Monocyte heterogeneity underlying phenotypic changes in monocytes according to SIV disease stage

Monocyte heterogeneity underlyingphenotypic changes in monocytes

according to SIV disease stageWoong-Ki Kim,*,1 Yue Sun,* Hien Do,† Patrick Autissier,* Elkan F. Halpern,‡

Michael Piatak Jr.,§ Jeffrey D. Lifson,§ Tricia H. Burdo,�� Michael S. McGrath,†,¶

and Kenneth Williams*,2

*Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, and ‡Department of Radiology, Massachusetts GeneralHospital, Harvard Medical School, Boston, Massachusetts, USA; †Pathologica, LLC, San Francisco, California, USA; §AIDSVaccine Program, SAIC Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland, USA; ��Department of

Biology, Boston College, Chestnut Hill, Massachusetts, USA; and ¶Department of Laboratory Medicine, Positive HealthProgram, University of California, San Francisco, California, USA

RECEIVED FEBRUARY 16, 2009; REVISED AUGUST 28, 2009; ACCEPTED AUGUST 31, 2009. DOI: 10.1189/jlb.0209082

ABSTRACTInfection by HIV is associated with the expansion ofmonocytes expressing CD16 antigens, but the signifi-cance of this in HIV pathogenesis is largely unknown. Inrhesus macaques, at least three subpopulations of bloodmonocytes were identified based on their expression ofCD14 and CD16: CD14highCD16�, CD14highCD16low, andCD14lowCD16high. The phenotypes and functions of thesesubpopulations, including CD16� monocytes, were inves-tigated in normal, uninfected rhesus macaques and ma-caques that were infected with SIV or chimeric SHIV. Toassess whether these different monocyte subpopulationsexpand or contract in AIDS pathogenesis, we conducteda cross-sectional study of 54 SIV- or SHIV-infected ma-caques and 48 uninfected controls. The absolute num-bers of monocyte populations were examined in acutelyinfected animals, chronically infected animals with no de-tectable plasma virus RNA, chronically infected animalswith detectable plasma virus RNA, and animals that diedwith AIDS. The absolute numbers of CD14highCD16low andCD14lowCD16high monocytes were elevated signifi-cantly in acutely infected animals and chronically in-fected animals with detectable plasma virus RNAcompared with uninfected controls. Moreover, a sig-nificant, positive correlation was evident between thenumber of CD14highCD16low or CD14lowCD16high mono-cytes and plasma viral load in the infected cohort.

These data show the dynamic changes of bloodmonocytes, most notably, CD14highCD16low mono-cytes during lentiviral infection, which are specific todisease stage. J. Leukoc. Biol. : 000–000; 2010.

IntroductionHIV infection in humans results in hematologic abnormalitiesand immune suppression, which is well represented bychanges in the CD4/CD8 T cell ratio. As obviously seen in Tcell populations, HIV-induced immune perturbation is alsoeasily observed within the mononuclear phagocyte system in-cluding macrophages and monocytes. Circulating monocytesin HIV disease display marked phenotypic and functional alter-ations associated with progression to AIDS [1–5]. Notably, cellsurface antigens that exhibit a bimodal expression on all ofthe resting monocyte populations (thereby indicating the pres-ence of subpopulations) have been recognized as the mostaffected by HIV infection. For example, an increase in Fc�RIII(CD16) expression and decrease in Fc�RI (CD64) and L-selec-tin (CD62L) expression on blood monocytes were significantin HIV-infected patients compared with uninfected controls[6–12]. It is likely that these changes in HIV-infected patientsare largely a result of expansion and contraction of monocytesubpopulations expressing these markers. Indeed, it has beenshown repeatedly that CD14� monocytes, expressing CD16,expand during HIV infection [13–18] and SIV infection [19–21]. We have observed recently that monocytes leave the bonemarrow and enter the blood in response to monocyte/macro-phage apoptosis in monkeys that are SIV-infected [22].

The presence of phenotypically definable subpopulationsof human monocytes has been recognized for over 20 years.

1. Current address: Department of Microbiology and Molecular Cell Biology,Eastern Virginia Medical School, Norfolk, VA 23507, USA.

2. Correspondence at current address: Department of Biology, Boston Col-lege, Chestnut Hill, MA 02467, USA. E-mail: [email protected]

Abbreviations: APC�allophycocyanin, CCR2�C–C motif chemokine re-ceptor 2, CD14highCD16� monocytes�monocytes with a high density ofCD14 and no CD16 antigen, CD14highCD16low�monocytes that coexpresshigh levels of CD14 and low levels of CD16, CD14lowCD16high monocytes�monocytes that coexpress low levels of CD14 and high levels of CD16,CD62L�CD62 ligand, CX3CR1�C–X3–C motif chemokine receptor 1,Cy5.5�cyanin 5.5, DC�dendritic cell, HAART�highly active antiretroviraltherapy, mDC�myeloid dendritic cell, pDC�plasmacytoid dendritic cell,p.i.�postinfection, SHIV�SIV/HIV

The online version of this paper, found at www.jleukbio.org, includessupplemental information.

Article

0741-5400/10/00-0001 © Society for Leukocyte Biology Volume , MONTH 2010 Journal of Leukocyte Biology 1

Epub ahead of print October 20, 2009 - doi:10.1189/jlb.0209082

Copyright 2009 by The Society for Leukocyte Biology.

Using two-color flow cytometry of human monocytes for thecorrelated expression of CD14 (LPS receptor) and CD16,Ziegler-Heitbrock et al. [23] first showed two monocyte sub-populations: CD14highCD16� and CD14lowCD16high. Follow-ing this initial description, refined analyses of chemokinereceptors and adhesion molecules have better defined sub-populations of monocytes and shown additional phenotypicheterogeneity of human monocytes [24 –27]. These datashow a CD14highCD16� subpopulation of monocytes that ex-press CD62L, CD64, and CCR2 with low levels of CX3CR1expression. In contrast, the CD14lowCD16high subpopulationexpresses no detectable CD62L, CD64, or CCR2 but highlevels of CX3CR1.

