Macrophages sustain HIV replication in vivo independently of T cells

Abstract

Macrophages have long been considered to contribute to HIV infection of the CNS; however, a recent study has contradicted this early work and suggests that myeloid cells are not an in vivo source of virus production. Here, we addressed the role of macrophages in HIV infection by first analyzing monocytes isolated from viremic patients and patients undergoing antiretroviral treatment. We were unable to find viral DNA or viral outgrowth in monocytes isolated from peripheral blood. To determine whether tissue macrophages are productively infected, we used 3 different but complementary humanized mouse models. Two of these models (bone marrow/liver/thymus [BLT] mice and T cell-only mice [ToM]) have been previously described, and the third model was generated by reconstituting immunodeficient mice with human CD34+ hematopoietic stem cells that were devoid of human T cells (myeloid-only mice [MoM]) to specifically evaluate HIV replication in this population. Using MoM, we demonstrated that macrophages can sustain HIV replication in the absence of T cells; HIV-infected macrophages are distributed in various tissues including the brain; replication-competent virus can be rescued ex vivo from infected macrophages; and infected macrophages can establish de novo infection. Together, these results demonstrate that macrophages represent a genuine target for HIV infection in vivo that can sustain and transmit infection.

Figure 1. Absence of HIV DNA in monocytes but not in T cells isolated from peripheral blood of infected patients.

(A) Schematic of monocyte and T cell purification strategy. Total MNCs were isolated from the peripheral blood of 8 HIV-infected patients (from Table 1). T cells were depleted using positive magnetic selection, and monocytes were enriched using negative magnetic selection. (B) Flow cytometric analysis of total MNCs, T cells, non–T cells, non-monocytes, and monocytes revealed very pure populations of both T cells and monocytes isolated using this protocol (data from patient 04 are shown). (C) One million purified T cells or monocytes (Mono) were analyzed for the presence or absence of HIV-gag DNA using nested PCR analysis of untreated and treated patients. The lower limit of detection for these analyses was 2 copies of HIV DNA. Purified T cells or monocytes from untreated patients 01, 02, and 03 were injected into BLT mice. (D) vDNA analysis demonstrated systemic infection of BLT mice injected with patients’ purified T cells but not monocytes. LN, lymph node; Org, human thymic organoid; Pt, patient.

Figure 2. NOD/SCID mice transplanted with hCD34⁺ hematopoietic stem cells are reconstituted with human B cells and myeloid cells but lack T cells.

(A) Flow cytometric analysis of the peripheral blood of mice showed a sustained presence of 20% to 30% hCD45⁺ cells over time (28 weeks shown, n = 52). (B) Flow cytometric analysis of the bone marrow, spleen, liver, lung, and peripheral blood of a representative mouse demonstrated the presence of human B cells (hCD19⁺) and human myeloid cells (hCD33⁺), with a complete absence of human T cells (hCD3⁺). (C) Average levels of human cell reconstitution in bone marrow (n = 36), spleen (n = 36), liver (n = 35), lung (n = 35), and peripheral blood (n = 18) and the distribution between myeloid and B cells of the human cells. (D) Phenotypic characterization of the human monocytes and macrophages in the tissues of a mouse reconstituted with hCD34⁺ cells (gating scheme: live → hCD45⁺ → hCD33⁺/hCD11b⁺). (E) Flow cytometric analysis of the brains of MoM demonstrated the presence of human B cells and macrophages, with a lack of human T cells. Percentages in B, D, and E signify the percentage of cells expressing the indicated surface markers for the cell populations designated in the gating strategy. PB, peripheral blood.

Figure 3. HIV replication can be sustained over time in human macrophages in vivo, but it is limited to very few HIV strains.

(A) Efficient replication of all HIV strains tested in BLT mice as determined by viral load analysis. (B) Efficient replication of all HIV strains tested in ToM mice as determined by viral load analysis. (C) Plasma viral load of infected MoM. Only HIV-1 ADA, CH040, and CH040-4013 env (a patient envelope–derived chimeric virus) were detectable in the plasma over time. (D) Increased numbers of CD14⁺CD16⁺ cells (intermediated phenotype monocytes) were detected in the blood of infected MoM (n = 17) during the first few weeks of infection compared with the blood of uninfected MoM (n = 11). VL, viral load.

Figure 4. Systemic replication of HIV-1 in the tissues of MoM.

(A) Cell-associated vDNA copies per 100,000 total cells and (B) cell-associated vRNA copies per 100,000 total cells were measured from cells isolated from bone marrow (BM), spleen, liver, and lung of MoM infected with ADA (n = 2), CH040 (n = 10), or CH040-4013 env (n = 9). (C) Electron micrograph of infected MoM bone marrow shows the presence of viral budding (black arrows) as well as free virions (green and red arrows) in the tissue. Virions in which the viral core was visible are denoted with red arrows. Original magnification, ×20,000 to ×100,000, as indicated in each image. (D) ISH analysis of the tissues of a MoM infected with CH040 demonstrated the presence of human macrophages (expressing CD68, brown staining) producing HIV RNA transcripts (black granules). Original magnification, ×100. (E) Cells from infected MoM tissues (bone marrow [n = 20], spleen [n = 11], liver [n = 11], and lung [n = 8]) were cultured with allogenic donor CD8–depleted feeder cells. Viral outgrowth was determined by the presence (black squares) or absence (white squares) of vRNA in the culture supernatants after 10 days. The number of human macrophages plated in each culture is graphed on the y axis. We were able to measure outgrowth in 41 of 50 total samples (All tissues).

Figure 5. Human cells are present in the brains of MoM and increase in numbers during HIV infection.

(A) MNCs from the brains of infected (n = 25) and uninfected (n = 35) MoM were analyzed via flow cytometry, and the absolute numbers of human cells (hCD45⁺) and human macrophages (hCD33⁺/hCD11b⁺) were calculated per whole brain. There was a significant increase in the overall number of human cells (P = 0.0009) and human monocytes and macrophages (P = 0.0055) in the brains of infected mice compared with numbers detected in uninfected mice. P values were determined by Mann-Whitney U test. (B) Immunohistochemical analysis of the brain was used to confirm the presence of human macrophages (hCD68) in the brains of infected MoM. (C) Cell-associated vRNA copies per 100,000 total cells were measured in the brains of infected MoM. We were able to detect virus in the brains of 8 of 14 animals (lower limit of detection, 30 copies/10⁵ cells). (D) Immunohistochemical analysis demonstrated the presence of HIV p24 in the brains of infected MoM. (B and D) Original magnification, ×10 and ×40 (insets). The location of CD68⁺ or p24⁺ cells is indicated by an abbreviation in each panel. BS, brain stem; CB, cerebellum; CC, cerebral cortex; CP, caudate putamen; CpC, corpus callosum; MB, midbrain; and VS, ventral striatum.