HIV-1 infection can interfere with the hematopoiesis and thus result in the onset of different types of cytopenia, which are more often described in the late stages of the disease [1–3]. Thrombocytopenia is the most common of the HIV-associated cytopenias, accounting for about 30% of cases . Possible pathogenical mechanisms include the immune-mediated destruction of platelets occurring in the periphery [5–10], HIV-1 infection of megakaryocytes and/or bone marrow stromal cells with reduced platelet production [11–13], and indirect inhibition of progenitor cell activity by HIV-1 [14,15]. Unlikely anemia or leukopenia, thrombocytopenia can occur early in the course of HIV disease  as the only hematological finding in patients not showing the bone marrow abnormalities described in the advanced stages of infection [17,18]. It is still unclear whether the HIV-associated megakaryocytopoietic dysfunction is present before the onset of overt platelet reduction. In addition, further investigation is needed to understand the mechanisms underlying the impact of HIV replication on megakaryocytopoiesis and the effects of antiretroviral therapy (ART) suppression of viral replication on development of megakaryocytopoietic colony-forming units (CFU-MK).
The present study assessed whether the impairment in megakaryocytopoiesis precedes the onset of peripheral thrombocytopenia and examined the relationship between HIV-1 viral load levels, in-vitro development of CFU-MK and platelet counts. It also examined whether the administration of ART is associated with better megakaryocytic lineage development from CD34 cells. The use CD34 cell samples from both peripheral blood and bone marrow allowed evaluation of peripheral blood as possible source of hematopoietic progenitors for studies focusing on megakaryocytopoiesis or, more generally, on hematopoiesis.
Forty HIV-infected subjects were studied; of these, 20 had not been treated with ART and 20 had been taking ART for at least 6 months with varying regimens (seven taking a protease inhibitor-containing regimen, seven taking a non-nucleoside reverse transcriptase inhibitor-containing regimen and six taking only nucleoside reverse transcriptase inhibitors). Twenty healthy HIV-negative subjects formed a control group. Peripheral blood samples were collected from all the study participants; bone-marrow aspirates were obtained from posterior iliac crest of 10 HIV-infected (five untreated and five ART-treated) and 16 HIV-negative individuals (seven bone-marrow donors and nine undergoing elective surgery for hip replacement, cells being collected from the ablated bone). Platelet counts for follow-up assessment were obtained retrospectively from the patient's medical records. The study was approved by the Institutional Ethical Committee of Marche Polytechnic University and all participants gave written informed consent (according to Helsinki protocol) prior to the initiation of any study procedure.
Lymphocyte phenotype was determined in fresh whole blood samples by multiparametric flow cytometry using the following monoclonal antibodies: anti-CD3 conjugated with peridinin–chlorophyll a complex protein or allophycocyanin (APC), anti-CD4 conjugated with fluoroscein isothiocyanate or APC, APC-conjugated anti-CD8, and phycoerythrin (PE)-conjugated anti-DR. All the monoclonal antibodies were purchased from Becton Dickinson (San Jose, California, USA). After the erythrocytes were stained, they were lysed with hypotonic buffer solution and four-color cytofluorimetric analysis was performed on FACScalibur (Becton Dickinson), using the CellQuest software. An electronic gate was set in the peripheral blood lymphocyte region, and a minimum of 20 000 events/sample were acquired. To quantify the CD4 or CD8 T cell subpopulations, CD4 or CD8 T lymphocytes were gated by combining side-scatter parameter and reactivity with anti-human monoclonal antibody for CD4 or CD8, respectively.
Plasma HIV-1 RNA levels were measured using the branched-DNA technique (b-DNA, Quantiplex HIV-1 RNA 3.0 assay, Chiron Diagnostics Corp., East Walpole, Massachusetts, USA), according to the manufacturer's instructions.