Studies in mice have demonstrated in addition to their pheno-typic differences differential homing potential of i.v.-injectedCX3CR1lowCCR2�Gr1� and CX3CR1highCCR2�Gr1� monocytes(two rodent counterparts of human CD14highCD16� andCD14lowCD16high monocytes, respectively) [28, 29]. A morerecent study by Auffray et al. [30] showed that mouseCD11a�CX3CR1highCCR2�Gr1� monocytes (equivalent tohuman CD14lowCD16high monocytes) are the first to exitinto inflamed tissues. In this study, Auffray et al. [30] dem-onstrated that this subset crawls on normal endothelium ofblood vessels, invades tissues rapidly upon tissue damage,and becomes tissue macrophages. This study is also in linewith the work showing that the human counterpartCD14lowCD16high monocytes preferentially reside in themarginal pool and are mobilized quickly [31].

Functional in vitro studies analyzing endocytotic and phago-cytotic functions and the ability to produce inflammatory cyto-kines in response to LPS also confirmed the existence of twodistinct monocyte subsets in humans [14, 32]. It is importantto note the presence of a third, less well-described subpopula-tion that expresses high levels of CD14 and low levels of CD16(CD14highCD16low monocytes). Adding one additional layer ofcomplexity to monocyte heterogeneity, these monocytes withan “intermediate” or “transient” phenotype may also representa subset with distinctive functions. Their role in HIV and SIVpathogenesis is understudied.

SIV- or chimeric SHIV-infected rhesus macaques are the primemodel to study HIV pathogenesis. In this study, we investigatedthe phenotypic and functional differences of monocyte subpopu-lations in normal, uninfected and SIV- or SHIV-infected monkeys.We initially identified by flow cytometry four distinct subpopula-tions of blood mononuclear phagocytes in rhesus monkeys basedon differential expression of CD14 and CD16: CD14highCD16�,CD14highCD16low, CD14lowCD16high, and CD14�/lowCD16�. Tobetter characterize these subpopulations, we have further exam-ined the expression of a broad spectrum of other monocyte- andDC-associated markers. The differential expression of these mark-ers was largely confirmed by gene array studies using FACS-sortedmonocyte subpopulations. To investigate the relative contributionof subsets that replicate virus in vivo, we examined levels of SIVRNA associated with freshly sorted monocyte subpopulations ininfected animals and found their differential ability to sustainSIV infection. Changes in monocyte subpopulations during lenti-virus infection were assessed by a cross-sectional study of the un-infected and infected cohorts of rhesus macaques. In this report,

by performing a comprehensive analysis of the association be-tween lentiviral infection and changes in monocytes, we identifydisease-specific changes in monocyte subpopulations.

MATERIALS AND METHODS

AnimalsOne hundred and two rhesus macaques (Macaca mulatta) were recruitedfor this study, including 48 uninfected control macaques and 54 animalsinfected with SIV or SHIV. The infected cohort of monkeys consisted ofanimals infected with SIVmac239 (n�3), SIVmac251 (n�30), SHIV-89.6P(n�14), and SHIV-SF162P3 (n�7), all of which have similar magnitudes ofpeak viremic, chronic infection and AIDS-related plasma virus. Most bloodsamples were collected once at necropsy, from killed animals that were notallowed to develop AIDS and death or from animals that died with AIDS.Four animals were used for serial blood samples, two to three times overthe course of disease.

Flow cytometric immunophenotypingPeripheral blood samples were used to determine the immune phenotypeof rhesus monocytes. We and others [33, 34] found that heparinized bloodis not suitable to assess expression of some monocyte-associated antigens asa result of high, nonspecific background staining (e.g., CCR2) or inconsis-tency of staining (e.g., CD163). Similarly, as circulating immune complexesbind CD16 that mask recognition by anti-CD16 antibodies [35], it is essen-tial to remove plasma to detect CD16 consistently. Therefore, all subse-quent immunofluorescence staining for flow cytometry was performed us-ing EDTA-anticoagulated, plasma-depleted blood. For this, aliquots of 100�L EDTA-anticoagulated whole blood were washed with PBS, and the pel-lets were incubated at room temperature for 15 min with a mixture of anti-bodies conjugated with FITC, PE, PerCP/PerCP-Cy5.5, or APC. Three four-color immunophenotyping panels were used: One panel included anti-CD14-FITC (clone M5E2, BD PharMingen, San Diego, CA, USA), anti-CD16-PE (3G8, BD PharMingen), and anti-HLA-DR-PerCP (L243, BectonDickinson, San Diego, CA, USA); the second panel included anti-CD16-PE,anti-HLA-DR-PerCP, and anti-CD14-APC; and the third panel included anti-CD16-FITC, anti-CD14-PerCP-Cy5.5, and anti-HLA-DR-APC. Using thesethree basic panels, monocyte subpopulations were examined further forexpression of other monocyte- and DC-associated antigens. The mAb usedfor this included: CD1c-APC (AD5-8E7, Miltenyi Biotec, Auburn, CA, USA),CD11b-APC (M1/70.15.11.5, Miltenyi Biotec), CD11c-APC (S-HCL-3, Bec-ton Dickinson), CD31-APC (WM59, eBioscience, San Diego, CA, USA),CD45RA-FITC (2H4, Beckman Coulter, Fullerton, CA, USA), CD45RB-FITC (PD7/26, Dako, Denmark), CD62L-PE (SK11, BD PharMingen),CD64-FITC (10.1, BD PharMingen), CD163-FITC (Mac2-158, Trillium Diag-nostics, Brewer, ME, USA), CD123-PE (7G3, BD PharMingen), CD141-PE(1A4, BD PharMingen), CCR2-APC (48607, R&D Systems, Minneapolis,MN, USA), CCR8 (191704, R&D Systems), and CX3CR1-APC (rabbit poly-clonal, Torrey Pines Biolabs, Houston, TX, USA). Isotype control mAbwere used in all panels. These included: mouse IgG2b-APC (27-35, BDPharMingen), rat IgG2b-APC (141945, R&D Systems), mouse IgG1-APC(MOPC-21, BD PharMingen), mouse IgG1-FITC (MOPC-21), mouse IgG2a-FITC (G155-178, BD PharMingen), and mouse IgG2a-PE (G155-178). Afterstaining, cells were fixed in 2% paraformaldehyde and analyzed on a FACS-Calibur (BD Biosciences, San Jose, CA, USA). To establish the flow cyto-metric identification of monkey monocytes in blood, a gate was set basedon light-scatter properties of blood monocytes to contain predominantlymonocytes. The monocyte gate constituted 80–90% CD14� cells in all sam-ples tested, and contaminating CD3� T cells were consistently found to be�5%. Isotype controls were used to set gates for positive cells, allowing�1% positive cells in the negative control samples. Absolute counts ofmonocytes and monocyte subsets were calculated by multiplying the per-centage of each subpopulation within the blood monocyte gate by thenumber of monocytes/�L blood, as determined by complete blood cellcounts (ADVIA 120 hematology analyzer, Bayer Diagnostics, Germany).

