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Monocytes/macrophages are critical components of HIV and SIV encephalitic lesions. We used in vivoBrdU labeling and markers specific to stages of macrophage differentiation or inflammation to define macrophage heterogeneity and to better define the role of macrophage populations in lesion formation and productive infection. Lesions were heterogeneously composed of resident macrophages (CD68+HAM56+), perivascular macrophages (CD163+CD68+MAC387−), and recently infiltrated MAC387+CD68−CD163−monocytes/macrophages. At 24 and 48 hours after BrdU inoculation, 30% of MAC387+monocytes/macrophages were BrdU+, consistent with their being recently infiltrated. In perivascular cuffs with low-level SIV replication, MAC387+monocytes/macrophages outnumbered CD68+macrophages. Conversely, lesions with numerous SIV-p28+macrophages and multinucleated giant cells had fewer MAC387+monocytes/macrophages. The MAC387+cells were not productively infected nor did they express detectable CCR2, unlike perivascular macrophages. Overall, we found that the proportion of MAC387+cells tends to be higher than the proportion of CD68+macrophages in the brain of animals with mild encephalitis; the ratio was reversed with more severe encephalitis. These results suggest that development of SIV and HIV encephalitis is an active and ongoing process that involves the recruitment and accumulation of: i) nonproductively infected MAC387+monocytes/macrophages that are present with inflammation (potentially M1-like macrophages), ii) CD163+perivascular macrophages (consistent with M2-like macrophages), and iii) CD68+or HAM56+resident macrophages. The latter two populations are cellular reservoirs for productive infection.

Monitoring changes in human immune cell populations such as lymphocytes, monocytes, and dendritic cells DCs during infectious diseases like human immunodeficiency virus HIV is crucial. However, difficulties to identify rare or heterogeneous cell populations can be limiting. For example, to accurately measure DC subsets, eight flow cytometry parameters are ideal. The aim of this work was to analyze the phenotype of human lymphocyte, monocyte, and DC subsets using a single 12color flow cytometry panel. After erythrocyte lysis, blood from healthy human volunteers was washed and labeled with a cocktail of 12 antibodies. Samples were analyzed on a BectonDickinson FACSAria™ equipped with three lasers. Data were compared with lineagespecific panels using 5–8 Ab combinations per lineage. Acquired data were analyzed using FlowJo software. Our 12color panel allows for the identification of the following major subsets of circulating cells in a single tube: CD4 and CD8 T lymphocytes, B lymphocytes, NK cells, NKT cells, monocyte subsets CD14 andor CD16, and five nonoverlapping HLADRLin− subsets: CD34 hematopoietic stem cells, CD123 plasmacytoid DC, and three subsets of CD11c myeloid DC expressing either CD16, CD1c BDCA1, or CD141 BDCA3. We have developed a single flow cytometry panel that allows for simultaneous detection of the lymphocyte and monocyte cell populations and all known DC subsets. Studying these major players of the immune system in one single panel may give us a broader view of the immune response during HIV infection and the ability to better define the role of individual cell types in Acquired Immune Deficiency Syndrome AIDS pathogenesis. © 2010 International Society for Advancement of Cytometry

Studies in rodents have shown that brain perivascular macrophages are derived from bone marrow precursors. Less is known about the origin and turnover of perivascular cells in the human central nervous system. We took advantage of non-human primates reconstituted with autologous CD34+ hematopoietic stem cells that had been transduced with a lentiviral vector expressing the enhanced green fluorescent protein (EGFP) to study the ontogeny of brain macrophages of rhesus macaques. Flow cytometry and immunohistochemistry/fluorescence microscopy showed long-term reconstitution of monocytes/macrophages in the blood, lymphoid, and brain tissues 4 years post-transplant. In the brain, EGFP+ cells were detected in the choroid plexus, cerebellum, and cerebrum, where the percent engraftment between animals reflected the percentage of EGFP+ monocytes in the blood. Morphology and location of brain EGFP+ cells exclusively in the vicinity of blood vessels were consistent with perivascular macrophages. Up to 85% of brain EGFP+ cells expressed CD163, a marker of perivascular macrophages, and greater than 70% were CD68+ macrophages. These findings clearly demonstrate that a subpopulation of CD163+/CD68+ brain perivascular macrophages in rhesus macaques are renewed by CD34+ hematopoietic stem cell-derived precursors and exhibit a continuous long-lasting turnover. Because perivascular macrophages are significant targets of productive HIV/simian immunodeficiency virus infection in the brain, these observations point to hematopoietic stem cells as targets of both HIV/simian immunodeficiency virus infection and potential gene therapy.