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Original Research Effects of Simian Betaretrovirus Serotype 1 (SRV1) Infection on the Differentiation of Hematopoietic Progenitor Cells (CD34+) Derived from Bone Marrow of Rhesus Macaques (Macaca mulatta)

Nestor A Montiel,1,2,* Patricia A Todd,2 JoAnn Yee,2 and Nicholas W Lerche2

Peripheral blood cytopenias, particularly persistent anemia and neutropenia, are commonly associated with simian betaretrovirus infection of Asian monkeys of the genus Macaca. The pathogenetic mechanisms underlying these hematologic abnormalities are not well understood. The current study investigated the in vitro tropism of simian betaretrovirus (SRV) for both hematopoietic progenitor (CD34+) and stromal cells obtained from rhesus macaque bone marrow and assessed the effects of infection on hematopoietic progenitor cell differentiation in vitro. After in vitro exposure, SRV proviral DNA could be demonstrated by real-time PCR in cells and the reverse transcriptase assay in supernatants from SRV-exposed progenitor-associated stroma, but not in differentiated colonies derived from SRV-exposed progenitors. Furthermore, in vitro exposure involving cell–cell contact of uninfected CD34+ progenitor cells with SRV-infected stromal cells resulted in a statistically significant reduction in granulocyte–macrophage colony formation in absence of detectable SRV-infection of progenitor cells. Reduction in colony formation occurred in a ‘dose-dependent’ fashion with increasing contact time. No effects on erythroid lineages and RBC differentiation were noted. Our results suggest that hematologic abnormalities observed during SRV disease (natural or experimental) of rhesus macaques may not result from direct effects of viral infection of progenitor cell populations, but rather be (at least in part) a consequence of SRV infection of supportive bone marrow stroma with secondary effects on differentiation of associated progenitor cells.

Abbreviations: E, erythroid; GEMM, granulocytic–erythroid–monocytic–megakaryocytic; GM, granulocytic–macrophage; rh, recombinant human; SRV1, simian type D retrovirus 1.

Simian betaretrovirus (formerly simian type D retrovirus; SRV) naturally acquired SRV infection of macaques20,37 and are general- comprises a group of closely related exogenous retroviruses for ly more pronounced in animals with active viremia than in persis- which the natural hosts are Asian monkeys of the genus Maca- tently infected but nonviremic animals.19 Bone marrow changes ca. SRV is now classified in the genus Betaretrovirus subfamily accompanying peripheral cytopenias have been described as Orthoretrovirinae and is the etiologic agent of an immunosup- hypercellular in early stages becoming hypocellular only in the pressive syndrome in various species of macaques used in bio- late stages of SRV infection.37 The specific mechanisms respon- medical research.9,15,27,36,38,39,42 SRV exhibits a broad cellular tropism sible for the origin SRV-associated hematologic abnormalities are for cells of both lymphoid and nonlymphoid tissues.40 Clinical not known, and relevant studies of this phenomenon are few. By and pathologic manifestations of SRV infection range from sub- analogy with other viral infection models exhibiting hematologic clinical (carrier state) to fatal immunosuppressive disease.20,30 SRV abnormalities, a variety of underlying pathogenetic mechanisms is an important pathogen in macaques maintained for use in bio- acting at the bone marrow level may contribute to peripheral cy- medical research, and undetected infections represent a potential topenias, including virus-induced dysregulation of cytokine or confounding variable in research protocols. As a result, SRV is chemokine production, production of soluble factors that inhibit one of several persistent viruses targeted for elimination in SPF normal hematopoiesis, direct viral infection of hematopoietic colony development.28,31 progenitor cells that leads to altered function and metabolism, Hematologic abnormalities, particularly anemia and neutro- and infection and alteration of cells comprising the bone mar- penia, are common laboratory findings in both experimental and row microenvironment that could indirectly impair the ability of progenitors to differentiate into lineage-committed cells.34,35,46,54 Anemia and leukopenias associated with reduced progenitor Received: 16 May 2011. Revision requested: 28 Jun 2011. Accepted: 07 Sep 2011. cell proliferation (by either direct susceptibility of progenitors to 1Center for Comparative Medicine and 2California National Primate Research Center, University of California, Davis, California. infection or indirect effects of the infected microenvironment) and *Corresponding author. Email: [email protected] impaired iron utilization have been observed in several diseases 61 Vol 62, No 1 Comparative Medicine February 2012