Sorting of CD34 cells
To purify CD34 cells derived from peripheral blood and bone marrow for the clonogenic experiments, peripheral blood mononuclear cells (PBMC) or bone marrow-derived mononuclear cells were isolated on density gradient Histopaque-1077 (Sigma Chemicals Co., St. Louis, Missouri, USA); CD34 cells were purified by sorting with immunomagnetic beads system (MiniMACS, Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were washed, resuspended in phosphate-buffered saline (pbi international, Milan, Italy) and counted. A sample of CD34 cells was labeled with PE-conjugated anti-CD34 monoclonal antibody (Becton Dickinson), and the purity of the cell suspension was assessed by flow cytometry. Mean purity was 87% (SD, 7; range 74–98) in treatment-naive patients, 87% (SD, 7; range, 70–96) in ART-treated patients, and 87% (SD, 12; range, 58–97) in uninfected controls. The remaining CD34 cells were resuspended at final concentration of 1.1 × 105 cells/ml in Iscove's Modified Dulbecco's Medium (IMDM; Biological Industries, Kibbutz Beit Haemek 25115 Israel) and used for the culture.
Assay of colony-forming units
CD34 cells were cultured in collagen-based medium (5000 cells in 1.5 ml; MegaCult-C, Stem Cell Technologies, Vancouver, British Columbia, Canada) containing thrombopoietin (TPO, 50 ng/ml), interleukin-3 (IL-3, 10 ng/ml), and interleukin-6 (IL-6, 10 ng/ml), according to the manufacturer's instructions. After incubation for 10–12 days at 37°C under 5% CO2, cultures were first dehydrated and fixed in 1:3 methanol:acetone solution and then labeled by MegaCult-C Staining Kit (Stem Cell Technologies), according to the manufacturer's instructions. In brief, the following reagents were added in sequence: (a) mouse IgG2a monoclonal antibody anti-human GPIIb/IIIa; (b) biotin-conjugated goat anti-mouse IgG antibody; (c) avidin–alkaline phosphatase conjugate; (d) alkaline phosphatase substrate; (e) Evans Blue counterstain. CFU-MK growth was assessed by counting the colonies under light microscopy. All the experiments were performed in duplicate.
Samples of 3000–5000 CD34 cells were labeled with the following PE-conjugated monoclonal antibodies: mouse anti-human IL-3-Rα (anti-CD123, Becton Dickinson); mouse anti-human c-Mpl (anti-thrombopoietin receptor, Pharmingen, San Diego, California, USA); mouse anti-human IL-6-R (anti-CD126, Pharmingen). Analysis was performed at flow cytometry by QuantiBRITE system (Becton Dickinson). The system used tubes containing beads coated with different (and known) amounts of PE to generate a calibration curve. The percentage of positive cells and number of receptor molecules per single cell were calculated using QuantiCALC software (Becton Dickinson).
Data were elaborated by SPSS software (SPSS, Chicago, Illinois, USA); statistical comparisons were performed by ANOVA test plus Tukey's HSD post-hoc analysis for parametric data, and by Kruskal–Wallis test plus Dunn's post-hoc test for non-parametric data. HIV-1 viral load levels were compared by Mann–Whitney's U test. To assess the correlation coefficients between the variables, Spearman's rank correlation coefficients were calculated. Differences were considered statistically significant when P < 0.05.
Clinical and hematological features of the study population
Table 1 summarizes the baseline characteristics of the study population. One single patient, in the ART-treated group, had prior diagnosis of AIDS-related wasting syndrome [stage C3 in the Centers for Disease Control and Prevention (CDC) classification 1993]; stage B diagnoses included 10 cases of oral Candida albicans (three among untreated and seven among ART-treated subjects). Plasma HIV-1 RNA was below the detection limit (50 copies/ml, 1.70 log copies/ml) in 13 ART-treated HIV-infected subjects; in the others, HIV-1 viremia ranged between 93 and 1700 copies/ml (1.97–3.23 log copies/ml). Mean HIV-1 viral load among untreated individuals was 16 051 copies/ml (4.20 log copies/ml). As expected, mean HIV-1 plasma RNA concentration was significantly higher in the untreated than in the ART-treated group (P < 0,001). Platelet counts were also compared in the two groups of HIV-infected patients. Of note, no statistically significant differences were found between controls and either the ART-treated or the untreated HIV-infected subjects. Moreover, no differences were observed between treated and untreated HIV-infected individuals with respect to CD4 and CD8 T cell count and percentage. No major abnormalities were detected at light microscope examination in bone marrow smears obtained from both the HIV-infected individuals and the controls.
