*Laboratory of Cellular Immunology, National Institute for Infectious Diseases “Lazzaro Spallanzani” I.R.C.C.S., Rome, Italy;
†Clinical Department, National Institute for Infectious Diseases “Lazzaro Spallanzani” I.R.C.C.S., Rome, Italy;
‡Department of Haematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy; and
§Virology Laboratory, National Institute for Infectious Diseases “Lazzaro Spallanzani” I.R.C.C.S., Rome, Italy.
Correspondence to: Veronica Bordoni, PhD, Laboratory of Cellular Immunology, National Institute for Infectious Diseases “Lazzaro Spallanzani” I.R.C.C.S., Via Portuense, 292—00149 Rome, Italy (e-mail: firstname.lastname@example.org).
Supported by Italian Ministry of Health (RF-IMI-2009-1304187 and Programma di Ricerca Corrente IRCCS).
The authors have no conflicts of interest to disclose.
Received May 31, 2013
Accepted July 08, 2013
Dendritic cells (DCs) are heterogeneous population of rare hematopoietic cells that are essential mediators of innate and adaptive immune responses.1,2 DC populations are thought to be derived from blood-borne precursors that develop in the bone marrow (BM).3–5
A number of previous studies have demonstrated a reduction in peripheral blood DC number and function, both early during acute HIV infection and in the later stages of infection, being the DCs depletion associated with HIV disease progression and opportunistic infections.6–8
The possible mechanism accounting for the decreased circulating DCs and for their dysfunction is the current topic of intense investigation. It has been suggested that the decreased DC frequency in the blood may be because of a redistribution to secondary lymphoid tissue,9,10 because of direct infection,6,11 or because of indirect mechanism involving DC differentiation from monocytes.12
The key question of this study was to investigate whether DCs impairment in HIV infection could in part result from a failure of BM hematopoietic progenitor cells (HPCs) to produce an adequate number of DCs.
PATIENTS AND METHODS
CD34+ HPCs were isolated from BM of 6 combination antiretroviral therapy (cART)-treated (>24 months) chronic HIV-infected patients with undetectable viremia and from 3 HIV-negative controls. Patients’ data are summarized in Figure 1A. All those patients required BM aspirate for differential diagnosis purposes. Ethics committee approval was obtained for the project, and individual signed consents were obtained from enrolled subjects. The pathologic analysis of BM samples resulted normocellular to hypercellular with reactive plasmacytosis being not pathologic in controls. No patients received previously hematotoxic therapy. Freshly isolated CD34+ HPCs were purified from BM mononuclear cells by a 2-step magnetic procedure involving a lineage depletion, followed by a CD34 isolation (CD34 Diamond isolation kit; Miltenyi Biotec, Bologna, Italy).
Unilineage CD34+ HPC liquid suspension culture and generation of DCs were performed as previously described.13 Briefly, highly pure CD34+ HPCs were cultivated in monocytic condition with the addition of interleukin-6, Flt3, and M-CSF (density, 8 × 104−2 × 105/cm2) on 96- to 48-well plates; after 10 days, the differentiation toward DCs was induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 treatment for 5 days. To evaluate DCs maturation, cells were stimulated by 1 microgram per milliliter lipopolysaccharide (LPS) (Sigma-Aldrich, Steinheim, Germany) for 16 hours.
Phenotype analysis of CD34-derived DCs from BM patients was performed by flow cytometry on 1 × 105 viable cells. Samples acquisition and data analysis were performed by a FACS Canto II flow cytometer (Becton Dickinson, San Jose, CA) by using Diva software.
Quantification of HIV-RNA in cultured CD34+ samples was performed by using the Abbott Real-time HIV-1 assay (Abbott Molecular, Inc., Des Plaines, IL) according to manufacturer’s instructions.
