Human Microglia Extensively Reconstitute in Humanized-BLT Mice With Human Interleukin-34 Transgene and Support HIV-1 Brain Infection

Abstract

Humanized bone marrow-liver-thymic (hu-BLT) mice develop a functional immune system in periphery, nevertheless, have a limited reconstitution of human myeloid cells, especially microglia, in CNS. Further, whether bone marrow derived hematopoietic stem and progenitor cells (HSPCs) can enter the brain and differentiate into microglia in adults remains controversial. To close these gaps, in this study we unambiguously demonstrated that human microglia in CNS were extensively reconstituted in adult NOG mice with human interleukin-34 transgene (hIL34 Tg) from circulating CD34+ HSPCs, nonetheless not in hu-BLT NOG mice, providing strong evidence that human CD34+ HSPCs can enter adult brain and differentiate into microglia in CNS in the presence of hIL34. Further, the human microglia in the CNS of hu-BLT-hIL34 NOG mice robustly supported HIV-1 infection reenforcing the notion that microglia are the most important target cells of HIV-1 in CNS and demonstrating its great potential as an in vivo model for studying HIV-1 pathogenesis and evaluating curative therapeutics in both periphery and CNS compartments.

Keywords: HIV-1; Hu-BLT mice; NOG mice; interleukin-34; microglia.

Figure 1

Experimental timeline and Flow Cytometric evaluation of human immune cells reconstitutions in the peripheral blood of hu-BLT-hIL34 and hu-BLT mice. (A) Experimental design and timeline for the humanization of hIL34 transgenic mice and NOG mice by engrafting with human CD34+ hematopoietic stem and progenitor cells (HCSPs) and human fetal liver and thymic tissues, HIV-1 Ada infection of some mice in each group, and the evaluation of human myeloid and microglial cell reconstitution and HIV-1 infection in the CNS of euthanized mice using immunohistochemical staining (IHCS) and RNAscope in situ hybridization (ISH). (B) The representative flow cytometric dot plots of the peripheral blood mononuclear cells of a hu-BLT-hIL34 mouse (#1703) at the 4 months after transplantation, which were gated with hCD45+, hCD3+, hCD9+, and hCD14+ cells. (C) The percentage of hCD14+ myeloid cells in the peripheral blood of hu-BLT-hIL34 mice is significantly higher than that hu-BLT mice at the 4 months post transplantation. Each symbol represents an individual mouse, hu-BLT-hIL34 mice n=6, hu-BLT mice n=10; ****p < 0.0001.

Figure 2

The hIba-1+ myeloid cell reconstitutions in the brain tissues of hu-BLT-hIL-34 and hu-BLT mice. The upper panel (A–E) shows a representative whole brain tissue section from the third coronal brain slice of a hu-BLT-hIL34 mouse (#1703) that was stained immunohistochemically for hIba-1+ (brown) and counterstained with hematoxylin. LV stands for lateral ventricle. The blue and red boxed regions in the Figure C were highlighted at a higher magnification (A, D). In turn, the blue and red boxed regions of the (A, B) were further highlighted (B, E). The hIba-1+ cells are morphologically ramified and mainly distributed in brain parenchyma. The lower panel (F–J) shows a representative whole brain tissue section (H) of a hu-BLT mouse (#1717) that was stained immunohistochemically for hIba-1+ cells (brown). The blue and red boxed regions in the Figure H was highlighted at a higher magnification (F, I). In turn, the blue and red boxed regions of the (F, I) were further highlighted (G, J). There were no detectable parenchymal hIba-1+ cells.

Figure 3

The hCD14+ myeloid cell reconstitutions in the brain tissues of hu-BLT-hIL34 BLT and hu-BLT mice. The upper panel (A–C) shows representative whole brain tissue sections and the highlighted images form the corresponding boxed regions from hu-BLT-hIL34 mouse (#1708) that were stained immunohistochemically for hCD14+ cells (brown) and counterstained with hematoxylin. There were abundant hCD14+ cells (A–C) in the brain of the hu-BLT-hIL34 mouse. The lower panel (D–F) in contrast shows no detectable hCD14+ myeloid cells in the brain parenchyma and a very limited number of hCD14+ cells in meninges in a representative whole brain tissue section of a hu-BLT mouse (#1723).

Figure 4

The microglia-specific marker hTMEM119+ microglia in the brain tissues of hu-BLT-hIL34 and hu-BLT mice and humans. A representative whole brain tissue section (C) from the third coronal brain slice of a hu-BLT-hIL34 mouse (#1703) that was stained immunohistochemically for hTMEM119+ cells (brown) and counterstained with hematoxylin. LV stands for lateral ventricle. The blue and red boxed regions in the Figure (C) were highlighted at a higher magnification (A, D). In turn, the blue and red boxed regions of the Figures (A, D) were highlighted (B, E). The hTMEME119+ cells (brown) are morphologically ramified and distributed in brain parenchyma. The histogram of quantitative images analysis of hTMEM119 + cells in the cerebral cortex of the hu-BTL-hIL34 mice (F) (304.08 ± 131.93 hTEME119+ cells/mm2, n=6), where there were an absent of these cells in the hu-BLT mice. The middle panel (G–I) shows hTMEM119 + microglia in the cerebral cortex of a HIV-1 non-infected individual with no registered medical complications (G–I). The frequency, distribution, and morphology of hTMEM119+ human microglia in hu-BLT-hIL34 mice are similar to this human individual. The lower panel (J–L) shows no detectable hTMEM119+ cells in a representative whole brain section of a hu-BLT mouse (#1721).

Figure 5

The HIV-1 infection of human microglia of hu-BLT-hIL34 mice. The Upper panel shows HIV-1 RNA+ cells (A, B, red) in a representative whole brain tissue section from a hu-BLT-hIL34 mouse (#1708) that was detected using RNAscope in situ hybridization with HIV-1 clade B anti-sense probe and counterstained with hematoxylin. The highlighted boxed region from the Figure (A) was shown at a higher magnification of HIV-1 RNA+ cells (B, red). HIV-1 p24+ cells (C, brown) in the cerebral cortex of the same mouse were detected using IHCS. The colocalization of HIV-1 vRNA and human myeloid cell marker hIba-1 (D, arrows) indicating the reconstituted human myeloid cells could support HIV-1 replication. The lower panel (E, F) shows there were no detectable HIV-1 RNA+ cells in a representative whole brain tissue section from a hu-BLT mouse (#1724) using RNAscope in situ hybridization.