Growth of megakaryocytopoietic colony-forming units
CD34 progenitor cells derived from peripheral blood or bone marrow were cultured in medium suitable for megakaryocytic lineage development and differentiation. CFU-MK growth was detectable in all the cultures, with a level of variability in the number of recovered colonies that was similar in HIV-infected individuals and controls. Generation of CFU-MK colonies from peripheral blood-derived CD34 cells was significantly impaired in HIV-infected untreated individuals compared both with ART-treated subjects [19.1/5000 CD34 cells (SD, 15.2) versus 42.3/5000 CD34 cells (SD, 25.1); P = 0.032] and controls [19.1/5000 CD34 cells (SD, 15.2) versus 47.7/5000 CD34 cells (SD, 39.2); P = 0.006]. By comparison, no difference was found between ART-treated patients and controls (P = 0.818). A similar picture was observed by in-vitro culturing of bone marrow-derived CD34 cells, although differences between groups were not statistically significant, probably because of the limited sample size (Fig. 1). In general, CD34 cells derived from peripheral blood tended to yield slightly higher numbers of CFU-MK colonies than bone marrow-derived progenitors, but differences among groups were similar in the two compartments. Consequently, peripheral blood would provide a suitable source of CD34 progenitors for studies focusing on megakaryocytopoiesis. The perturbations in the ability of CD34 cells to develop into CFU-MK colonies detected here were occurring in individuals who had yet to show any overt reduction in platelet numbers in the periphery. As normal megakaryocytic lineage differentiation and proliferation was preserved or restored in those HIV-positive individuals receiving ART, use of ART could possibly avoid platelet loss later in the course of the disease.
Impact of viral replication and immune activation on megakaryocytopoiesis
Platelet loss during the course of HIV disease could occur by direct infection of developing megakaryocytes and/or by HIV-mediated disruption of the bone marrow environment that supports CFU-MK differentiation [11–15]. The current study did not address whether the described abnormalities were related to direct HIV infection of CFU-MK. Instead, the overall effect of viral replication on CFU-MK development in the whole organism was examined. To this end, untreated HIV-infected individuals were stratified according to their viral loads. There was significantly reduced growth of peripheral blood-derived megakaryocytic colonies in subjects with plasma HIV RNA > 104 copies/ml compared with those with plasma HIV RNA < 104 copies/ml (Fig. 2a; P < 0.01) and with controls (P < 0.001). Conversely, no differences were observed between controls and untreated HIV-1-infected individuals who had low viral load (P > 0.05). Moreover, there was a significant negative correlation among untreated HIV-infected individuals between their plasma viral load and CFU-MK growth in culture (Fig. 2c) and between their HIV viremia and platelet counts (Fig. 2d). These correlations were not observed among ART-treated individuals (Fig. 2e,f). Finally, there was significantly higher expression of the activation marker HLA-DR on the CD8 T cells from untreated HIV-infected subjects compared with ART-treated individuals and controls (Fig. 2b). These findings support the hypothesis that HIV-1 is directly involved in the pathogenesis of the abnormal megakaryocytes development and that there is a possible link between viral replication, immune activation, and reduced CFU-MK colony formation.
