Abstract
To our knowledge, this study demonstrates for the first time that the AIDS virus differentially impacts two distinct subsets of lung macrophages. The predominant macrophages harvested by bronchoalveolar lavage (BAL), alveolar macrophages (AMs), are routinely used in studies on human lung macrophages, are long-lived cells, and exhibit low turnover. Interstitial macrophages (IMs) inhabit the lung tissue, are not recovered with BAL, are shorter-lived, and exhibit higher baseline turnover rates distinct from AMs. We examined the effects of SIV infection on AMs in BAL fluid and IMs in lung tissue of rhesus macaques. SIV infection produced massive cell death of IMs that contributed to lung tissue damage. Conversely, SIV infection induced minimal cell death of AMs, and these cells maintained the lower turnover rate throughout the duration of infection. This indicates that SIV produces lung tissue damage through destruction of IMs, whereas the longer-lived AMs may serve as a virus reservoir to facilitate HIV persistence.
This article is featured in In This Issue, p.4547
Introduction
Drugs and vaccines for curing and preventing pandemic HIV/AIDS, respectively, remain elusive as the result of an incomplete understanding about AIDS pathogenesis. The SIV/rhesus macaque model is often used to study pathogenesis because of similarities to human HIV infections, including the loss of CD4+ T cells and general immune activation, opportunistic infections, and clinical symptoms (1). Although the decline in CD4+ T cells and/or immune activation are considered primary causes of AIDS and HIV-associated non-AIDS (HANA) morbidity and mortality, neither declining CD4+ T cells levels nor immune activation always correlate with AIDS disease progression (2). We recently reported that increased blood monocyte turnover rate predicted the onset of rapid progression to AIDS and the destruction of tissue macrophages in SIV-infected rhesus macaques (2), suggesting that blood monocytes and tissue macrophages play critical roles in AIDS pathogenesis. This was consistent with and supported by findings from Burdo et al. (3) that increasing monocyte turnover rates correlated with SIV encephalitis progression, as well as our recent observation that increased monocyte turnover was associated with the accumulation of interstitial lung macrophages in SIV-infected rhesus macaques (4).
Macrophages and monocytes are primary phagocytic cells of the innate immune system and function as major regulatory cells for tissue homeostasis and wound healing (5). Like CD4+ T cells, macrophages are targeted by HIV and SIV that bind to surface coreceptors, such as CCR5 and CCR3 (6, 7). Macrophages also play a critical role in innate immune responses to environmental exposures in the lung and support homeostasis and resistance to respiratory infections. We recently reported that macrophages make up ∼70% of immune cells in the lungs of healthy rhesus macaques and that there are at least two major populations of lung macrophages, alveolar macrophages (AMs) and interstitial macrophages (IMs) (8), consistent with reports about lung macrophages in humans (9). We also reported that, following bronchoalveolar lavage (BAL) to harvest and remove AMs, there occurred a rapid differentiation of blood monocytes and IMs to putatively replace AMs in the alveoli (8).
Evidence of lung pathology associated with HIV infection is observed in ∼85% of AIDS lung autopsies (10), and AIDS-defining respiratory opportunistic infections of macrophages, such as Pneumocystis and tuberculosis, also contribute to morbidity and mortality in HIV-infected patients (11–14). SIV replication in the BAL fluid (BALF) and lung tissue was reported as early as 7 and 14 d after inoculation of rhesus macaques, respectively, and was further confirmed within AMs of the BALF by in situ hybridization (15). Microscopically, SIV-associated interstitial pneumonia has been observed as early as 2 wk postinoculation, and it increased in incidence over time after inoculation (16). In addition, decreased expression of the mannose receptor CD206 on the AMs of HIV-1–infected patients correlated with defective binding and phagocytosis of Pneumocystis jiroveci (17). AMs from AIDS patients also exhibited dysregulated secretion of the proinflammatory cytokine IL-12 after challenge with Salmonella spp. in vitro (18). These findings suggest that, during HIV/SIV infection, macrophage dysfunction contributes to pathogenesis, but it is still unclear how the distinct macrophage populations in the lung contribute to HIV/AIDS or HANA conditions.
BALF can be obtained from humans to obtain AMs, but IMs must be recovered by lung biopsy, which can be accomplished more readily in SIV-infected rhesus macaques than in HIV-infected humans (19). The purpose of this study was to use the SIV/AIDS rhesus macaque model to relate blood monocyte turnover as a measure of disease progression, with SIV infection of distinct lung macrophage populations to better understand the mechanisms of SIV-induced pulmonary pathogenesis.