BM mononuclear cells from HIV-infected patients were found significantly reduced compared with the HIV-negative controls [mean, 57 × 106 (range, 30–150 × 106/10 mL) vs mean, 221 × 106 (range, 200–240 × 106/10 mL), P = 0.02] (Fig. 1B). A lower differentiation capability of highly pure BM-derived CD34+ HPCs (Fig. 1C) toward CD14–CD1a+ DCs was observed in HIV-infected patients in comparison with HIV-negative controls [mean, 4.5% (range, 0–10.6) vs 21.7% (range, 10.3–42.3), P = 0.04] (Figs. 1A, D–F).
We next analyzed the baseline DCs activation profile by measuring the expression of CD80, CD83, and CD86 markers. As reported in Figure 1G, an increased CD80 expression in the baseline activation status was found in HIV-infected patients compared with controls [% CD80+ cells, mean, 74.9 (range, 47.7–95.8) vs 24.0 (range, 15–41.8), respectively, P = 0.03]; for CD83 and CD86, an increase trend was found [% CD83+ cells, mean, 58.6 (range, 43.2–83.9) vs 43.3 (range, 28.9–61.9); % CD86+ cells, mean 68.0 (range, 44.5–97.5), vs 36.9 (range, 33.8–40.8)] although not significant, probably because of sample size. Moreover, as expected, LPS stimulation was able to significantly increase the expression of the CD80, CD83, and CD86 in HIV-negative controls (Fig. 1G); in contrast, DC from HIV-infected patients failed to respond to LPS stimulation, probably because of their higher baseline activation status (Fig. 1G).
Finally, no HIV-RNA was found in culture supernatants of CD34+ HPC from treated HIV-infected patients at different times of culture (data not shown).
To our knowledge, this is the first study specifically examining the unilineage differentiative capability toward mature DCs of CD34+ HPCs purified from BM of HIV-infected patients. In fact, several studies have reported a reduction of circulating DCs number in HIV-infected patients at various stages of disease and have indicated that HIV infection is associated with DC defective maturation and functions.6–8,14 Nevertheless, no data are available on the involvement of differentiation from BM-derived CD34+ HPCs.
Our results suggest that the HIV-induced modulation of CD34 differentiation capability may play a key role in the decreased frequency of DCs observed in patients. Moreover, response of DCs to LPS stimulus in chronically infected patients was hampered compared with HIV-negative controls, probably because of a higher baseline activation status.
Results seem to exclude a direct role of HIV replication in this process; however, its integrated quiescent form may itself modulate HPC activity.
Thus, the debated issue of HIV presence in CD34+ BM HPC from infected individuals still needs a clear answer.15–17 Although possibly not directly infected, BM CD34+ HPC function is indeed affected during HIV infection.18 Many possible indirect mechanisms, such as chronic systemic immune activation and altered cytokine milieu, may explain this issue and deserve further work.
Our research is limited first by the small sample size, although this limitation is shared by most studies of BM CD34+ HPC in HIV; second, in this study, we used only surface markers because detailed information regarding functional response of DCs in our patients was not available. A larger study with an expected accrual of 40 patients yearly is ongoing, so that in a near future, we will be able to strengthen and expand our findings and, in particular, to verify how impairment of differentiation/maturation of DCs reflects on their functional capability in vitro.
In conclusion, our results suggest that the damage in CD34+ HPC differentiation into DC may, in part, explain DCs number reduction and impairment observed in HIV-infected patients. Additional studies with larger sample sizes are needed to analyze HPC impairment in correlation to the different stages of HIV disease and to identify which HIV-driven direct or indirect mechanism may affect CD34+ HPC differentiation capability.
The authors gratefully acknowledge the patients who made this study possible. V.B. and F.M. designed the study. M.B. and A.A. were in charge of patients. V.B. and D.V. performed experiments. V.B., A.S., and C.A. analyzed data. G.C. contributed reagents/materials/tools. A.A. and I.A. performed HIV quantification. V.B., F.M., and M.B. drafted and revised the article.
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