of viral origin, including simian parvovirus of macaques,44 hu- Suspension cultures of hematopoietic progenitor cells were man parvovirus B19 infection,6,43 simian and human immunode- maintained for either 7 or 20 d at 3 × 105 cells/mL in 16-mm wells ficiency syndromes,12,26,46,51,54 and feline retroviral infections.23,35,47,53 in either 10% FBS in Iscove-modified Dulbecco medium (Gibco) Limited studies of SRV-associated cytopenias have suggested that or in serum-free expansion medium (StemSpan SFEM, StemCell adverse hematologic effects of SRV infection may originate at the Technologies) containing recombinant human (rh) cytokines IL3 bone marrow level.37,42 The objectives of the current study were to (20 ng/mL), IL6 (20 ng/mL), SCF (100 ng/mL), and Flt3 ligand determine 1) the in vitro tropism of SRV1 for both CD34+ hema- (100 ng/mL; StemCell Technologies). Long-term stromal cell cul- topoietic progenitors and supportive stroma cell components of tures were maintained for 4 to 8 wk in either 35- or 16-mm wells rhesus macaque bone marrow and 2) the effects of SRV infection in 20% FBS in DMEM (Gibco) containing 10-6 M hydrocortisone of either or both marrow constituent cell populations on in vitro (StemCell Technologies). Primary stromal cells were obtained differentiation of erythrocytic and granulocytic precursor cells. from the CD34+ retention column wash fractions (CD34–). Stromal cells were counted, seeded onto 9.5- or 1.91-mm2 wells (35- and Materials and Methods 16-mm dishes, respectively) in 20% FBS in DMEM (Gibco) containing 10-6 M hydrocortisone (StemCell Technologies) at initial Animals and bone marrow collection. Twelve healthy adult concentrations of 5 × 105 or 3 × 105 cells/mL, and incubated at 37 rhesus macaques (Macaca mulatta) maintained as blood donors °C in humidified 5% CO until layers were completely confluent. and representative of the normal adult rhesus population at our 2 Stromal cell cultures were maintained for a maximum of 12 wk. institution (California National Primate Research Center, Univer- Infection of progenitor and stromal cells with SRV1. Infection of sity of California, Davis, California) were used as bone marrow rhesus bone marrow CD34+ hematopoietic progenitor cells with donors for these experiments. One rhesus macaque (MMU 22396) chronically infected with SRV1 (confirmed by PCR and serology) SRV1 was performed by exposure of progenitor cells to infec- but without clinical signs of SRV disease served as a donor of tious supernatant obtained from selected SRV1-infected Raji cell bone marrow cells and was used as a positive control for some cultures presenting high reverse transcriptase activity (greater assays during the study. All donors (except MMU 22396) were than 20-fold background). Reverse transcriptase activity was de- determined to be free of SRV infection by serology and RT-PCR. termined by using a commercially available detection kit (Cavidi Our facility is fully AAALAC-accredited, and all animals were Tech AB, Uppsala, Sweden). The multiplicity of infection used housed and handled in accordance with the Animal Welfare ranged from 0.1 to 1. After 1- or 4-h incubation at 37 °C, with Act and the Guide for the Care and Use of Laboratory Animals.24 All equal volumes of infected or uninfected cell supernatant, cells procedures associated with this research were approved by the were resuspended in PBS (Gibco) and seeded onto culture dish- IACUC of the University of California, Davis. Macaques were es in semisolid medium to assay progenitor cell differentiation 5 anesthetized with ketamine HCl (10 mg/kg IM; Vetamine, Scher- as previously described. Semiconfluent stromal cultures were ing-Plough, Kenilworth, NJ). The bone marrow extraction site incubated twice (with a 24-h interval between exposures) with was prepared aseptically, and 0.5 mL lidocaine (Abbott Laborato- SRV-infected