Materials and Methods
Animals and virus inoculations
A total of 55 adult male Indian rhesus macaques (Macaca mulatta), between 3.4 and 22 y of age, was used in the study and housed at the Tulane National Primate Research Center. Six monkeys served as uninfected controls. The remaining 49 monkeys were infected with seven strains of SIV as follows: 24 monkeys received SIVmac251, 10 monkeys received SIVmac239, 3 monkeys received SIVmac239/316e*, 2 monkeys received SIVmac239∆GY, 3 monkeys received SIVmac239∆Nef, 4 monkeys received SHIV89.6P, and 3 monkeys received SIV0302-2 (Supplemental Table I). All of the rhesus macaques infected with SIVmac251, SIVmac239, SHIV89.6P, or SIV0302-2 strains exhibited increased monocyte turnover rates during disease progression to AIDS. Animal procedures were performed according to the National Institute of Health’s Guide for the Care and Use of Laboratory Animals and were approved by the Tulane University Institution Animal Care and Use Committee.
BrdU/5-ethynyl-2′-deoxyuridine injection and sample collection
The thymidine analog BrdU (Sigma-Aldrich, St. Louis, MO) or 5-ethynyl-2′-deoxyuridine (EdU; Molecular Probes, Carlsbad, CA) was injected i.v. at 60 or 50 mg/kg, respectively. BALF samples were obtained by rinsing the lung with two aliquots of PBS (20 ml each) under a pediatric fiber optic bronchoscope. Open chest lung biopsies ∼1.5 cm3 were collected during the longitudinal studies and, at necropsy, ∼4-cm3 sections of lung tissue were obtained for flow cytometry and (immuno)histochemistry analyses.
Isolation of macrophages and lymphocytes from lung tissue
Single-cell suspensions from lung tissues (including biopsy and necropsy samples) were prepared using enzymatic digestion. Briefly, lung tissues were sliced into 0.5-mm-thick sections after removing bronchi and resuspended in 30 ml RPMI 1640 (Cellgro, Manassas, VA) supplemented with 5% FBS (catalog no. 26140-079; Life Technologies, Grand Island, NY), 100 IU/ml penicillin/streptomycin (EMD Millipore, Billerica, MA), 2 mM l-glutamine (Cellgro), 25 mM HEPES (Molecular Probes), 200 U/ml type IV collagenase (catalog no. 4189; Worthington Biochemical, Lakewood, NJ), and 0.05 mg/ml DNase I (catalog no. 10104159001; Roche Applied Science, Indianapolis, IN). The suspensions were incubated at 37°C for 30 min, followed by pipetting, incubation for an additional 10 min at 37°C, and enrichment by discontinuous density centrifugation over layers of 24 and 50% Percoll (catalog no. 17-0891-01; GE Healthcare, Boston, MA) at 2000 rpm for 20 min (Allegra X-12R; Beckman, Brea, CA). Cells were recovered from the 24–50% Percoll interface, washed with PBS containing 2% FBS, suspended in BAMBANKER serum-free cell culture freezing medium (catalog no. 302-14681; Wako Laboratory Chemicals, Richmond, VA), and stored in liquid nitrogen until further analyses.
Histochemical staining of BALF cells and lung tissue
A total of 200,000 BALF cells was subjected to cytospin centrifugation (Shandon Cytospin 3; Thermo Electron) at 400 × g for 3 min and stained with Wright-Giemsa. Differential counting was performed by light microscopy at original magnification ×200. Lung pieces ∼1 cm3 collected during necropsy were processed for H&E staining. Briefly, lung tissues were fixed in 10% neutral-buffered formalin, sectioned to 6 μm thickness, stained with H&E using an automated Leica Autostainer XL (Leica Biosystems, Buffalo Grove, IL), and viewed under regular light microscopy at original magnification ×400.
Flow cytometry
For Ab staining, 200 μl whole blood or 106 cells from BALF were prepared and stained, as previously reported (2). Staining for BrdU uptake was conducted using a BD Pharmingen BrdU Flow Kit (catalog no. 559619; Becton Dickinson, San Jose, CA) following surface Ab staining, according to the manufacturer’s protocol. EdU staining was performed using a Click-iT EdU Pacific Blue Flow Cytometry Assay kit (catalog no. C-10418; Invitrogen, Carlsbad, CA) following surface Ab staining, also according to the manufacturer’s protocol. A three-laser FACSAria (Becton Dickinson) was used to detect the surface markers or intracellular BrdU/EdU incorporation in the multicolor-stained cells. Abs used in these analyses are shown in Supplemental Table II. AMs were defined as cells HLA-DRhi, CD11bdim CD163+ CD206+ cells, and IMs were defined as HLA-DR+, CD11bhi CD163+ cells, as previously described (8). Results were analyzed using FlowJo software (Version 9.6.2; TreeStar).