2 Journal of Leukocyte Biology Volume , MONTH 2010 www.jleukbio.org

Lymphocyte immunophenotyping was performed using combinations offollowing mAb: CD3-PerCP-Cy5.5 or -APC (SP34, BD PharMingen); CD4-FITC (19thy5d7, Beckman Coulter) or CD8-FITC or -PE (SK1, Becton Dick-inson); and CD20-PerCP-Cy5.5 (L27, Becton Dickinson), CD28-PE (28.2,Beckman Coulter), and CD95-APC (DX2, BD PharMingen). Naı̈ve T lym-phocytes were determined by their CD28�CD95� phenotype in CD3�CD4�

or CD3�CD8� cells, whereas central and effector memory T cell subsetswere defined as being CD28�CD95� and CD28�CD95�, respectively.

Isolation of monocyte subpopulationsFACS sorting of monocyte subpopulations was performed as described pre-viously [20]. Briefly, Ficoll-separated PBMC isolated from monkey bloodwere stained with anti-CD14-FITC, anti-CD16-PE, anti-CD4-PerCP-Cy5.5, andanti-CD3-APC. Cell sorting was done using a BD FACSVantage flow cytome-ter in a Biosafety Level 2� facility. CD3-negative cells in the monocyte gatewere fractionated based on CD14 and CD16 expression into CD14high

CD16�, CD14highCD16low, and CD14lowCD16high cells. Sorted cells werewashed and snap-frozen rapidly for gene array or SIV-DNA and -RNA analy-sis described below. In addition, CD3�CD4� lymphocytes were sorted forcomparison. Purity of all FACS-sorted populations was confirmed by flowcytometry and was routinely �95%.

Intracellular cytokine stainingThe phenotype and frequency of nonstimulated monocytes spontaneouslyproducing TNF-� and granzyme B in normal, uninfected and SIV-infectedanimals were determined by intracellular cytokine staining followed by sev-en-color flow cytometry. PBMC were isolated from heparinized blood byFicoll gradient centrifugation and then incubated at 37°C in a 5% CO2 in-cubator for 3 h in the presence of monensin (GolgiStop, BD Biosciences).Cultured cells were stained with mAb specific for cell surface molecules,including anti-CD3-Pacific Blue (SP34-2, Becton Dickinson), anti-CD4-Am-Cyan (L200, Becton Dickinson), anti-CD8-PerCP-Cy5.5 (RPA-T8, BectonDickinson), anti-CD16-PE (3G8, BD PharMingen), and anti-CD14-PE-TexasRed (RMO52, Beckman Coulter). Cells were fixed and permeabilized withCytofix/Cytoperm solution (BD Biosciences) and stained with anti-TNF-�-FITC (mAb11, BD PharMingen) and anti-granzyme B-Alexa Fluor 700(GB11, BD PharMingen). Labeled cells were resuspended and fixed with1% formaldehyde in PBS. Samples were collected and analyzed on a BDLSR II instrument using FlowJo software (Tree Star, Ashland, OR, USA).Events (500,000–1 million) were collected/sample.

Determination of plasma viral RNA andcell-associated viral RNA and DNACopies of plasma SIV and SHIV RNA and cell-associated SIV RNA andDNA were determined using a quantitative real-time RT-PCR assay as de-scribed previously [20].

Statistical analysisThe Kruskal-Wallis test was used for nonparametric-independent groupcomparisons of the medians. Only if the test were significant (P�0.05)were comparisons of each of the infected groups with an uninfected con-trol group performed using the two-tailed Wilcoxon test. All combinationsof the independent variables were analyzed with 95% confidence level foreach variable. Spearman correlation coefficients were computed betweentwo of all variables.