cell-free supernatant for 4 h at 37 °C each time, at ries, Alameda, CA) was administered subcutaneously. Bone mar- an estimated multiplicity of infection of 0.5. After the second in- row (5 to 10 mL) was aspirated from either the humerus or iliac cubation, cells were washed twice in PBS (Gibco) and then resus- −6 crest of each animal by using a heparin-coated (0.1 mL) syringe pended in 10% FBS in DMEM containing 10 M hydrocortisone with a 20-gauge, 1.5-in. spinal needle (Becton Dickinson, San (StemCell Technologies) for long-term maintenance. Jose, CA). A 50-µL aliquot of each sample was used to determine Progenitor cell differentiation with recombinant cytokines. the nucleated cell count and to estimate the extent of peripheral CD34+ progenitor cell differentiation into colonies was assessed blood contamination. quantitatively in vitro by colony-forming unit assay.5 The analysis Purification of CD34+ progenitor cells and establishment of sus- included the quantitation of granulocytic–erythroid–monocytic– pension and stromal cell cultures. Each bone marrow sample was megakaryocytic (CFU-GEMM), granulocytic–macrophage (CFU- filtered by using a 40-µm strainer (BD Falcon, San Jose, CA) and GM), and erythroid (CFU-E) colonies in semisolid medium. After diluted in 3 volumes of PBS (pH 7.2, Gibco Invitrogen, Grand inoculation with SRV-infected cell supernatants for 1 or 4 h, 300 Island, NY). Bone marrow mononuclear cells were separated by µL of each cell suspension was added to 3 mL of methylcellulose Ficoll–Hypaque gradient centrifugation (density 1.077 g/mL; basic medium (MethoCult SF, StemCell Technologies) containing ICN Biomedicals, Costa Mesa, CA). CD34+ cells were isolated the following cytokines and growth factors (R and D Systems, by using a magnetic cell-sorting system (MiniMACS, Miltenyi Minneapolis, MN): FBS (100 µL/mL), rhSCF (200 ng/mL), rhIL3 Biotec, Auburn, CA) and a StemSep separation system (StemCell (50 ng/mL), rhIL6 (50 ng/mL), rhG-CSF (50 ng/mL), rhGM-CSF Technologies, Vancouver, BC, Canada) in accordance with the (50 ng/mL), and erythropoietin (4 U/mL). Increasing concentra- manufacturers’ recommendations. The purity of cell fractions tions of progenitor cells (500, 1000, and 2000 cells per dish) were was evaluated throughout the selection process by specific immu- assayed in duplicate. At days 10 and 12, dishes were examined nolabeling. Stained cells were fixed by using Q-Prep (Beckman by light microscopy by using an inverted microscope and the Coulter, Fullerton, CA) and later analyzed by flow cytometry number and type of colonies were determined. Colonies of more (FACSCalibur system, Becton Dickinson) and the FlowJo soft- than 50 hemoglobinized cells with defined borders were scored ware platform (Tree Star, Ashland, OR). Antibody incubations as CFU-E; colonies of more than 50 colorless cells with poorly were performed at 4 °C for 30 min. Gating of forward and side defined and diffuse borders were scored as CFU-GM; colonies of scatter excluded blood cells, granulocytes, and monocytes.41 Con- more than 200 mixed cells with a red hue in the center and a dif- trol procedures included omission of the primary antibody and fuse border were scored as CFU-GEMM.11 substitution of a heavy-chain-matched monoclonal antibody of Effects of stromal cell supernatants or stromal cell contact on irrelevant specificity (isotype control). progenitor cell differentiation. To assess the ability of SRV-infected 62 Simian betaretrovirus infection of macaque bone marrow cells

and uninfected stromal cell supernatants to support differentiation to the manufacturer’s recommendations. Protein concentration of CD34+ progenitor cells in the absence of recombinant cytokines was measured in representative subsets of cell supernatants (n = 4) and growth factors, progenitor cells were cultured in semisolid by using the BCA assay.48 media in the presence of SRV-infected or noninfected stromal cell Statistical analysis. Data were analyzed and graphs generated supernatants. Briefly, 500, 1000, and 2000 CD34+ progenitor cells by using Excel 2007 (Microsoft, Redmond, WA). Differentiated were resuspended in 300 µL of either infected or noninfected colony counts were expressed as the mean ± SEM of independent stromal cell supernatant and added to 3 mL of methylcellulose assays performed in duplicate. Counts of CFU-E, CFU-GM, and basic medium (Methocult SF