SIV RNA in situ hybridization
To detect SIV RNA in tissues, in situ hybridization was performed using riboprobes, as described previously (20). Briefly, 7-μm-thick formalin-fixed, paraffin-embedded tissue sections were treated sequentially with a series of xylene, ethanol, and distilled water containing diethylpyrocarbonate (catalog no. D5758; Sigma-Aldrich) for deparaffinization and rehydration before Ag retrieval. The tissue sections were blocked with saline sodium citrate hybridization buffer containing 50% formamide with denatured herring sperm DNA and yeast tRNA (10 mg/ml each) in a humidified chamber at 45°C for 1 h. SIV-digoxigenin–labeled anti-sense riboprobes (Lofstrand Labs, Gaithersburg, MD) were applied to the tissue sections (10 ng/slide) in hybridization buffer and incubated overnight at 45°C. After hybridization, slides were washed sequentially with 2×, 1×, and 0.1× sodium saline citrate, followed by the application of blocking solution. Alkaline phosphatase–conjugated sheep anti-digoxigenin Ab diluted at 1:200 (catalog no. 11093274910; Roche, Penzberg, Germany) was used to detect hybridized digoxigenin-labeled probes. The Dako Cytomation Liquid Permanent Red substrate-chromogen system (catalog no. 0640; Dako, Carpinteria, CA) was prepared, according to the manufacturer’s instructions, and added to the tissue for 20 min at room temperature to develop the reaction. Controls included matched positive and negative tissues hybridized with digoxigenin-labeled sense RNA-labeled probes that were processed in parallel. Rinsing with TBS was used to stop the reaction, followed by Ab staining of the tissue sections.
Confocal microscopy imaging
Imaging was performed with a Leica TCS SP2 confocal microscope equipped with three lasers (Leica Microsystems) at original magnification ×800 or ×630 and with a resolution of 512 × 512 pixels. Adobe Photoshop software (version 7.0; Adobe Systems) was used to process and assemble the images. Quantification of AMs and IMs was performed by manually counting 20 fields of each slide at original magnification ×200.
Quantification of plasma SIV RNA and DNA in infected cells
Quantification of SIV RNA in plasma, SIV-integrated provirus, and extrachromosomal (episomal) DNA (21) in FACS-sorted cells or lung tissue was performed using the TaqMan real-time PCR method, as described previously (22). SIV DNA extraction from sorted cells or lung tissue was performed using NucleoSpin Tissue (catalog no. 740952; Macherey-Nagel, Bethlehem, PA), following the manufacturer’s instructions. Amplification targeted a 74-bp fragment in the gag region and was performed in the RT-PCR Unit of the Pathogen Detection and Quantification Core at the Tulane National Primate Research Center (23). A TaqMan RNase P Control Reagents Kit (catalog no. 4316844; Life Technologies, Carlsbad, CA) was used to calibrate the cellular input for SIV DNA detection. All real-time PCR assays were carried out using an ABI Prism 7900HT Sequence Detection System (Applied Biosystems). Absolute viral RNA and DNA copy numbers were deduced by comparing the signal strength with corresponding values obtained from six 10-fold dilutions of standardized RNA controls that were reverse transcribed and amplified or from standardized DNA controls that were amplified in parallel, respectively.
Statistical analysis
Comparisons between mean values were analyzed by the Student t test. The Spearman test was used for correlation analysis. Data were analyzed and graphed using GraphPad Prism 5 software. The p values < 0.05 were considered statistically significant.
Results
Increased monocyte turnover correlates with severity of lung tissue damage in SIV-infected monkeys progressing to AIDS
To determine whether blood monocyte turnover correlates with tissue damage in the lung, we examined lung tissue integrity in SIV-infected monkeys exhibiting different rates of blood monocyte turnover but expressing similarly low levels of circulating CD4+ T cells and high viral loads, using a histological scoring system, as previously described (4, 16). Microscopically, the pulmonary architecture, cellular morphology, and alveolar content in SIV-infected monkeys with low blood monocyte turnover (≤30%) were indistinguishable from uninfected animals. The lung tissue in these animals exhibited a thin alveolar septum lined by a single layer of flattened epithelial cells with rare macrophages in the alveoli (Fig. 1A, 1B). The pulmonary lesions associated with SIV infection were evident in the monkeys with ≥30% blood monocyte turnover (Fig. 1C, 1D). The alveolar septa from these animals exhibited diffuse, mild to moderate thickening characterized by an increased number of mononuclear cells, capillary dilation, fibrin deposition, edema, and hyperplastic type II pneumocytes. The alveolar spaces were filled with increased numbers of foamy macrophages and multinucleated giant cells (Fig. 1C, 1D).
The severity of pulmonary lesions correlates with the rate of monocyte turnover (RMT) in SIV-infected rhesus macaques. (A) Normal lung tissue from an uninfected monkey (RMT = 1.61%). (B) Lung tissue from an SIV-infected animal (RMT = 22.7%) demonstrates minimal interstitial accumulation of a few mononuclear cells (shown by arrows) and rare AMs (<). Lung tissues from SIV-infected animals with RMT = 40.5% (C) and RMT = 55% (D) exhibited mild (C) to moderate (D) interstitial pneumonia characterized by alveolar septa thickening, capillary dilation (C), increased numbers of mononuclear cells (shown by arrows), and hyperplastic type II pneumocytes (||). Low (C) to moderate (D) numbers of AMs (< or >) and multinucleated giant cells (G) were noted in the alveolar spaces (H&E, original magnification ×400). Lung injury was assessed using a histological scoring system, as previously described (4, 16). *, alveolar septum.