RESULTS

Phenotypic heterogeneity of monkey mononuclearphagocytes in bloodWithin the blood, the mononuclear phagocyte system is repre-sented by monocytes, which can be identified by flow cytom-

etry as cells within a “gate” of high forward-scatter and inter-mediate side-scatter. This light-scatter gate contained cells dis-playing different levels of CD14 expression (Fig. 1A). Flowcytometric analysis of cells within the gate for the correlatedexpression of CD14 and CD16 delineated four distinct sub-populations. A major subpopulation is CD14highCD16�. Twoadditional subpopulations express variable levels of CD14 andCD16: CD14highCD16low and CD14lowCD16high. A fourth popu-lation has CD14low/�CD16�. This CD14low/�CD16�populationis heterogeneous, containing immature monocytes,

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Figure 1. Phenotypic heterogeneity of blood mononuclear phagocytesin monkeys. (A) Definition of mononuclear phagocytes in peripheralblood of rhesus macaques. (Left) Typical light-scatter contour map ofwhole blood cells; Ly, lymphocytes; Mo, monocytes; G, granulocytes(middle) overlay of gated mononuclear phagocytes (Mo; green) onside-scatter versus CD14 contour profile (red); (right) correlated ex-pression of CD14 and CD16 defines four distinct subpopulations ofmononuclear phagocytes in normal monkey blood. The regions indi-cated in the CD14 versus CD16 contour plot of gated mononuclearphagocytes were used to differentiate the following monocyte and DCpopulations in the blood. (B) Phenotypic analysis of blood monocytesubpopulations. The expression of indicated molecules was deter-mined by four-color flow cytometry. The histograms exhibit the phe-notypic characteristics of the three monocyte subpopulations understudy: CD14highCD16�, CD14highCD16low, and CD14lowCD16high. Themean fluorescence intensity is shown. The discriminators shown wereset at the foot of the isotype control. The results for each marker arerepresentative of at least 10 uninfected animals. (C) Phenotypic char-acterization of CD14low/�CD16� cells (CD14–CD16–). These cells areheterogeneous, containing CD11c� mDCs and CD123� pDCs (leftpanel). CD11c� mDCs express another DC marker CD1c (middle).Some CD14low/�CD16� cells express CD40 antigen, unlike monocytecells (right).

Kim et al. Monocyte heterogeneity in SIV infection

www.jleukbio.org Volume , MONTH 2010 Journal of Leukocyte Biology 3

CD1c�CD11c� mDCs, and CD1c�CD123� pDCs, as well aslymphocytes (Fig. 1C). Monocyte subpopulations,CD14highCD16�, CD14highCD16low, and CD14lowCD16high, innormal, uninfected macaques (n�48) comprised 65% � 8%(mean�sd), 8% � 4%, and 9% � 4%, respectively, of the to-tal monocytes, gated on forward- versus side-scatter.

To better define the cells of these subpopulations, we ex-amined their expression of monocyte- and DC-associatedmarkers including CD1c, CD11b, CD11c, CD31, CD40,CD45RA, CD64, CD68, CD86, CD123, CD163, CCR2,CX3CR1, HLA-DR, MAC387, and TLR2 (Fig. 1B). CD1c(blood DC antigen-1) expression in the CD14highCD16�

population was bimodal and demonstrated CD1c� or CD1c�

phenotypes. Virtually, all of the CD14highCD16low cells wereCD1c�, whereas all CD14lowCD16high cells were CD1c�. CD11bexpression was uniformly high on CD14highCD16� andCD14highCD16low cells but low on CD14lowCD16high cells. CD11cexpression was bimodal in the CD14highCD16low population withCD11c� and CD11c� subsets. Essentially, all of the CD14low

CD16high cells were CD11c�, and all of the CD14highCD16� cellswere CD11c�. PECAM-1 (CD31) was expressed on nearly all cellsin the CD14highCD16�, CD14highCD16low, and CD14lowCD16high

populations, with the highest levels on CD14lowCD16high cells.CD45RA expression was limited to a subset of the CD14low

CD16high population. All CD14highCD16� cells and a majority ofCD14highCD16low cells were CD64�, but no cells expressing thismarker were detected in the CD14lowCD16high population. A sub-set of CD86-positive cells was detected in all of the populations,where the highest levels of CD86 were found on CD14high

CD16low cells. CD163, a macrophage marker, was expressed onall CD14highCD16� and CD14highCD16low cells but not onCD14lowCD16high cells. The expression of CCR2 followed the pat-tern of CD64. High CX3CR1 was found on all cells in CD14high

CD16low and CD14lowCD16high, whereas CX3CR1low cells weredetected in about one-half of the CD14highCD16� population;the level of CX3CR1 expression on CD14highCD16low cells wasgreater than that on CD14lowCD16high cells. Virtually, all of theCD14highCD16�, CD14highCD16low, and CD14lowCD16high cellswere HLA-DR�, and CD14highCD16low cells had the highest lev-els. Overall, these data demonstrate that the CD14highCD16�,CD14highCD16low, and CD14lowCD16high populations comprisethe majority of blood monocytes/macrophages, and theCD14low/�CD16� cells are heterogeneous and consist of mDCand pDC, CD3� T lymphocytes, and likely, monocyte precursors.Table 1 summarizes the data described above with regard to theCD14highCD16�, CD14highCD16low, and CD14lowCD16high pheno-types.

It has been suggested that within blood, especially duringdisease states, populations of monocytes are present with phe-notypes similar to maturing or mature macrophages. We ex-amined the different monocyte populations for two such mark-ers: intracellular expression of CD68 (a pan-macrophagemarker) and MAC387 (a heterodimer of S100A8 and S100A9,expressed by monocytes, which is down-regulated and lost dur-ing macrophage differentiation) antigens. We found two mu-tually exclusive subsets: a CD68�MAC387� subset andCD68�MAC387� subset. All CD14highCD16� and CD14high

CD16low cells were CD68�MAC387�, and a majority

of CD14lowCD16high cells were CD68�MAC387�, suggestingthat compared with CD14highCD16� and CD14highCD16low

cells, the CD14lowCD16high populations represent more matureand macrophage-like cells.

