-2 participates to a lesser degree in the apoptosis mechanisms in single-positive thymocytes. Furthermore, the bulk of negative selection occurs during the double-positive stage. Double-positive thymocytes also comprise the major population of cells in the thymus, so disruption of this subset will most likely result in substantial effects at the immature stage, but depletion of these cells will have obvious consequences on the generation of single-positive T cells.
It is well documented that Fas contributes to the induction of apoptosis in single-positive thymocytes and peripheral T cells (14, 26, 27, 28). In general, Fas is expressed at low levels in normal thymocytes. When we examined bulk thymocytes, modest differences in Fas expression were evident. However, when we examined the level of Fas in single-positive thymocytes, we detected a striking difference, with much greater increases in surface Fas on CD4+ single-positive thymocytes. This is in fact consistent with what might be expected based on the fact that Fas contributes to cell death of single-positive and peripheral T cells and not double-positive thymocytes, as well as the fact that CD4+ thymocytes are the primary target of HIV and SIV infection (26, 27, 28). It would be predicted that this is again a consequence of an indirect mechanism, as the frequency of cells with increased surface Fas expression appears to exceed the number of SIV-infected cells. The contribution of proliferating cells to alterations in Fas expression cannot be excluded.
It has recently been reported that Nef is capable of inducing Fas expression in T lymphoid cells (14). Although it is unlikely that all CD4+ single-positive thymocytes with increased Fas expression are directly infected, it is possible that this may be one of the mechanisms responsible for increased Fas expression in this subset of cells, as both intracellular and extracellular Nef have been shown to induce Fas expression. As predicted by studies in normal animals, SIV infection results in disruption of the antiapoptotic pathway governed by bcl-2 in double-positive thymocytes and the Fas apoptosis-inducing pathway in single-positive CD4+ thymocytes.
It has been demonstrated that apoptosis may be a favorable response in HIV infection where the virus is induced to replicate in dying cells (29). Alternatively, it has been postulated that the induction of Fas expression may result in death of Fas ligand-positive CTL to either SIV or HIV infection (14). This may then facilitate immune evasion of CD4+ SIV or HIV-infected single-positive thymocytes, and these cells could enter the peripheral circulation, contributing to the dissemination of virally infected cells to distant sites. An additional mechanism of immune evasion that has recently been reported in PBLs is the down-regulation of MHC class I molecules (30, 31). This has been shown to be a direct consequence of HIV or SIV infection, predominately as a consequence of Nef. The down-regulation of MHC class I prevents complete recognition of the cell by Ag-specific CTL, allowing them to escape immunosurveillance. This is similar to our observation in thymocytes of SIV-infected neonates where we detected a decrease in MHC class I expression, although in this in vivo model it is unlikely to only be a direct effect as the frequency of SIV-infected cells is too low to account for the global down-regulation of MHC class I. In addition, this was not detected in animals infected with SIVmac239ΔNef, a less pathogenic deletion mutant of SIV (data not shown). It is feasible that an indirect mechanism contributes to this phenomenon in SIV-infected monkeys and that this in turn contributes to evasion of the immune response. This is in contrast to a recent report that demonstrated an increase in MHC class I expression in the thymus during HIV-1 infection (7). It should be noted that this observation was in the SCID-hu model in the context of HIV and the absence of an immune response. The situation in vivo in an animal with a relatively intact immune system (albeit immature) in the context of acute SIV infection may in fact be different due to differences in the microenvironment of the thymus in the two animal models. The consequences of SIV infection of the thymus result in thymocyte depletion due to increased apoptosis, and this may contribute to the ultimate failure of the regenerative response of the immune system.
Acknowledgments
We thank Xavier Alvarez for assistance with confocal microscopy, Jean Charles Grivel and Lonja Margolis for assistance with the Phi Phi Lux assay, Doug Pauley and Heather Knight for assistance with immunohistochemistry and in situ hybridization, Jeffrey Lifson and Michael Piatak for performing viral RNA analyses, Kristen G. Toohey for assistance in preparation of figures, and Carolyn A. O’Toole for manuscript preparation.
Footnotes
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↵1 This work was funded by Grants AI-139423 and RR-00168 from the National Institutes of Health and Grant NS-30769 from the Pediatric AIDS Foundation. A.A.L. and R.P.J. are Elizabeth Glaser Scholars and are supported by the Elizabeth Glaser Pediatric AIDS Foundation.
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↵2 Address correspondence and reprint requests to Dr. Michael Rosenzweig, Division of Immunology, New England Regional Primate Research Center, Harvard Medical School, One Pine Hill Drive, Southborough, MA 01772. E-mail address: michael_rosenzweig{at}hms.harvard.edu
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↵3 Abbreviations used in this paper: FSC, forward scatter; SSC, side scattter.
- Received April 5, 2000.
- Accepted July 5, 2000.
- Copyright © 2000 by The American Association of Immunologists
References
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