The severity of pulmonary lesions correlates with the rate of monocyte turnover (RMT) in SIV-infected rhesus macaques. (A) Normal lung tissue from an uninfected monkey (RMT = 1.61%). (B) Lung tissue from an SIV-infected animal (RMT = 22.7%) demonstrates minimal interstitial accumulation of a few mononuclear cells (shown by arrows) and rare AMs (<). Lung tissues from SIV-infected animals with RMT = 40.5% (C) and RMT = 55% (D) exhibited mild (C) to moderate (D) interstitial pneumonia characterized by alveolar septa thickening, capillary dilation (C), increased numbers of mononuclear cells (shown by arrows), and hyperplastic type II pneumocytes (||). Low (C) to moderate (D) numbers of AMs (< or >) and multinucleated giant cells (G) were noted in the alveolar spaces (H&E, original magnification ×400). Lung injury was assessed using a histological scoring system, as previously described (4, 16). *, alveolar septum.
Increased turnover of blood monocytes in SIV-infected macaques is not reflected in the BALF
BALF specimens routinely harvested to examine innate immune responses of the lung in humans are composed mainly of AMs but not IMs (24, 25). To determine whether the increased blood monocyte turnover rates that predict onset of disease progression to AIDS (2) also correlated with increased turnover of lung macrophages, we first harvested AMs from BALF specimens during acute, chronic, and terminal stages of SIV infection and evaluated turnover by differential staining and flow cytometry for incorporation of BrdU/EdU. Interestingly, there was no correlation between turnover rates of AMs in BALF and turnover rates of blood monocytes (p = 0.3389, p = 0.7664, p = 0.8717, Fig. 2A–C). In addition, AMs failed to incorporate BrdU/EdU 24 h (Fig. 2A), 48 h (Fig. 2B), or 7 d (Fig. 2C) after BrdU/EdU injection, indicating that only negligible replication or replacement of AMs occurred during SIV infection. Moreover, by differential cell counting, there was no significant difference in the mean percentage of macrophages in BALF of SIV-infected monkeys compared with uninfected control monkeys (p = 0.5964, Fig. 2D). The mean absolute number of AMs in BALF specimens from SIV-infected monkeys was significantly lower than from uninfected monkeys (p = 0.0001, Fig. 2E), but there was no correlation between blood monocyte turnover rates and BALF AM levels in SIV-infected monkeys (p = 0.1375, Fig. 2F).
Increased turnover of blood monocytes in SIV-infected macaques is not reflected in BALF. BALF samples were collected prior to and during different stages of SIV infection from rhesus macaques. BrdU or EdU was injected i.v. into SIV-infected rhesus macaques, and the turnover of blood monocytes and AMs recovered from BALF was analyzed by flow cytometry. There was no statistically significant correlation between blood monocyte turnover and AM turnover at 24 h [(A), n = 21], 48 h [(B), n = 12], or 7 d [(C), n = 38] after BrdU/EdU injection using Spearman correlation analysis. There also was no difference in the percentage of macrophages recovered in BALF from SIV-infected versus uninfected macaques (D), although there was a significant decrease in the absolute number of macrophages recovered from BALF of SIV-infected macaques compared with uninfected macaques by the Student t test (E). (F) The numbers of macrophages recovered from BALF of SIV-infected monkeys did not correlate with blood monocyte turnover by Spearman correlation analysis.
Increased turnover of blood monocytes in SIV-infected macaques is not reflected in BALF. BALF samples were collected prior to and during different stages of SIV infection from rhesus macaques. BrdU or EdU was injected i.v. into SIV-infected rhesus macaques, and the turnover of blood monocytes and AMs recovered from BALF was analyzed by flow cytometry. There was no statistically significant correlation between blood monocyte turnover and AM turnover at 24 h [(A), n = 21], 48 h [(B), n = 12], or 7 d [(C), n = 38] after BrdU/EdU injection using Spearman correlation analysis. There also was no difference in the percentage of macrophages recovered in BALF from SIV-infected versus uninfected macaques (D), although there was a significant decrease in the absolute number of macrophages recovered from BALF of SIV-infected macaques compared with uninfected macaques by the Student t test (E). (F) The numbers of macrophages recovered from BALF of SIV-infected monkeys did not correlate with blood monocyte turnover by Spearman correlation analysis.