To investigate whether CD16� monocytes are comprised ofdistinct subpopulations of monocytes rather than a continuum ofCD14� monocytes with differing levels of cell activation, we usedgene array analysis that compared overall gene expression pro-files of FACS-sorted CD14highCD16�, CD14highCD16low, andCD14lowCD16high cells (Supplemental Data Section). Microarrayanalysis confirmed the phenotypic differences between these sub-populations, differentiated on the basis of markers describedabove (Supplemental Fig. 1) and discerned further between thesesubpopulations by revealing additional differences (SupplementalTables). In comparison with CD14highCD16� cells, a large num-ber of genes (9098/29361, 30.9%) were expressed differentiallyin CD14highCD16low and CD14lowCD16high cells, underscoringthe fundamental differences between CD16� and CD16� mono-cytes (Supplemental Table 1). Thirty-one genes and 94 geneswere associated specifically with CD14highCD16low andCD14lowCD16high subpopulations (Supplemental Tables 2 and 3).A relatively small set of genes that was expressed differentiallybetween the two CD16� subpopulations highlights similarity be-tween the two cell types, but differentially expressed genes offunction observed in CD14highCD16low and CD14lowCD16high

subpopulations suggest different roles that these two subpopula-tions may play in vivo.

Blood monocyte subpopulation dynamics during SIVinfectionIn an effort to understand how the distribution of monocytesand monocyte subpopulations changes in HIV/SIV infection,we analyzed SIV- or SHIV-infected (n�54) and naı̈ve (n�48)

TABLE 1. Monkey Monocyte Subpopulations: PhenotypicCharacteristics of Three Identified Subpopulations

Determined by Flow Cytometry Are Summarized

Subpopulation

Marker CD14highCD16– CD14highCD16low CD14lowCD16high

CD14 �� CD16 – � ��CD1c �/– � –CD11b �� CD11c – �/– ��CD31 � �� CD45RA – – �/–CD64 �� –CD68 – – �CD163 � � –CCR2 � �/– –CX3CR1 �/– �� HLA-DR � �� MAC387 � � –TNF-� � � –

CD14 and CD16 were listed first, as these markers were used to differ-entiate the monocyte populations.


rhesus monkeys (Table 2). The percentage of total monocyteswas significantly higher in infected animals than in uninfectedcontrols (mean�sd; infected, 7.5%�5.3% vs. uninfected,4.5%�1.7%; P�0.01, unpaired t-test). Additionally, the expan-sion of monocytes in blood, termed monocytosis (defined as�500 cells/�L), was seen more frequently in infected animals(20/54, 37.4%) than in uninfected animals (6/48, 12.5%).However, the mean absolute numbers of total monocytes werenot significantly different between the infected and uninfectedgroups (506�582 and 327�170 counts/�L, respectively). Thepercentage and absolute number of monocytes in the infectedcohort were highly variable and displayed a more positivelyskewed distribution as compared with uninfected controls(skewness of the absolute numbers, 5.58 and 1.06, for the in-fected vs. uninfected animals). For these reasons, we analyzedthe data from these cohorts using nonparametric tests of me-dians.

To characterize in detail distribution patterns of monocytesubpopulations reflecting the progression of the disease, westratified infected monkeys using the following disease stagesat the time of specimen collection: (1) acute phase (2 weeksp.i.; n�6) and chronic phase (�12 weeks p.i.); (2) without(n�16) or (3) with (n�26) detectable plasma virus RNA; and(4) terminal AIDS (at necropsy; n�6; Table 2A). The absolutenumbers of total monocytes were significantly higher in chron-ically infected monkeys with detectable plasma virus RNA (me-dian 370 counts/�L, P�0.0392, Wilcoxon rank-sum test) andmonkeys with terminal AIDS (median 685 counts/�L, P�0.0083, Wilcoxon rank-sum test) than in normal, uninfectedcontrols (median 285 counts/�L). In acutely infected animals,the monocyte number (median 265 counts/�L) was compara-ble with that in uninfected controls, and the median monocytepercentage was significantly higher than in uninfected controls(P�0.0014, Wilcoxon rank-sum test). In acutely infected ani-mals, monocytes expressed lower levels of CD14 (data notshown), in confirmation of previous reports [19, 36]. The me-dian absolute numbers of both CD16� monocyte subpopula-tions (CD14highCD16low: 66 counts/�L; and CD14lowCD16high:47 counts/�L) were significantly greater in acutely infectedanimals than in uninfected controls (CD14highCD16low:21 counts/�L; and CD14lowCD16high: 24 counts/�L; Table2A). In parallel to these increases, the relative percentage ofthe CD14highCD16� population decreased significantly(P�0.0003, Wilcoxon rank-sum test). This may indicate that arapid shift in the circulating monocyte pool occurred 2 weeksp.i. from the CD14highCD16� subpopulation toCD14highCD16low and CD14lowCD16high subpopulations.Whether this occurs in blood or from cells emerging frombone marrow is not clear.

The distribution of monocyte subpopulations in chronicallyinfected animals with no detectable plasma virus is comparablewith that in uninfected controls (Table 2A). In contrast, inchronically infected animals with detectable plasma virus,CD14highCD16low and CD14lowCD16high subpopulations in-creased significantly (medians 53 and 41 counts/�L, respec-tively), and the absolute number of the CD14highCD16� sub-population (median 194 counts/�L) was similar to that in un-infected controls (median 172 counts/�L). This indicates that

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CD14highCD16low and CD14lowCD16high subpopulations wereexpanded during this stage and that the marginal rise in thetotal monocyte count could be exclusively a result of the ex-pansion of CD14highCD16low and CD14lowCD16high subpopula-tions. This chronically infected group had highly variableplasma viral load among animals, and these variations may re-sult in variations in the distribution of monocyte subpopula-tions. Therefore, chronically infected animals with detectableplasma virus were stratified further into two potential sub-groups, according to median viral load (70,000 copies/mL):those with viral load �70,000 copies/ml (the low viral loadsubgroup, n�14) and those with viral load �70,000 copies/ml(the high viral load subgroup, n�12; Table 2B). Absolutenumbers of total monocytes and CD14highCD16low andCD14lowCD16high subpopulations were significantly and mark-edly higher in the high viral load subgroup than in the lowviral load subgroup. In fact, in the low viral load subgroup, thedistribution of monocyte subpopulations is similar to that inthe uninfected cohort.