StemCell Technologies). Semisolid CFU-GEMM colonies were compared between SRV-exposed and cultures were performed in 1.1-mL duplicates after adjusting cell mock-exposed cultures by using paired t tests. A P value less than numbers per volume and the remaining media-containing cells 0.05 was considered statistically significant. discarded. After 12 d, culture dishes were examined and evaluated for colony formation by light microscopy as described ear- Results lier. Similarly, to assess the ability of primary stromal cell cultures In vitro tropism of SRV for bone marrow constituents. To in- to support hematopoiesis, SRV-infected and uninfected stromal vestigate the in vitro tropism of SRV for bone marrow cell con- cell layers were reconstituted by adding 2000 CD34+ progenitor stituents, CD34+ progenitor cells and stromal cell subsets were cells extracted from SRV-free rhesus macaque bone marrow as inoculated with SRV1-infected Raji cell supernatants. As expect- described earlier. Positive control experiments were performed on ed, proviral DNA (residual DNA from infectious supernatants) methylcellulose complete media in the presence of recombinant was detected in SRV viral stocks and in CD34+ progenitors cell cytokines (Methocult GF+, StemCell Technologies). A chronically aliquots exposed to SRV at 1 and 4 h after inoculation. Similarly, infected SRV-positive rhesus macaque (MMU 22396) was used as reverse transcriptase activity ranged from 5 to more than 20-fold a bone marrow donor for positive control assays. Negative control background levels in progenitor cell aliquots at 1 and 4 h after experiments were performed in the absence of preformed stroma inoculation. However, samples of progenitor cells cultures super- to assess both survival and clonogenic potential of progenitor cells natants obtained at 3, 5, and 7 d after inoculation did not show in methylcellulose basic medium without recombinant cytokines. consistent presence of proviral DNA, and no reverse transcriptase + Stromal cultures reconstituted with CD34 progenitors were main- activity was detectable. When colonies (pooled or tested individ- tained at 37 °C in humidified 5% CO for 24 to 72 h. Stromal cul- 2 ually) originating from SRV-exposed progenitors were analyzed ture-derived supernatants containing hematopoietic progenitor by real-time RT-PCR after 18 d in culture, no proviral DNA could cells were assayed for colony formation as described earlier. be detected. Stromal cell cultures examined at 7, 14, 21, and 28 d Detection of SRV provirus and reverse transcriptase activity in after inoculation contained moderate levels of proviral DNA (Ct cultured cells. Real-time RT-PCR was used to determine presence = 33.1 to 39) and demonstrated significantly elevated RT activity + of SRV1 proviral DNA in differentiated progenitor (CD34 ) and (more than 20-fold, P < 0.05; Table 1). However, no evidence of stromal cells. DNA from suspension and semisolid progenitor cell SRV infection could be demonstrated in CD34+ progenitor cells in cultures was extracted and processed for PCR analysis by using contact with SRV-infected stromal cells for as long as 14 d (data the Gentra Puregene DNA Purification kit (Qiagen, Valencia, CA) not shown). These results suggest that CD34+ progenitor cells do according to the manufacturer’s recommendations. The major- not support SRV infection after exposure to cell-free virus or after ity (> 90%) of the colonies from both SRV-exposed and mock-ex- direct contact with SRV-infected stromal cells. Our results confirm posed control plates were collected individually, pooled by type, the in vitro tropism of SRV1 for marrow-derived stromal cells. and processed. Infection of cultured stromal cells was confirmed Effects of SRV on CD34+ progenitor cell differentiation. To de- 12 to 14 d after exposure by real-time RT-PCR and the reverse termine the effects of SRV infection on the clonogenic potential transcriptase activity assay in culture supernatants. The reverse of CD34+ hematopoietic progenitor cells, SRV-exposed and unex- transcriptase activity assay, a confirmatory test for infectivity, posed aliquots of enriched CD34+ cells were assayed for colony was performed in culture supernatants by using a commercially formation in cytokine-enriched semisolid medium. The mean available kit (Cavidi Tech AB) according to the manufacturer’s percentage colony formation (total, CFU-GM, and CFU-E) for recommendations. All samples were maintained at −70 °C before cultures exposed to SRV for 1 and 4 h are shown in Figure 1. No analysis. Samples were read by using an ELISA plate reader with statistically significant differences were observed in the clono- an A405 filter. Control samples included SRV1-infected and unin- genic potential of lineage-committed progenitors (CFU-GM and fected Raji cells propagated in tissue culture. CFU-E) between paired SRV- exposed and unexposed cultures. Detection of soluble factors in stromal cell culture supernatants. These results indicate that in vitro exposure to cell-free SRV has The supportive role of stromal cells in the differentiation of he- no apparent detrimental effects on the ability of bone marrow matopoietic precursor cells is thought to be due in part to the CD34+ progenitor cells to differentiate into colonies. These find- elaboration by stromal cells of soluble factors, including various ings are consistent with our observation that CD34+ progenitors cytokines and growth factors.32,50 To assess the presence of soluble appear resistant to direct infection with SRV in vitro. factors, Th1 (INFγ, TNFα, and IL2) and Th2 (IL4, IL10, and IL6) Effects of SRV on marrow-derived stromal cells. In stromal cytokine levels and growth factor (G-CSF, MIP1α, MIP1β, and cell cultures exposed to cell-free SRV, cytopathogenic effect was TGFβ1) levels were measured in stromal cell culture superna- clearly visible in semiconfluent cultures at 7 d after inoculation, tants obtained throughout the study (n = 11) by using quantitative represented by a general unhealthy appearance, massive detach- multiplex bead reagents (Luminex, Invitrogen, Carlsbad, CA)14 ac- ment from the plate surfaces, cytoplasmic vacuolization, and cording to the manufacturer’s recommendations. GM-CSF was de- presence of scattered multinucleated cells (Figure 2 A). Healthy tected and measured by using a monkey-specific sandwich ELISA uninfected stromal cell cultures are shown in Figure 2 B. Superna- kit (U-CyTech Biosciences, Utrecht, The Netherlands) according tants from SRV-infected and uninfected stromal cell cultures were 63 Vol 62, No 1 Comparative Medicine February 2012

Table 1. Intensity of SRV1 proviral DNA and reverse transcriptase (RT) signals in various cultures of hematopoietic progenitor cells and stimulated progenitor-derived colonies Proviral DNAa RT activityb Viral stocks 18–25 >20 1-h exposed progenitors 18–25 5–20 4-h exposed progenitors 18–25 5–20 3-d progenitor cell cultures negative negative 5-d progenitor cell cultures negative negative 7-d progenitor cell cultures negative negative 18-d pooled colonies negative not done 18-d individual colonies negative not done 18-d intercolony fluid 39.1–45 not done Stromal cell cultures 33.1–39 >20 (7, 14, 21, and 28 dpi) aProviral DNA signal intensity is relative to cycle threshold (Ct) values obtained by real-time PCR and compared with SRV plasmid standards. Strong signal, 18 to 25; intermediate, 25.1 to 32; weak, 33.1 to 39; undetermined, 39.1–45. Ct values exceeding 45 are considered negative. bRT activity in supernatants is measured as optical density (OD) relative to background of negative samples and reported as fold activity above background.

Figure 2. Cytopathogenic effect in SRV-infected bone marrow stromal cells in culture. Images are representative of 7-d cell cultures of (A) SRV- Figure 1. Effects of 1- and 4-h SRV1 incubation on colony formation of infected and (B) healthy (uninfected) stromal cells. A general unhealthy bone marrow CD34-derived progenitor cells. White columns represent appearance, with cellular debris and dead cells (arrows), and dying cells percentage total colony formation, gray columns correspond to percent- that are detaching from the tissue culture plates (arrow heads) can be age CFU-GM, and black columns represent percent CFU-E. Frequencies seen in panel A. In contrast, a healthy semiconfluent culture of fibrob- are calculated as the ratio of number of colonies scored to progenitor cell last-like stromal cells is depicted in panel B. Images were produced by concentration. There were no statistically significant (P > 0.05) differenc- phase-contrast microscopy; magnification, 40×. es between paired groups. Assays were performed using bone marrow samples from 6 different experimental subjects, with each experiment (13.12 ± 1.16 pg/mL) were significantly (P = 0.03) lower than corresponding to a single experimental subject. For 1-h incubation as- those of control cultures (19.42 ± 3.04 pg/mL; Table 2). says, 6 different animals were used as donors. For 4-h incubation assays, Effect of SRV-infected stromal cell supernatants on CD34+ 3 of the same 6 donors were chosen randomly and used. Results are expressed as the mean ± SEM of independent experiments performed progenitor differentiation. To evaluate the effects of stromal cell in duplicate. supernatants on in vitro colony formation, SRV-infected and uninfected stromal cell supernatants were collected and used as the only source of cytokines and growth factors in the semisolid analyzed for the presence of soluble factors, including Th1 (INFγ, colony-forming assay. The percentage GM colony formation for TNFα, and IL2) and Th2 cytokines (IL4, IL10, and IL6), and solu- SRV-infected cultures and uninfected controls were 0.99% ± 0.18% ble growth factors (GM-CSF, MIP1α, MIP1β, and TGFβ1). All and 1.36% ± 0.17%, respectively (Figure 3 A). GM-CSF levels in cytokines and growth factors were below the level of detection supernatants used for colony-forming assays (Figure 3 B) dem- with the exception of IL6, GM-CSF, and TGFβ1. No statistically onstrated that supernatants from both SRV-infected (13.12 ± 1.16 significant differences between infected and uninfected stromal pg/mL) and uninfected (19.42 ± 3.04 pg/mL) stromal cell cultures cell cultures were apparent for TGFβ1 and IL6; however, concen- are capable of supporting differentiation and proliferation of he- trations of GM-CSF in SRV1-infected stromal cell supernatants matopoietic progenitor cells in vitro. Although GM-CSF levels 64 Simian betaretrovirus infection of macaque bone marrow cells

Table 2. Detection of soluble factors (pg/mL; mean ± SEM) in superna- factors, but this decrease is insufficient to impair progenitor cell tants from SRV-infected stromal cell cultures differentiation in vitro. SRV Control P Effect of SRV-infected stromal cell contact on CD34+ progenitor GM-CSF 13.12 ± 1.16 19.42 ± 3.04 0.03 differentiation. Progenitor cells reconstituted with SRV-infected stroma were monitored at increasing cell–cell contact time for IL6 2092.12 ± 237.41 22314.84 ± 267.40 0.61 their clonogenic potential by using the colony-forming assay. TGFβ1 0.37 ± 0.02 0.42 ± 0.03 0.08 Statistically significant (P < 0.05) decreases in both percentage 200,000 cells per 1 mL of medium were incubated undisturbed in tissue total and GM-specific colony formation were found in cultures culture wells at 37 ºC, 5% CO , 95% humidity until the total supernatant + 2 of CD34 progenitor cells that were in contact with SRV-infected was collected for assays. stromal cells. This effect was not observed until after 24 h of cell– aGM-CSF, n = 11 (in triplicate); IL6 and TGFβ1, n = 33 to 36 cell contact. In particular, the percentage total colony formation for 72 h cell–cell contact (Figure 4 A) was significantly (P = 0.009) reduced in SRV-infected stromal cell cultures (0.31% ± 0.018%) compared with uninfected controls (0.42% ± 0.038%). Percentage CFU-E formation did not differ between progenitor cultures exposed to SRV-infected stroma (0.008% ± 0.0035%) compared with uninfected controls (0.0056% ± 0.0037%). For GM-committed progenitors, however, significant P( = 0.006) differences in percentage colony formation occurred between SRV-infected stromal cell cultures (0.30% ± 0.017%) and uninfected stromal cell cultures (0.41% ± 0.037%). Individual colonies analyzed for the presence of SRV proviral DNA were consistently negative by real-time RT-PCR. In addition to this finding, a ‘dose–response’ type effect was noted with increasing contact time associated with greater decreases in GM-specific colony formation when stroma from an SRV ex- perimentally infected animal (positive control) was reconstituted with healthy progenitors. At 24-, 48-, and 72-h contact, the percentage colony formation was 0.38% ± 0.071%, 0.31% ± 0.060%, and 0.19% ± 0.070%, respectively (Figure 4 B). The reduction in percentage colony formation at 72-h contact was significantly P( = 0.02) different from that at 24- or 48-h contact. These data demonstrate that direct contact of progenitor cells with SRV-infected stroma is sufficient to cause significant reductions in cell proliferation and differentiation.