Increased turnover rate of lung IMs correlates with increased blood monocyte turnover in SIV-infected macaques
Lung tissue also was composed of a distinct subset of IMs that were smaller, located exclusively in the interstitium, and phenotypically more similar to blood monocytes than AMs (8). In vivo BrdU labeling also demonstrated that lung IMs exhibited higher baseline turnover rates, similar to those of blood monocytes in uninfected macaques, unlike AMs, which exhibited very low turnover rates and, thus, appeared to be longer lived (8). Thus, we compared turnover rates of lung tissue IMs and blood monocytes during various stages of SIV infection and observed that increasing blood monocyte turnover rates during disease progression to AIDS (2) also significantly correlated with increasing turnover rates of IMs 24 h (r = +0.9023, p < 0.0001, Fig. 3A) and 48 h (r = +0.6434, p < 0.0278, Fig. 3B) after BrdU injection. Immunohistochemistry and confocal microscopy also demonstrated higher numbers of BrdU-labeled IMs compared with AMs in lung tissue of SIV-infected monkeys (Fig. 3E, 3F).
Increased turnover rate of lung IMs correlates with increased blood monocyte turnover in SIV-infected macaques. (A and B) BrdU or EdU was injected i.v. into SIV-infected or uninfected rhesus macaques, and lung IMs were analyzed by flow cytometry. A significant correlation was observed between blood monocyte turnover and IM turnover 24 h [(A), n = 35] and 48 h [(B), n = 12] after BrdU/EdU injection using Spearman correlation analysis. Paraffin-embedded lung tissue sections obtained after necropsy from uninfected macaques [(C and D), representative of four animals] and SIV-infected rhesus macaques [(E and F), representative of four animals] were stained with anti-CD163 Ab (macrophages; green), anti-BrdU Ab (turnover; red), and Topro-3 (nucleic acid; blue). Images were captured with a Leica TCS SP2 confocal microscope equipped with a three-laser system (Leica Microsystems) using an oil-immersion objective (original magnification ×63, Fluotar/NA 1.0) for a final magnification of ×1260. White arrows indicate CD163-staining IMs (green), yellow arrows indicate CD163-staining IMs that incorporated BrdU (green and red), and asterisks indicate CD163-staining AMs (green).
Increased turnover rate of lung IMs correlates with increased blood monocyte turnover in SIV-infected macaques. (A and B) BrdU or EdU was injected i.v. into SIV-infected or uninfected rhesus macaques, and lung IMs were analyzed by flow cytometry. A significant correlation was observed between blood monocyte turnover and IM turnover 24 h [(A), n = 35] and 48 h [(B), n = 12] after BrdU/EdU injection using Spearman correlation analysis. Paraffin-embedded lung tissue sections obtained after necropsy from uninfected macaques [(C and D), representative of four animals] and SIV-infected rhesus macaques [(E and F), representative of four animals] were stained with anti-CD163 Ab (macrophages; green), anti-BrdU Ab (turnover; red), and Topro-3 (nucleic acid; blue). Images were captured with a Leica TCS SP2 confocal microscope equipped with a three-laser system (Leica Microsystems) using an oil-immersion objective (original magnification ×63, Fluotar/NA 1.0) for a final magnification of ×1260. White arrows indicate CD163-staining IMs (green), yellow arrows indicate CD163-staining IMs that incorporated BrdU (green and red), and asterisks indicate CD163-staining AMs (green).
Increased death rate of lung IMs correlates with increased blood monocyte turnover rate in SIV-infected rhesus macaques
To determine whether the increased IM turnover during SIV infection was a consequence of increased cell death, a TUNEL technique was applied. There was a higher number of TUNEL+ IMs in lung tissues of SIV-infected animals exhibiting higher blood monocyte turnover rates than in uninfected monkeys or SIV-infected monkeys exhibiting lower blood monocyte turnover rates (Fig. 4A, 4B). The Spearman rank correlation further demonstrated statistically significant relationships between blood monocyte turnover rates and death of IMs (r = 0.9505, p < 0.0001, Fig. 4A) and death of newly recruited CD163+ BrdU+ TUNEL+ IMs (r = 0.5874, p = 0.0489, Fig. 4B). A representative confocal microscopy image of lung tissue from an SIV-infected monkey is shown in Fig. 4C.
Increased death rate of lung IMs correlates with increased blood monocyte turnover rate in SIV-infected rhesus macaques. Spearman correlation analyses were performed to relate blood monocyte turnover (BrdU) and percentage of TUNEL+ IMs (cell death) from lungs of uninfected and SIV-infected monkeys collected 24 h post-BrdU injection [(A), n = 14] and 48 h post-BrdU injection [(B), n = 12]. A minimum of 400 cells was counted in at least five microscopic fields of lung tissues from SIV-infected (●) and uninfected (★) monkeys. (C) Representative image of IMs in the lung tissues from an SIV-infected monkey with high monocyte turnover (55% BrdU-staining monocytes at 24 h). Lung tissues from 4 uninfected rhesus macaques and 10 SIV-infected monkeys with different monocyte turnover rates were stained with anti-CD163 (green) and anti-BrdU (turnover; red) Abs. Cell death/apoptosis was measured by TUNEL (blue) staining. Images were captured with a Leica TCS SP2 confocal microscope (original magnification ×1260).
