HIV-1 in peripheral blood monocytes: an underrated viral source

Tuofu Zhu*

Department of Laboratory Medicine, University of Washington School of Medicine, Box 358070, 960 Republican Street, Seattle, WA 98109-8070, USA


    Evidence for HIV-1 persistence and replication in peripheral blood monocytes
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 Evidence for HIV-1 persistence...
 Models of HIV-1 persistence...
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Mononuclear phagocytes can be divided into two groups: the circulating mononuclear phagocytes, or monocytes, in the peripheral blood and the tissue macrophages in organs such as the spleen, lymph nodes, liver (Kupffer cells), the lung (alveolar macrophages), brain (microglial cells) and intestine. Tissue macrophages originate from circulating blood monocytes, and monocytes in turn are generated from precursor cells in the bone marrow. Peripheral blood monocytes are heterogeneous in size, morphology and cell-surface molecule expression, each of which serve as parameters for isolating and defining monocytes.1 In the earliest studies monocytes were isolated by gradient centrifugation and counterflow centrifugation (elutriation), which allowed for the separation of monocyte subsets by size and density. Adherence of peripheral blood mononuclear cells (PBMC) has also been used to generate monocytes and monocyte-derived macrophages (MDM). In recent years the combination of beads conjugated to monoclonal antibodies and flow cytometry has provided a powerful tool to purify and characterize blood monocytes, although there appears to be no reagent that reacts exclusively with monocytes. Antibodies against CD14 are among the most commonly used reagents to purify blood monocytes. CD14, a receptor for the lipopolysaccharide (LPS)-binding protein, is present on mononuclear phagocyte lineages, and is expressed on the cell surface of 80–90% of blood monocytes. CD14 expression is lower, but detectable, in macrophages from certain tissues, such as lung, and some Langerhan cells.2 However, CD14 is absent in mucosal macrophages from the small intestine, the largest reservoir of macrophages in the body.2 CD14 is also absent from T cells, B cells and natural killer cells,2 making it a good marker to distinguish blood monocytes from these cells.

HIV-1 infects and replicates primarily in CD4+ T cells and tissue macrophages, which accounts for many of the major aspects of HIV-1 pathogenesis in vivo. Previous studies showed that HIV-1 could be detected in blood monocytes.35 It was, however, not known whether the virus was produced or was maintained latently in monocytes in vivo.5 In vitro studies have shown that HIV-1 replication in freshly isolated blood monocytes and resting CD4+ T cells was blocked prior to the completion of reverse transcription and integration.6,7 However, treating, but not activating, resting CD4+ T cells with IL-2, IL-4, IL-7 and IL-15 was able to overcome this block, resulting in HIV-1 replication in resting CD4+ T cells.8 Thus, it is likely that replication of HIV-1 occurs in vivo in resting CD4+ T cells that are exposed to cytokines at sites of infection or in tissues.810 Whether cytokines similarly render monocytes susceptible to HIV-1 infection in vivo is possible, but still unknown. Sonza et al.11 showed recently that HIV-1 could be isolated when a patient’s monocytes had differentiated into MDM. These results indicate that monocytes harbour replication-competent HIV-1, and that HIV-1 could be produced upon the differentiation of monocytes into macrophages.6 Whether or not HIV could replicate in undifferentiated monocytes remained to be determined.

To examine the extent of HIV-1 replication in vivo in monocytes, and its role as a potential source of virus, we isolated highly purified CD14+ monocytes by a two-step procedure of immunoprecipitation depletion and cell sorting.10,12 The CD14+ monocytes purified by this assay represent the majority of heterogeneous blood monocytes circulating in vivo, because blood monocytes isolated by this method are not likely to have differentiated into macrophages as is the case during conventional adherence of PBMC in vitro.6 We measured the levels of cell-associated viral DNA, messenger RNA (mRNA) and genetic evolution of HIV-1 in patient samples obtained longitudinally during the course of HIV-1 infection.10,12 HIV-1 DNA was detected in CD14+ monocytes throughout the course of study in both patients without antiretroviral therapy and patients whose plasma viraemia had been <200 copies/mL for 3 years during highly active antiretroviral therapy (HAART). We analysed the decay of HIV-1 DNA in CD14+ monocytes in patients on effective HAART, and then compared the proviral decay in CD14+ monocytes with that in purified resting CD4+ T cells and activated CD4+ T cells. While significant variation in the decay slopes of HIV-1 DNA was seen between treated patients, viral decay in CD14+ monocytes was on average slower than that in activated or resting CD4+ T cells.10 We estimated the half-life of HIV-1 DNA in each cell compartment during the period when patients’ plasma virus was maintained at levels below the detection limit of conventional assays. Although there was a significant variation in all three cell compartments and between individual patients, the mean half-life of HIV-1 DNA in CD14+ monocytes [41.3 months, 95% confidence interval (CI): 17.9–infinite] was longer than that in resting CD4+ T cells (23.5 months, CI: 12.5–196.6) or activated CD4+ T cells (19.8 months, CI: 11.9–59.8).10 These apparent half-lives of HIV-1-infected cells from our patients were much longer than the estimated mean intermitotic lifespans of monocyte-macrophages (14 days), activated (2 days) and resting memory (6 months) CD4+ T cells, suggesting that these reservoirs may be renewed as a result of recent infection and/or ongoing virus replication in monocytes or in their precursor cells. To further assess these possibilities, we looked for the signs of viral replication by examining HIV-1 mRNA and sequence evolution in CD14+ monocytes. We determined HIV-1 transcription activity by assessing the levels of cell-associated multiply spliced (MS) (tat) and unspliced (US) (gag) viral mRNA in samples that were taken after plasma virus had been ‘undetectable’ for more than 1 year, which would indicate continuous HIV-1 transcription in CD14+ monocytes.13,14 Both MS and US HIV-1 mRNA were detected consistently in CD14+ monocytes from each time point. The mean concentrations of gag and tat mRNA in CD14+ monocytes were significantly higher than those in resting CD4+ T cells. We also estimated HIV-1 transcriptional activity by measuring the ratio between viral DNA and RNA. The mean ratios of tat and gag mRNA/DNA for CD14+ monocytes were similar to those for activated CD4+ T cells, and were significantly higher than that for resting CD4+ T cells.10 These results indicate ongoing higher levels of viral transcription in CD14+ monocytes and activated CD4+ T cells compared with resting CD4+ T cells in patients with suppressive HAART. While the above data indicate ongoing viral transcriptional activity, the production of infectious virus could still be blocked at assembly by the protease inhibitor included in HAART. We therefore evaluated HIV-1 sequence evolution in CD14+ monocytes at time points pre-antiretroviral therapy and 3 years post-HAART, since mutational changes accumulate as a result of completed rounds of viral replication in vivo. We observed that sequence evolution varied by patient, as reported by others,15,16 and by cell population. When HIV-1 sequences from all treated patients were analysed together, we found significant genetic evolution in CD14+ monocytes. However, the sequence evolution in the two CD4+ T cell populations was not statistically significant,10 although HIV tends to evolve in activated and resting CD4+ T cells. Further comparison of HIV-1 sequences among CD14+ monocytes, activated and resting CD4+ T cells, and peripheral blood plasma by phylogenetic analyses showed that after prolonged HAART the viral populations related or identical to those found only in CD14+ monocytes were seen in plasma from three of the seven patients, suggesting that CD14+ monocytes appear to be a potential in vivo source of HIV-1.