The animals that died with AIDS had significantly greaternumbers of monocytes than uninfected controls. Absolutenumbers of the CD14highCD16�, CD14highCD16low, andCD14lowCD16high populations were found increased markedlyin this group as compared with the uninfected cohort. In theanimals that died with AIDS, the median number of theCD14highCD16low subpopulation was highest among all of thegroups, and the difference with the uninfected controls almostachieved statistical significance (P�0.0592, Wilcoxon rank-sumtest). The median number of the CD14lowCD16high populationin this group was also highest, but this increase was not statisti-cally significant (P�0.1965). Two animals with terminal AIDShad strikingly low counts of CD14lowCD16high cells (five andsix counts/�L), whereas the other four animals had highCD14lowCD16high cell numbers of 279, 150, 106, and 95counts/�L, respectively.

To minimize interindividual variations in the numbers ofmonocyte subpopulations, the ratio of CD14highCD16low,CD14lowCD16high, and both to CD14highCD16� in individualmonkeys was calculated. The median CD14highCD16low/CD14highCD16� ratio in uninfected controls was 0.12. In theacutely infected animals, this ratio increased and was changedsignificantly in favor of the CD14highCD16low population (1.02,P�0.0002). The CD14highCD16low/CD14highCD16� ratio inchronically infected animals with no detectable plasma virus(0.19) or with low viral load (0.18) is comparable with that ofuninfected controls. Then, the ratio increased to median levelsof 0.38 in the high viral load subgroup and 0.36 in the AIDSgroup. The CD14lowCD16high/CD14highCD16� ratio followedthe similar pattern, but an increase of this ratio in the AIDSgroup was not significant. Collectively, these data indicate thatthe distribution of monocyte subpopulations was altered in amanner specific to disease stages, where an expansion of cellswith more mature macrophage-like properties emerges.

Immunologic correlations with monocyteheterogeneityFor each animal, important immunological parameters of HIVinfection, including CD4� and CD8� T-lymphocyte counts, the

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number of CD4� and CD8� central memory T cells, andplasma viral load, were analyzed in parallel to monocyte sub-sets. We sought to determine in this cross-sectional studywhether any immunologic parameter correlates with thechanges in distribution of monocyte subpopulations. Initially,we found significant correlations between some of these pa-rameters and monocyte subpopulations, especially theCD14highCD16low, when we used a pool of all of the animalsstudied. Upon further investigation, we realized that these cor-relations were mostly attributed to disease-specific clustering ofdata. Thus, correlations were sought separately in the unin-fected and infected cohorts (Table 3). In the uninfected con-trols, the CD4 and CD8 T cell counts were positively corre-lated with numbers of total monocytes and CD16� monocytes(CD14highCD16low and CD14lowCD16high). However, we foundsignificant negative correlations between the CD4/CD8 ratio andmonocyte subpopulations. Interestingly, CD8� central memory Tcells were negatively correlated with monocytes and monocytesubpopulations with strong Spearman correlation coefficients,suggesting a role of CD8� central memory T cells in the controlof the numbers of monocytes. We have found recently similarobservations in HIV-infected humans [37]. In infected animals,viral load is the only parameter amongst those tested in our studythat correlated with monocytes and monocyte subpopulations.Statistically significant, positive correlations were found betweenthe CD14highCD16low count and the viral load (r�0.3757,P�0.0085) or between the CD14lowCD16high count and the viralload (r�0.3781, P�0.0081), suggesting that virus or viral proteinscould play a role in controlling monocyte dynamics.

Functional heterogeneity of monocyte subpopulationsin SIV infectionThe phenotypic and transcriptional heterogeneity of monocytesubpopulations and their dynamics in SIV infection suggestthat there exist functionally distinct subsets. We examined theexpression of TNF and the replication of SIV in these subsets.

Differential ex vivo expression of TNF in monocyte subsets.CD14� monocytes/macrophages are the dominant source of

TNF-� in vivo, and it has been shown that the CD14lowCD16high

monocyte is a major source of TNF in response to LPS in vitro.To determine which subsets express this cytokine, intracellularcytokine staining was used to compare the phenotype and fre-quency of monocytes spontaneously producing (without invitro stimulation) TNF-� as well as granzyme B in monkeyswith subacute (n�5; 8 weeks p.i.) and chronic (n�8; 2.6 yearsp.i.) SIV infection and uninfected animals as controls (n�5).Monocytes from normal, uninfected controls expressed little-to-no TNF-� but produced granzyme B at low frequency.Monocytes from infected monkeys produced TNF-� spontane-ously, where expression of TNF was preferentially associatedwith CD14highCD16� and CD14highCD16low cells but not withthe CD14lowCD16high (Fig. 2). The percentage of TNF-express-ing cells in the CD14highCD16� population was 7.5–25 timesgreater than that in the CD14lowCD16high. The frequency ofmonocyte subsets spontaneously producing TNF-� was in-creased significantly in infected monkeys compared with unin-fected controls. The magnitude of the increase in frequencywas greater in chronically infected animals than in subacutelyinfected animals. These results support SIV infection-depen-dent, spontaneous production of TNF-�, which may reflect theimmune activation in infected monkeys. Granzyme B was ex-pressed exclusively in CD14lowCD16high cells, and the fre-quency of granzyme B-expressing cells in the CD14lowCD16high

population in infected animals was not significantly higherthan that in uninfected controls (infected, mean�sd,17.25%�13.38% vs. uninfected, mean�sd, 9.43%�7.43%). Itappears that granzyme B expression is not associated specifi-cally with SIV infection.