Increased death rate of lung IMs correlates with increased blood monocyte turnover rate in SIV-infected rhesus macaques. Spearman correlation analyses were performed to relate blood monocyte turnover (BrdU) and percentage of TUNEL+ IMs (cell death) from lungs of uninfected and SIV-infected monkeys collected 24 h post-BrdU injection [(A), n = 14] and 48 h post-BrdU injection [(B), n = 12]. A minimum of 400 cells was counted in at least five microscopic fields of lung tissues from SIV-infected (●) and uninfected (★) monkeys. (C) Representative image of IMs in the lung tissues from an SIV-infected monkey with high monocyte turnover (55% BrdU-staining monocytes at 24 h). Lung tissues from 4 uninfected rhesus macaques and 10 SIV-infected monkeys with different monocyte turnover rates were stained with anti-CD163 (green) and anti-BrdU (turnover; red) Abs. Cell death/apoptosis was measured by TUNEL (blue) staining. Images were captured with a Leica TCS SP2 confocal microscope (original magnification ×1260).
Increased SIV levels in lung tissues of infected rhesus macaques correlate with increased IMs and blood monocyte turnover rates but not with SIV plasma viral loads
To determine whether SIV levels in lung macrophages correlated with death and turnover rates of IMs, quantitative PCR was used to measure SIV DNA copies in lung tissue. SIV DNA levels in the lung significantly correlated with turnover of IMs represented by the percentage of BrdU labeling at 24 h post-BrdU injection (r = +0.6703, p = 0.0087, Fig. 5A). SIV DNA levels also correlated with SIV RNA viral copies in lung tissue (r = +0.7212, p < 0.0234, Fig. 5B), suggesting that active SIV replication was occurring in the lung. Interestingly, plasma viral loads (pVLs) did not correlate with levels of SIV DNA and RNA in the lung (Fig. 5C). Taken together, these results suggest that the increasing virus replication destroys lung macrophages, especially IMs, which may promote blood monocyte turnover to replace the killed pulmonary macrophages.
Increased SIV levels in lung tissues of infected rhesus macaques correlate with increased IMs and blood monocyte turnover rates but not with SIV pVLs. Lung tissues were collected from SIV-infected macaques at different stages of disease and quantitated for SIV DNA and RNA. (A) The number of SIV DNA copy equivalents in lung tissue directly correlated with IM turnover in SIV-infected macaques (n = 14). (B) SIV DNA copy numbers directly correlated with SIV RNA copy numbers in the lungs of SIV-infected macaques (n = 10). (C) SIV DNA copy numbers in lung did not correlate with the pVL in SIV-infected macaques (n = 12). The Spearman correlation was applied for statistical analysis.
Increased SIV levels in lung tissues of infected rhesus macaques correlate with increased IMs and blood monocyte turnover rates but not with SIV pVLs. Lung tissues were collected from SIV-infected macaques at different stages of disease and quantitated for SIV DNA and RNA. (A) The number of SIV DNA copy equivalents in lung tissue directly correlated with IM turnover in SIV-infected macaques (n = 14). (B) SIV DNA copy numbers directly correlated with SIV RNA copy numbers in the lungs of SIV-infected macaques (n = 10). (C) SIV DNA copy numbers in lung did not correlate with the pVL in SIV-infected macaques (n = 12). The Spearman correlation was applied for statistical analysis.
Infection and replication of SIV in pulmonary IMs and AMs of rhesus macaques exhibiting high monocyte turnover
In situ hybridization of SIV RNA and confocal microscopy were performed and demonstrated that SIV infected AMs and IMs in lung tissue (Fig. 6A, 6B). A higher percentage of animals with monocyte turnover rates >30% exhibited foci of SIV in both IMs and AMs in lung tissue, whereas SIV foci were primarily found in IMs of animals exhibiting monocyte turnover rates ≤30% (Supplemental Table III). Quantitative PCR was performed to quantify and compare SIV infection levels in AMs, IMs, and CD4+ T cells captured by FACS of lung tissue cell suspensions (Fig. 6C). Significantly higher levels of SIV DNA were measured in IMs from animals exhibiting higher monocyte turnover rates >30% compared with animals exhibiting monocyte turnover rates ≤30% (p = 0.0273, Fig. 6D). SIV DNA levels also were higher in AMs of animals with higher monocyte turnover, but these did not achieve statistical significance compared with the mean level of SIV DNA in animals with lower monocyte turnover (p = 0.0895, Fig. 6D). Interestingly, SIV DNA levels in lung CD4+ T cells were similar between animals with high and low monocyte turnover rates (p = 0.2042, Fig. 6D). The levels of SIV RNA in plasma also did not significantly correlate with blood monocyte turnover rates (r = −0.1193, p = 0.6599, Fig. 6E). The mean number of CD4+ T cells in lungs of SIV-infected monkeys was significantly lower than in lungs of uninfected monkeys (p < 0.0001, Fig. 6F), but the number of lung CD4+ T cells in SIV-infected monkeys did not correlate with the degree of blood monocyte turnover rates indicative of disease progression (r = −0.4818, p = 0.1375, Fig. 6G).