Expression of cytokine receptors involved in megakaryocytopoiesis
Defective CD34 cell responsiveness to megakaryocytopoietic stimuli could contribute to the decreased CFU-MK growth among untreated HIV-infected individuals. The cytokines TPO, IL-3, and IL-6 are main regulators of megakaryocytic differentiation [19–21] and were added to the culture media in a fixed dose. To determine whether a lack of response to TPO, IL-3, and IL-6 may be involved in the genesis of the HIV-associated abnormal megakaryocytopoiesis, the expression of the receptors for these cytokines were analyzed on the surface of peripheral blood-derived CD34 cells. The percentages of peripheral blood-derived CD34 cells expressing the different receptors were calculated, as well as the number of receptor molecules expressed per single CD34 cell. No significant differences were observed between groups in the percentages of CD34 cells expressing receptors for IL-3, TPO, or IL-6 (Fig. 3a), or in the absolute numbers of molecules per single cell (Fig. 3b). The expression of receptors for IL-3, TPO, and IL-6 was also assessed on bone marrow-derived CD34 cells (Fig. 3c,d); again, no major differences were detected, although in general the expression level of these receptors was slightly higher in untreated than in ART-treated HIV-positive individuals and in controls. Collectively, these data indicate that the reduced CFU-MK growth observed in untreated HIV-positive individuals is unlikely to be a result of defective expression of receptors for the main cytokines involved in megakaryocytopoiesis on the CD34 cell surface.
The present study was conducted to investigate the mechanisms underlying the perturbation of megakaryocytopoiesis that may follow HIV infection and to assess the differences between untreated and ART-treated subjects in terms of megakaryocyte and platelet development. The key findings of this study are that (a) perturbations of megakaryocytes development can occur before the onset of overt platelet reduction at peripheral blood level; (b) HIV replication plays a direct role in inducing abnormalities in CFU-MK development; and (c) ART may contribute to restore (or preserve) normal megakaryocytopoiesis. To the best of our knowledge, this is the first study demonstrating the possible impairment in CD34 cell differentiation toward megakaryocytic lineage in HIV-infected individuals with normal platelet numbers. In addition, our data suggest that ART may reduce the risk of onset of megakaryocyte differentiation defects, possibly lowering the risk of thrombocytopenia in the course of HIV disease. Longitudinal analyses are needed to address this point. The last aim of this study was to determine whether peripheral blood-derived CD34 cells could substitute for bone marrow-derived counterparts in functional studies on hematopoiesis. In this regard, our results suggest that peripheral blood is a reliable source of CD34 cells for studies focusing on megakaryocytopoiesis in the course of HIV infection.
Collectively, our findings are consistent with previously published data suggesting that the HIV-induced thrombocytopenia can develop earlier than neutropenia or multiple cytopenias, both more frequently observed in the advanced stages of the disease . The reported data are also consistent with the finding that platelet counts often increase after initiation of ART in HIV-infected thrombocytopenic individuals . Direct HIV infection of CD34 progenitors committed toward megakaryocyte differentiation and/or HIV-mediated perturbations of the bone marrow environment are among the possible mechanisms of impaired megakaryocytopoiesis [11–15]. Consequently, it is reasonable that HIV burden may influence CFU-MK development. Importantly, when HIV-infected untreated individuals were stratified according to HIV viral load (above or below 4 log copies/ml), decreased CFU-MK growth was identified in subjects with higher levels of HIV viral RNA. We also found a statistically significant negative correlation between HIV viral load and both CFU-MK growth and platelet numbers. Finally, levels of CD8 T cells expressing the activation marker HLA-DR were significantly increased in HIV-infected untreated individuals compared with those treated with ART. In all, these findings suggest that HIV replication probably plays a major role in the pathogenesis of the abnormalities in megakaryocytopoiesis, in agreement with published studies indicating HIV itself as the major cause of damage of the megakaryocyte developmental machinery at bone-marrow level [5–13]. In addition, we also hypothesized that in ART-naive individuals the presence of high levels of immune activation induced by HIV viral replication may also contribute to the impaired CFU-MK development. Defective production of cytokines and enhanced apoptosis of precursors are among the possible mechanisms of reduced bone-marrow clonogenic capability induced by chronic T cell activation [23,24]. In this situation, the ART-induced suppression of viral replication may contribute indirectly to turn off immune activation and thus allow functional recovery in bone marrow. An alternative explanation is that the ART-induced changes in CFU-MK development may be related to the presence of an immune reconstitution inflammatory syndrome .