Differential susceptibility to SIV infection in monocyte sub-sets. Peripheral blood monocytes are a major reservoir forHIV-1 in infected individuals on virally suppressive HAART[38, 39]. We examined levels of SIV-DNA and -RNA associatedwith monocyte subpopulations in acutely infected animals (12days p.i., n�4; Fig. 3). Although levels of SIV DNA were com-parable in all monocyte subpopulations (data not shown;P�0.05), significantly higher levels of SIV RNA were found in

TABLE 3. Correlations Between Immunologic Parameters and Monocyte Subpopulations

Animal group Monocyte countCD14highCD16�

countCD14highCD16low

countCD14lowCD16high

count

Uninfected control (n�48)CD4 T cells 0.3667a (0.0132)b 0.3715 (0.0120) 0.4211 (0.0040)CD8 T cells 0.5256 (0.0002) 0.4026 (0.0061) 0.5495 (�0.0001) 0.5936 (�0.0001)CD4/CD8 ratio –0.3795 (0.0101) –0.3085 (0.0330) –0.3156 (0.0347) –0.3924 (0.0077)CD4 CM T cellsCD8 CM T cells –0.7199 (0.0001) –0.5994 (0.0025) –0.5617 (0.0053) –0.5874 (0.0032)

Infected (n�54)CD4 T cellsCD8 T cells –0.2781 (0.0417)CD4/CD8 ratioCD4 CM T cellsCD8 CM T cellsPlasma viral loadc 0.4089 (0.0039) 0.3757 (0.0085) 0.3781 (0.0081)

aThe correlation coefficients were calculated using the Spearman’s rank test. bP values are shown in parentheses. The nonsignificant correlationcoefficients are not presented. cn � 48. CM, Central memory.



CD14highCD16low cells (P�0.05, paired t-test). However, thelevel of viral replication in CD14highCD16low cells was not com-parable with that of CD4� T lymphocytes. SIV replication,which occurred preferentially in CD14highCD16low cells, likelyinvolves high levels of cell-specific factors in CD14highCD16low

cells such as SerpinB2 (Supplemental Table 2).

DISCUSSION

The present study was designed to identify monkey monocytesubpopulations by gene array and flow cytometry and to char-acterize the distribution of these subpopulations during SIVinfection. Our data demonstrate for the first time large-scalegene expression differences between CD16� monocytes(CD14highCD16�) and the two CD16� subpopulations(CD14highCD16low and CD14lowCD16high). Between the twoCD16� subpopulations, differential expression of relatively fewbut distinct genes was found (Supplemental Data Section).Expansion of these CD16� monocytes during SIV infectionwas associated with SIV disease stages and correlated to plasmaviral load, suggesting effects of virus infection on monocyteheterogeneity.

Immature monocytes are derived from CD34� myeloid pro-genitor cells and become mature in the bone marrow. Mono-cytic maturation takes place in sequential stages, which is con-trolled by sequential expression of a specific set of transcrip-tion factors including PU.1, acute myelogenous leukemia 1,

C/EBP�, IFN regulatory factor-8, and MafB [40–42]. The se-quential expression and combination of these transcriptionfactors lead to gradual loss of CD34 phenotype and sequentialacquisition of CD11a, CD33, CD11b, CD14, and CD16 pheno-types [43, 44]. Mature CD14� monocytes continuously leavebone marrow, enter the circulation, and marginate to the ves-sel wall. The monocytes that pass through the vessel wall be-come tissue macrophages.

This transit population, however, appears not to be homoge-neous. It has been suggested that bone marrow monocytes arekinetically heterogeneous in their transit time through bone mar-row [45, 46]. Blood monocytes are heterogeneous in terms ofphysical and functional properties. Two or more subsets of mono-cytes that differ in size, density, or granularity [47–49] have beenisolated, and they show differential peroxidase and lysozyme pro-duction, cytokine production, phagocytotic and cytotoxic activi-ties, and responses to GM-CSF [50–53]. Phenotypically, the heter-ogeneity of human blood monocytes can be demonstrated by thepresence of CD16� monocytes. As CD16 is expressed only on aminor subset of mature CD14high monocytes in bone marrow[44, 54], CD14� monocytes that express CD16 in bone marrowand peripheral blood represent a subpopulation at the more ma-ture differentiation stages. Our genome-wide transcriptional pro-filing demonstrates the transcriptional heterogeneity of monkeymonocytes and identifies CD16� monocytes as even more maturecell types than mature CD16� monocytes (CD14highCD16�). Ourstudy differentiated CD16� monocytes further into two subpopu-

0

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Figure 2. Differential expression ofTNF-� and granzyme B in mono-cyte subpopulations. (A) The ex-pression of TNF-� and granzyme Bin monocyte subpopulations. Intra-cellular expression of TNF-� andgranzyme B was assessed with intra-cellular cytokine staining, followedby flow cytometry. CD3�, CD4�, orCD8� cells were excluded from theCD14� monocyte population toensure no contaminating cells. Thefigure shown was the analysis ofone chronically infected animalwith a detectable viral load; repre-sentative of n � 8. (B) The expres-sion of TNF-� in peripheral bloodmonocyte subsets defined by CD14and CD16 was evaluated in healthy,uninfected monkeys (n�5), mon-keys with subacute SIV infection(n�5), and monkeys with chronicSIV infection (n�8). Results areexpressed as means � sd; signifi-cant at *, P � 0.05, and **, P �0.01 (one-way ANOVA followed byTukey test).