Infection and replication of SIV in IMs and AMs contribute to the viral load in lung tissues of infected rhesus macaques exhibiting high monocyte turnover. Confocal microscopy was performed on lung tissues obtained from SIV-infected monkeys with low (≤30%) monocyte turnover [(A), n = 3]and higher (>30%) blood monocyte turnover [(B), n = 6]. Anti-CD163 (green) Ab was used to identify macrophages, and SIV RNA (red) was detected with anti-sense riboprobes. Arrows indicate SIV-infected IMs. The asterisk indicates an SIV-infected AM. (C) Gating strategy for analyzing and sorting AMs, IMs, and CD4+ T cells isolated from lung tissue. (D) IMs, AMs, and CD4+ T cells were sorted via FACS from single-cell suspensions of lung tissues from SIV-infected monkeys with low (n = 5) and high (n = 4) blood monocyte turnover, and SIV DNA levels were quantitated and standardized against RNase P levels. (E) pVL did not correlate with monocyte turnover in SIV-infected macaques. (F) CD4+ T cell depletion in the lung of all SIV-infected macaques was evident (p < 0.0001). (G) The levels of CD4+ T cells did not correlate with monocyte turnover rates in SIV-infected macaques. The Student t test was used in (D) and (F), and the Spearman correlation analysis was used in (E) and (G).
Infection and replication of SIV in IMs and AMs contribute to the viral load in lung tissues of infected rhesus macaques exhibiting high monocyte turnover. Confocal microscopy was performed on lung tissues obtained from SIV-infected monkeys with low (≤30%) monocyte turnover [(A), n = 3]and higher (>30%) blood monocyte turnover [(B), n = 6]. Anti-CD163 (green) Ab was used to identify macrophages, and SIV RNA (red) was detected with anti-sense riboprobes. Arrows indicate SIV-infected IMs. The asterisk indicates an SIV-infected AM. (C) Gating strategy for analyzing and sorting AMs, IMs, and CD4+ T cells isolated from lung tissue. (D) IMs, AMs, and CD4+ T cells were sorted via FACS from single-cell suspensions of lung tissues from SIV-infected monkeys with low (n = 5) and high (n = 4) blood monocyte turnover, and SIV DNA levels were quantitated and standardized against RNase P levels. (E) pVL did not correlate with monocyte turnover in SIV-infected macaques. (F) CD4+ T cell depletion in the lung of all SIV-infected macaques was evident (p < 0.0001). (G) The levels of CD4+ T cells did not correlate with monocyte turnover rates in SIV-infected macaques. The Student t test was used in (D) and (F), and the Spearman correlation analysis was used in (E) and (G).
Discussion
Results presented in an earlier publication using nonhuman primates (8) and data presented in this article demonstrate that there exist unique macrophage populations in the lung that display different turnover rates prior to SIV infection and different levels of SIV infection during disease progression to AIDS. HIV and SIV infect activated CD4+ T cells, but as primate lentiviruses, they also infect terminally differentiated macrophages (26–28). Declining levels of circulating CD4+ T cells are an indication of immunodeficiency, but our earlier reports demonstrated that increased turnover of circulating monocytes better predicted the onset of disease progression to AIDS in SIV-infected rhesus macaques (2, 29). The results of this study further extend these findings and demonstrate that the increased levels of SIV infection and cell death of IM in lung tissue also correlated with increased blood monocyte turnover and disease progression to AIDS (2). Furthermore, the turnover rates of IMs in lung tissue and blood monocytes were similar prior to infection (8) and similarly increased during SIV progression to AIDS. This suggests that the increase in blood monocyte turnover serves to replace dying SIV-infected IMs in the lung. Recruitment of monocytes to replace damaged tissue macrophages was reported during inflammation and immune responses to other infectious agents, as well (30–33). Thus, the results reported in this article further support a role for the newly recruited macrophages, such as IMs of the lung, in controlling infections and re-establishing tissue homeostasis (4).