Discussion The results of the current study indicate that 1) CD34+ hematopoietic progenitor cells appear resistant to infection by exposure to cell-free SRV and by direct contact with SRV-infected stromal cells, 2) bone marrow stromal cells are susceptible to SRV infection after exposure to cell-free virus in vitro, and 3) exposure of healthy CD34+ progenitor cells to SRV-infected stromal cells can significantly decrease colony formation in the absence of apparent progenitor infection resulting from cell–cell contact. Anemia and neutropenia are common features of SRV-associated disease. Although ample reports describe the clinical and pathologic manifestations of SRV infection in macaques8,21,22,27,37,39,40,42 few studies have attempted to address the pathogenic mecha- Figure 3. Effects of SRV-infected stromal cell supernatants on the production of CFU-GM. (A) Percentage colony formation in progenitor nisms underlying SRV-associated hematologic disorders. Most of cell cultures stimulated with supernatants from SRV-infected or unin- the mechanisms postulated to explain the origin of these abnor- fected stromal monolayers. (B) Concentration of GM-CSF (pg/mL) in malities are indirect and based on analogy with other viral mod- supernatants of SRV-infected and uninfected stromal cell cultures. Four els, such as HIV in humans,3,4,46 SIV in primates,51 MAIDS viral macaques were used as bone marrow donors, and assays were per- complex in mice,13 and FeLV in cats.10,34,45 SRV infection of cellular formed in triplicate. constituents of macaque bone marrow has been demonstrated by Mg+2-dependent reverse transcriptase activity in bone marrow in supernatants were significantly P( = 0.03) different between cultures and by electron microscopy15 and has been associated SRV-infected cultures and uninfected controls, colony formation with hyperplastic marrow in early SRV infection and hypoplastic did not differ (P = 0.19) between SRV-infected and uninfected marrow in late-stage infection.29,37 In our current cell-free viral controls. These results suggest that infection of marrow stromal exposure experiments, we did not find significant differences in cells with SRV reduces their ability to elaborate necessary soluble percentage colony formation in progenitor cell cultures exposed 65 Vol 62, No 1 Comparative Medicine February 2012

seropositive, nonviremic animals.19 Viremic animals would be expected to have higher virus loads than would SRV-infected nonviremic animals; consequently it is then possible that elevated virus levels in bone marrow are required to elicit more pronounced effects. Further, in vitro experiments with feline leukemia virus subgroup C (FeLV-C), a subgroup associated with profound anemia in infected cats, have shown that cell–cell contact of virus- infected marrow-derived fibroblasts with marrow target cells was required for impairment of erythroid burst-forming units, and this effect is thought to be associated with high virus titers pro- vided by cell-cell contact with infected fibroblasts.10 The bone marrow compartment contains a heterogeneous population of accessory cells (bone-marrow–associated stroma or stromal cells) that provide necessary regulatory factors for the differentiation and maintenance of hematopoietic stem cells.2,50 Stromal cell culture supernatants have been used as a source of growth factors necessary to support hematopoietic progenitor cells differentiation in vitro.17,18 Thus, similar to several retroviral models in which hematologic abnormalities can be induced by direct injury or by dysregulation of cytokines and growth factor levels after viral infection of bone marrow accessory cells,7,10,13,33,34,46 SRV-infected stromal cells may play a critical role by not supporting hematopoiesis due to lack of adequate production levels of soluble factors or by propagating SRV infection in the bone marrow microenvironment. In this regard, we have preliminary data that demonstrate altered cytokine mRNA levels in bone marrow progenitor and stromal cells from asymptomatic cynomolo- gus monkeys naturally infected with SRV2.42 The current results show that rhesus macaque marrow stromal cells are susceptible Figure 4. Effects of stromal–progenitor cell contact on percentage colony to SRV infection in vitro. We also demonstrated that direct contact formation in vitro. (A) Columns represent percentage colony formation of progenitor cells with infected stroma (72 h) was sufficient to of lineage-specific, differentiated progenitor cells (white, overall; gray, alter differentiation of lineage-committed progenitors into GM CFU-GM; black, CFU-E) after 72 h in contact with either SRV-infected lineages. Consequently, SRV-infected stromal cells may have, di- stroma or uninfected stroma. (B) Percentage colony formation of line- rectly or indirectly, altered progenitor cells homeostasis, leading age-specific progenitor cells after 24, 48, and 72 h of contact with stromal cells from an SRV-infected animal (positive control; n = 3). to abnormal production of more mature cell lineages, or shifted the normal cytokine milieu indirectly, disrupting the mechanisms of hematopoietic progenitor cell differentiation. to SRV when compared with controls. Furthermore, viral DNA Our data show limited or absence of deleterious effects of SRV was not present in individual colonies after 18 d in culture, thus on erythroid lineages (CFU-E). This observation is not surprising, minimizing the possibility of lack of detection