lations: CD14highCD16low and CD14lowCD16high. AlthoughCD14highCD16low cells appeared phenotypically intermediate be-tween CD14highCD16� and CD14lowCD16high in terms of expres-sion levels of CD11c, CD31, CD64, and CCR2, CD14highCD16low

cells do not seem to represent a transient subpopulation fromthe CD14highCD16� to the CD14lowCD16high. The highest levelsof CD1c, CD86, CX3CR1, HLA-DR, and TLR2 were expressed onCD14highCD16low cells. Our gene array data showed that thegene expression profile of the CD14highCD16low population issubstantially different from that of the CD14highCD16� andclosely shared with that of the CD14lowCD16high (SupplementalData Section). In addition, we showed that CD14highCD16low cellsare a functionally distinct subpopulation that preferentially takesup dextran polymers, produces TNF, and supports SIV replica-tion. Recently, it was shown that influenza virus infection andsevere asthma are associated with this particular subpopulation[55–57]. Our study also reports for the first time a “cytotoxic”phenotype (granzyme�lymphotoxinß�perforin�TRAIL�), whichspecifically defines CX3CR1highCD14lowCD16high cells. WhetherCD14highCD16low cells are the precursor of CD14lowCD16high

cells or whether CD14highCD16low and CD14lowCD16high cells aremacrophage-like cells with two different phenotypes remains tobe determined.

Monocytes are susceptible targets for HIV infection, andmacrophages, their tissue counterparts, constitute an impor-tant reservoir of virus, especially during the chronic phase ofinfection. A study of the relationship between monocyte differ-entiation and HIV replication demonstrated that restrictedHIV replication and no evident cytopathic effects in mono-cytes are important mechanisms of virus persistence [58]. In-deed, monocytes function as a major reservoir for persistingvirus during antiretroviral therapy [38]. In a cohort of 39 HIV-positive patients, increases in the number of HIV-positive

monocytes in the blood preceded the increase in the plasmaviral load, suggesting their contribution to viral load [59]. Onthe other hand, HIV infection appears to induce phenotypicand functional changes in monocytes. Myelomonoblastic cells,upon in vitro HIV infection, become a more mature, mono-cytic phenotype [60]. In HIV-infected patients, phenotypic andfunctional changes driven by HIV infection occurred in themonocyte population, which includes expansion of CD16�

monocytes [8, 13, 16, 61, 62]. It is important to note that onlya small fraction of the monocyte populations becomes infectedand that infection by HIV is not needed to develop such dys-functions. In vitro HIV gp120 stimulation is sufficient to in-duce dysfunctions and phenotypic changes in monocytes/mac-rophages [63–65]. HIV gp120 is known to induce IL-10 pro-duction in monocytes [66, 67] and induce the ability ofmonocytes to kill CD4� T cells [68], which may contribute toHIV-induced immune suppression in vivo. Notably, gp120 mim-ics the expansion of CD16� monocytes, which is observed inthe blood of HIV-infected patients, increasing CD16 expres-sion on cultured monocytes [64]. We suggest that HIV infec-tion induces differential monocyte maturation and kinetics,thereby affecting the heterogeneity of monocytes in the pe-ripheral blood.

Relevance of this heterogeneity in the pathogenesis of HIVis beginning to be understood. It has been shown recently thatin HIV-infected patients receiving HAART, CD16� monocytesare more susceptible to HIV infection and harbor preferen-tially HIV DNA [39]. A direct comparison between this humanstudy and our monkey study cannot be made, as Ellery et al.[39] measured HIV DNA detected in sorted CD16� monocytesincluding CD16low and CD16high subsets, whereas we exam-ined separately SIV RNA detected in CD16� monocytes, whichwere fractionated further into CD14highCD16low and CD14low

CD16high monocytes. We found that SIV RNA is detected in allthree monocyte subsets with a highest level in CD14high

CD16low monocytes. Our data in SIV-infected monkeys aresomewhat different than these human data. One reason forthis might be species differences, where SIV is detected readilyin monocytes, and HIV is less abundant in human monocytes[20, 21, 38, 39, 69, 70]. Another difference is that Ellery et al.[39] analyzed HIV DNA from patients on HAART, and we as-sessed SIV RNA from animals that were not on antiretroviraltherapy. The relationship between phenotypes and functionsof different monocyte subpopulations remains to be estab-lished. Interestingly, a recent mouse study by Auffray et al.[30] demonstrated CD11a�CX3CR1highGr1� monocytes(equivalent to human CD14lowCD16high monocytes) as a subsetthat crawls on normal endothelium of blood vessels, invadestissues rapidly upon tissue damage, and becomes tissue macro-phages. Whether such cells do so in the course of viral infec-tion has yet to be studied thoroughly.

We were interested in establishing the contribution of differ-ent monocyte subpopulations in HIV pathogenesis. In thisstudy, we have conducted a cross-sectional study of SIV- orSHIV-infected animals to investigate changes in the distribu-tion of monocyte subpopulations during infection. We reportthe positive correlations between plasma viral load and abso-lute numbers of total monocytes and the two CD16� subpopu-

-

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0

100000

200000

300000

400000

500000

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Figure 3. Differential levels of SIV replication in monocyte subpopula-tions, which with CD4� T cells, were FACS-sorted from the blood ofacutely infected animals (12 days p.i., n�4). Levels of SIV RNA associ-ated with each cell type were measured by quantitative RT-PCR; signifi-cant at *, P � 0.05, and **, P � 0.01 (paired t-tests).



lations. Expansion of CD16� monocytes was evident in theacute and chronic phase of infection with high plasma viralload, suggesting a biphasic increase (first phase early after pri-mary infection and second phase prior to development ofAIDS) in the absolute number of CD16� monocytes. Expan-sion or a depletion of CD16� monocytes was seen in cases ofterminal AIDS. This may signify impaired maturation at thelate chronic state of the disease. Collectively, our observationssuggest that monocyte maturation and/or activation are re-lated to plasma viral load.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grantsNS040237 (K. W.), NS037654 (K. W.), NS0500041 (K. W.),and MH81835 (K. W. and M. S. M.).

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KEY WORDS:subsets � macrophage � CD16 � HIV � AIDS



Monocyte heterogeneity underlying phenotypic changes in monocytes according to SIV disease stage

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