    Models of HIV-1 persistence and replication in peripheral blood monocytes
 Top
 Evidence for HIV-1 persistence...
 Models of HIV-1 persistence...
 References
 
Three models can be proposed to account for our findings of HIV-1 persistence in monocytes and the close association of HIV-1 in CD14+ monocytes and blood plasma. First, monocytes may serve as a direct source of plasma virus by producing infectious HIV-1 in peripheral blood. In patients without antiretroviral therapy, activated CD4+ T cells may produce up to 99% of all virus particles while the other 1% of virus may be generated primarily from tissue macrophages. In this situation, viruses produced by blood monocytes may be too minor to recognize. However, in patients on suppressive HAART in whom HIV replication in activated CD4+ T cells is blocked, viruses produced from monocytes become relatively dominant. This hypothesis is supported by our findings of higher levels of HIV-1 transcripts and sequence evolution in CD14+ monocytes than in resting CD4+ T cells of patients on HAART, which suggests a relatively higher level of HIV-1 replication in CD14+ monocytes compared with resting CD4+ T cells in these patients. Continued production of HIV in monocytes of treated patients may be explained by the inability of antiretroviral drugs to block viral replication in monocyte-macrophages as efficiently as in CD4+ T cells.17 Secondly, HIV-infected monocytes can be an indirect source of HIV-1 by carrying virus and differentiating into tissue macrophages where HIV may replicate productively. It is possible that infected monocytes produce relatively low amounts of virus while circulating in peripheral blood, but is a major carrier of virus into tissue sites where tissue macrophages may produce greater amounts of virus. In about 70% of the patients we studied, HIV-1 sequence populations in plasma were identical to those isolated from CD14+ monocytes, which were also identical to viral sequences from resting CD4+ T cells and activated CD4+ T cells, suggesting that not only CD14+ monocytes but also resting and activated CD4+ T cells, and probably also tissue macrophages could be potential viral sources in these patients.15,16 The third model to account for the persistence of HIV-1 in blood monocytes is that HIV-infected precursor cells of monocytes in bone marrow enter and renew HIV-1-infected monocytes in peripheral blood. In the bone marrow, a heterogeneous population of mononuclear phagocyte precursor cells, such as CD34+ progenitor cells, proliferate and differentiate. These cells may be infected with HIV, then enter the bloodstream and renew the viral pool in peripheral blood monocytes.

In summary, given the fact that monocytes may circulate in peripheral blood for only 1–3 days before differentiating into macrophages in tissues, our finding of persistent HIV-1 in blood monocytes itself suggests ongoing renewal of infected monocytes by virus replication and/or recent infection in monocytes or their precursor cells. Our findings of more evident HIV-1 replication in CD14+ monocytes suggest that the HIV-1 pool in monocytes could be renewed, as a result of viral replication, more frequently in CD14+ monocytes than in resting CD4+ T cells. Therefore, although the long half-life of HIV-infected CD14+ monocytes appears to be similar to that of resting CD4+ T cells, HIV-1 could turn over at a higher rate in CD14+ monocytes than in resting CD4+ T cells in the presence of HAART. However, the viral pool in CD14+ monocytes could also be renewed by viruses produced from resting and activated CD4+ T cells, and tissue macrophages. The source of HIV-1 in blood monocytes and the contribution they make to the overall pool of HIV-1 replication require more extensive studies. The establishment of HIV-1 infection in CD14+ monocytes in the early stage of infection and ongoing viral replication in CD14+ monocytes present a major challenge to HIV-1 eradication with current regimens of HAART. New therapies with greater potency against viral production in blood monocytes may enhance the efficacy of antiretroviral therapy.


    Footnotes
 
* Tel: +1-206-732-6079; Fax: +1-206-732-6055; E-mail: tzhu{at}u.washington.edu Back


    References
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 Evidence for HIV-1 persistence...
 Models of HIV-1 persistence...
 References
 
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