due to insufficient given that supplementation with erythropoietin is necessary to time in culture. These findings suggest that hematopoietic pro- induce differentiation of differentiated progenitors into erythroid genitor cells are not effective targets for in vitro infection by SRV. colonies.1 Erythropoietin, a hormone that is produced primarily Our apparent inability to infect CD34+ progenitor cells in vitro by the peritubular capillary endothelial cells in the renal cortex exposed, either directly to cell-free virus or indirectly by cell–cell of kidneys and secondarily in the liver, is necessary to regulate contact with SRV-infected stromal cells, was somewhat unex- RBC production in the bone marrow compartment.25,52 In conse- pected. The cell receptor for betaretroviruses, designated RDR, quence, the in vitro erythroid colony-forming assay is completely has been detected by direct immunohistochemical and flow cyto- dependent on exogenous erythropoietin, a soluble factor that we metric analyses on a range of normal human tissues and cells, in- did not include in our stromal cell-contact assays. Therefore, we cluding hematopoietic cell subsets expressing the CD34 antigen.16 attribute the apparent absence of negative effects on erythroid However, the presence or absence of this receptor on marrow- colony formation to the lack of cytokine supplementation in our derived CD34+ progenitor cells of rhesus macaques or other non- stromal contact assays rather than to true effects of SRV on cell human primates has, to date, not been demonstrated. In addition, differentiation. Tissue culture supplementation with appropriate CD34+ progenitors may be naturally resistant to infection with levels of erythropoietin and exposure of marrow-derived con- SRV due to their quiescence or slow rate of division.49 All in vitro stituent cells to higher titers of SRV in future in vitro experiments experiments in the current study were performed at relatively may yield more profound differences on CFU-E differentiation low virus doses (that is, low multiplicity of infection), and this between SRV-exposed and unexposed groups. practice might have accounted for the lack of more pronounced The results of our study suggest that bone marrow CD34+ deleterious effects (for example, reduction in colony formation) in progenitor cells from rhesus macaques are not primary targets SRV-infected cultures. Some authors have reported that anemia for SRV infection in vitro and that deleterious effects of SRV on and neutropenia are more severe in viremic macaques than in the hematopoietic pathway originating at the bone marrow level 66 Simian betaretrovirus infection of macaque bone marrow cells

may be due, at least in part, to direct viral infection of progenitor- 14. Giavedoni LD. 2005. Simultaneous detection of multiple cytokines associated stromal cells. We clearly demonstrated significant re- and chemokines from nonhuman primates using luminex technol- ductions in GM-committed differentiation of CD34+ progenitor ogy. J Immunol Methods 301:89–101. cells that had been in contact with SRV-infected stroma. However, 15. Gravell M, London WT, Hamilton RS, Sever JL, Kapikian AZ, + Murti G, Arthur LO, Gilden RV, Osborn KG, Marx PA, Henrickson in the absence of SRV infection of CD34 progenitors through RV, Gardner MB. 1984. Transmission of simian AIDS with type D cell–cell contact and in view of the demonstrated ability of SRV- retrovirus isolate. Lancet 1:334–335. infected stromal culture supernatants to support progenitor cell 16. Green BJ, Lee CS, Rasko JE. 2004. Biodistribution of the RD114/ differentiation, our understanding of the pathogenetic mecha- mammalian type D retrovirus receptor, RDR. J Gene Med 6:249– nisms underlying various peripheral cytopenias observed during 259. SRV infection of macaques remains incomplete. 17. Gupta P, Blazar BR, Gupta K, Verfaillie CM. 1998. Human CD34(+) bone marrow cells regulate stromal production of interleukin 6 and granulocyte colony-stimulating factor and increase the colony- Acknowledgments stimulating activity of stroma. Blood 91:3724–3733. We thank Abigail Spinner for her helpful assistance and guidance 18. Gupta P, Oegema TR Jr, Brazil JJ, Dudek AZ, Slungaard A, Verfaillie with flow cytometry and various other technical recommendations. We CM. 2000. Human LTC-IC can be maintained for at least 5 weeks also thank Drs Linda Lowenstine and Peter Barry for critically reading in vitro when interleukin 3 and a single chemokine are combined the manuscript. Ann Rosenthal and the Pathogen Detection Laboratory with O-sulfated heparan sulfates: requirement for optimal binding staff at the California National Primate Research Center are ac interactions of heparan sulfate with early-acting cytokines and matrix knowledged for excellent technical assistance and helpful discussions. proteins. Blood 95:147–155. Dr Stephen Barthold at the Center of Comparative Medicine, UC Davis, 19. Guzman RE, Kerlin RL, Zimmerman TE. 1999. Histologic lesions is especially acknowledged as principal investigator for the Animal in cynomolgus monkeys (Macaca fascicularis) naturally infected with Models of Infectious Diseases Training Program. This study was funded simian retrovirus type D: comparison of seropositive, virus-positive, in part by grants RR00169 and T-32 RR07038 from the National Center and uninfected animals. Toxicol Pathol 27:672–677. for Research Resources (NCRR), NIH. 20. 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