Our data suggest that HIV infection is associated with a relative early impairment in the CD34 capability to differentiate toward CFU-MK. While HIV-related reduction of CD34 precursor number in untreated individuals with high viral load may contribute to the impaired CFU-MK growth in vivo, this seems an unlikely occurrence in our experimental setting, since the initial number of plated CD34 cells was the same in all the experiments. It should be noted that the initial composition of the CD34 cell pools was not examined at single-subject level; therefore, we cannot completely exclude baseline differences between groups in the level of precursors already committed toward CFU-MK development. Further studies are necessary to test this possibility.
Expression of receptors for TPO, IL-3, and IL-6 was comparable between the studied groups of HIV-infected individuals, and in CD34 cells derived from both bone marrow and peripheral blood. As TPO, IL-3, and IL-6 were added to the culture media at fixed doses, it is unlikely that the observed differences resulted from defective production of cytokines and their receptors. In addition, previous published data suggest that TPO levels tend to be increased rather than reduced in HIV-infected individuals, especially in those with reduced platelet numbers . It should be noted that functional impairment of receptors or reduced expression of other receptors not included in our panel (e.g., interluekin-11 receptor, oncostatin M, leukemia inhibitor factor) may theoretically have contributed to the reduced CFU-MK growth observed among untreated HIV-infected individuals. Further studies are needed to investigate this potential mechanism of reduced CFU-MK growth.
The results described here suggest that HIV replication intereferes, either directly or indirectly, with the differentiation of bone marrow CD34 cell precursor toward megakaryocytopoiesis, and that the effects of this are detectable in circulating CD34 cells. Although some controversy still exists, it is now widely believed that HIV does not infect CD34 cells in vivo [27–31], whereas in-vitro infection of developing megakaryocytes by recombinant HIV-1 has been described [32,33]. While carry-over of HIV in our culture system is theoretically possible, we consider this event unlikely because of the multiple washing steps included in the CD34 cell purification procedure and the high purity of the final CD34 cell populations. In addition, HIV was not detected in CD34 cell suspensions analyzed before the culture (data not shown).
HIV-mediated infection and destruction of megakaryocytes may play a role in the pathogenesis of the defects in megakaryocyte lineage development. Zauli et al.  have reported a selective impairment in the in-vitro differentiation of purified CD34 into megakaryocytic lineage occurring in HIV-infected thrombocytopenic individuals. Our current findings complete and extend these results, showing that defects in CFU-MK differentiation can develop in HIV-infected patients before the occurrence of platelet loss in the periphery.
In summary, this study indicates that megakaryocytic differentiation can be impaired before the onset of an overt decrease in platelet numbers in the periphery and that the reduction of CFU-MK growth is observed in untreated but not in ART-treated HIV-infected individuals, suggesting that suppression of viral replication facilitates the reconstitution of a normal CFU-MK development and prevents further damage to megakarycytopoiesis. HIV viral replication also appear to impact on megakaryocytic development, since in patients with low viral load CD34 progenitor cells display better capacity to growth into CFU-MK colonies. These findings may have implications in selecting the optimal timing for ART initiation, which is still debated . Preservation of bone marrow and thymus function would be possible benefits of early ART; our data support the potential advantage of such early ART. Studies focusing on functionality of the hematopoietic cell precursors in the course of HIV infection might have implications for the future design of novel therapeutic strategies .
Sponsorship: This work has been partially supported by grant 40F.54, Fifth National AIDS Research Program, Istituto Superiore di Sanità, Italy.
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