In contrast, the turnover of pulmonary AMs was negligible during steady-state homeostasis prior to and during SIV infection. In addition, the longer-lived AMs initially were more resistant to virus infection but eventually became infected during end-stage disease, possibly via the close association or contact with SIV-infected IMs (Supplemental Fig. 1). The detection of BrdU-labeled AMs after BAL in our previous study supports the possibility that IMs serve as intermediates for differentiation into AMs and transport the virus into the alveoli (8). More importantly, the increased SIV DNA in AMs and IMs in SIV-infected monkeys exhibiting higher blood monocyte turnover strongly suggested that SIV replicated in the macrophages rather than being phagocytized for lysosomal degradation. The later infection of AMs, based on detection of SIV DNA, also implied that these cells could serve as a virus reservoir (34, 35) and suggests that successful control of HIV infection requires elimination of virus from both T cells and macrophages (Supplemental Fig. 1).
Various lineages and differentiation states of murine and human tissue macrophages have been compared and discussed (4, 36, 37), but further in vivo characterization of human tissue macrophages is necessary for a better understanding of different tissue macrophage subsets in humans. Longitudinal studies characterizing effects or shifts in monocyte and macrophage lineages during HIV infection in humans are difficult to accomplish, so studies using nonhuman primates are expected to provide basic information to guide such investigations. This study demonstrated that SIV affects at least two subsets of lung macrophages differently and strongly suggests that there exist functional differences between the shorter-lived IMs and longer-lived AMs of the lung. The direct link between the turnover of circulating monocytes and destruction of IMs, but not AMs, of the lung further support a role for IMs in homeostasis and/or the daily protection of the lung (4). Although not addressed in the current study, it will be important to understand the molecular mechanisms for SIV-induced cell killing of IMs, but not of AMs, in the lung tissues to include measurement for determining the level of cell death factors or cell survival factors (e.g., hexokinase-6) in different macrophage subsets, as previously described (38).
Studies on lung biology and innate immunity in humans typically rely on the use of BALF specimens. A limitation is that BALF specimens consist almost exclusively of AMs but not IMs. Evidence of viral infection of AMs, pneumonia, opportunistic infection, or neoplasms is observed both in humans and macaques (39), whereas the impact of HIV-1 infection of IMs in humans has been underexamined. AMs and IMs of the lung exhibit different kinetics in turnover and SIV infection rates, suggesting that BALF samples may provide an incomplete or limited representation of pulmonary immune responses. Therefore, a reliable and accessible marker of lung tissue damage produced by HIV/SIV infection, IM damage and higher monocyte turnover, is expected to monitor lung pathogenesis longitudinally during progression to AIDS and perhaps also for development of HANA conditions. It is important to note that the pathology of the lung observed in the SIV-infected macaques described in this study (Fig. 1) may differ somewhat from the lesions generally described in lung tissue of HIV-infected humans. This is mainly because lung tissue specimens collected in our study were based on an experimental timeline to relate blood monocyte turnover rate to lung tissue changes at time points that also were prior to clinical signs of pneumonia or the development of opportunistic infections. Lung tissues from HIV-infected humans were primarily obtained for purposes of diagnostics and treatment only after clinical signs, such as pneumonia arose, or from autopsy tissue to help determine cause of death. However, lung tissue of SIV-infected macaques obtained at the late stage with pneumonia produced similar lung lesions as previously reported from HIV-infected humans (16, 40, 41). Although more detailed studies are necessary and are underway, some candidate biomarkers of increasing monocyte turnover coincident with disease pathogenesis may include neopterin, IP-10, and sCD163 (data not sown). In addition, plasma CXCL13 could be considered because it was reported to correlate with the level of pVL in HIV-infected cohorts (42), although our study indicated that not all SIV-infected macaques with high viral load exhibited high monocyte turnover, so this is another area of exploration. Patro et al. (43) reported that apoptosis of monocytes correlated with HIV plasma levels; although SIV levels did not necessarily correlate with disease progression in our studies using rhesus macaques, monocyte apoptosis could also be considered for its relationship or impact on monocyte turnover. The nonhuman primate model of AIDS is especially amenable to examining these mechanisms and their impact on both populations of lung macrophages via BAL and tissue biopsy. Thus, it provides an important resource for studies about the mechanisms of HIV/AIDS and HANA pathogenesis, as well as for defining sites of viral reservoirs necessary to rationally target effective therapies.
Acknowledgements
We thank Toni P. Penny, Desiree K. Waguespack, Ashley N. Leach, Erin M. Haupt, Julie Bruhn, and Calvin Lanclos (Division of Immunology), Chris Monjure and Coty Tatum (Pathogen Detection and Quantification Core of the Division of Microbiology), and Dr. Jason P. Dufour (Division of Veterinary Medicine) at the Tulane National Primate Research Center for technical assistance.
Footnotes
This work was supported by National Institutes of Health Grants AI087302, AI091501, AI097059, AI110163, AI116198, and HL125054 (to M.J.K.) and P51OD011104 (to the Tulane National Primate Research Center), as well as a grant from Virginia’s Commonwealth Health Research Board (11-09 to W.-K.K.).
The online version of this article contains supplemental material.
References
Disclosures
The authors have no financial conflicts of interest.