Human Interleukin-34 facilitates microglia-like cell differentiation and persistent HIV-1 infection in humanized mice

Abstract

Background: Microglia are the principal innate immune defense cells of the centeral nervous system (CNS) and the target of the human immunodeficiency virus type one (HIV-1). A complete understanding of human microglial biology and function requires the cell's presence in a brain microenvironment. Lack of relevant animal models thus far has also precluded studies of HIV-1 infection. Productive viral infection in brain occurs only in human myeloid linage microglia and perivascular macrophages and requires cells present throughout the brain. Once infected, however, microglia become immune competent serving as sources of cellular neurotoxic factors leading to disrupted brain homeostasis and neurodegeneration.

Methods: Herein, we created a humanized bone-marrow chimera producing human "microglia like" cells in NOD.Cg-Prkdc^scidIl2rg^tm1SugTg(CMV-IL34)1/Jic mice. Newborn mice were engrafted intrahepatically with umbilical cord blood derived CD34+ hematopoietic stem progenitor cells (HSPC). After 3 months of stable engraftment, animals were infected with HIV-1_ADA, a myeloid-specific tropic viral isolate. Virologic, immune and brain immunohistology were performed on blood, peripheral lymphoid tissues, and brain.

Results: Human interleukin-34 under the control of the cytomegalovirus promoter inserted in NSG mouse strain drove brain reconstitution of HSPC derived peripheral macrophages into microglial-like cells. These human cells expressed canonical human microglial cell markers that included CD14, CD68, CD163, CD11b, ITGB2, CX3CR1, CSFR1, TREM2 and P2RY12. Prior restriction to HIV-1 infection in the rodent brain rested on an inability to reconstitute human microglia. Thus, the natural emergence of these cells from ingressed peripheral macrophages to the brain could allow, for the first time, the study of a CNS viral reservoir. To this end we monitored HIV-1 infection in a rodent brain. Viral RNA and HIV-1p24 antigens were readily observed in infected brain tissues. Deep RNA sequencing of these infected mice and differential expression analysis revealed human-specific molecular signatures representative of antiviral and neuroinflammatory responses.

Conclusions: This humanized microglia mouse reflected human HIV-1 infection in its known principal reservoir and showed the development of disease-specific innate immune inflammatory and neurotoxic responses mirroring what can occur in an infected human brain.

Keywords: HIV-1 infection; Hematopoietic stem cells; Humanized mice; Microglia.

Fig. 1

Generation and characterization of NOD.Cg-Prkdc^scid Il2rg^tm1Sug Tg (CMV-IL34)1/Jic (NOG-hIL-34) mice. a NOG-hIL-34 transgenic mice were created in NOD.Cg-Prkdc^scidil2g^tmlSug/Jic mice by inserting vector containing transgene (Tg), hIL-34, under CMV promoter. b NOG-hIL-34 mice were identified by PCR analysis of ear DNA that amplify hIL-34 (358 bp) in homozygous mice. No bands were detected in non-transgenic NOG controls. A representative gel is shown here. Analysis was done for all 17 NOG-hIL-34 being used in the study and confirmed with the presence of hIL-34 genomic DNA. c hIL-34 expression in plasma was confirmed by ELISA (NOG-hIL-34, n = 6; NOG control, n = 5). d Tissue specific expression of hIL-34 was observed by real time PCR using total RNA isolated from brain, spleen, lung, kidney, liver and skin of NOG-hIL-34 mice (n = 17, except for skin tissue n = 5) compared to NOG controls (n = 5). e, f Establishment of human peripheral hematolymphoid system in CD34-NOG-hIL-34 mice. e Flow cytometry analysis of peripheral blood at 6 months age and gating strategy Representative plots of human cluster of differentiation (CD) 45 positive cells and human CD3, CD19 and CD14 positive cells from human CD45⁺ gate. f Percentage of human cell subtypes in the peripheral blood of CD34-NOG-hIL-34 mice used in the study. Each symbol represents an individual mouse, n = 17

Fig. 2

Human microglial-like cells in CD34-NOG-hIL-34 mouse brains. a A comparison of tissue macrophage reconstitution between CD34-NOG-hIL-34 and CD34-NSG mice. Brain sections were stained for HLA-DR, and liver and spleen for human CD68 (Scale bar 10 μm). HLA-DR+ human cells were widely distributed in brain parenchyma of CD34-NOG-hIL-34 mice. b Magnified views of olfactory bulb (OB, 20×), cortex (CTX, 20×) and hippocampus (HC, 10×) from CD34-NOG-hIL-34 mouse brain sections stained for HLA-DR. c Microglial morphology of the human cells shown at higher magnification (Scale bar 2 μm). d Confocal images of brain stained for HLA-DR and Iba1. e Quantification of HLA-DR/Iba1 double positive human microglial cells from total Iba1 positive cells (sample size described in materials and methods, multiple reagions shown on Additional file 1: Figure S5)

Fig. 3

Expression of microglial markers by the human cells in mouse brain. a Immunohistology of 5 μm paraffin embedded CD34-NOG-IL-34 mouse brain sections stained for human microglial markers P2RY12, CD14, CD68 and CD163. Brown cells are positive for the respective protein (Magnification, 200×, Scale bar 20 μm) Inset shows 1000× magnification. b Transcriptomic analysis of RNA extracted from CD34-NOG-IL-34 mice brains by aligning the reads to human genome (h19) showed human myeloid specific gene expression. The graph shows gene names and expression levels of top classical microglial markers in CD34-NOG-hIL-34 mouse brains

Fig. 4

HIV infection in the humanized mouse brain. a RNAScope® assay for detecting HIV-1 RNA, using antisense probe V-HIV1- Clade-B, revealed single brown dots or cluster of dots. Immunohistology of brain regions showing the presence of HIV-1p24+ infected cells in corresponding regions. Original magnification of 400×, Scale bar 10 μm. b Peripheral viral load was determined by a COBAS Amplicor System after three and 6 weeks post infection. Each symbol represents an individual infected mouse. c Comparison of brain viral RNA levels determined by semi-nested RT-PCR from CD34-NSG and CD34-NOG-hIL-34 mice. d Immunofluorescence staining demonstrating mouse astroglia (GFAP, red) in control and HIV-1 infected, with astrogliosis near in HIV-1p24 positive (green) human microglia. Adjacent sections stained for human microglial cells (HLA-DR+ green) and astrogliosis (GFAP, red) indicating the presence of human microglial cells in the same brain region. Original magnification of 400×

Fig. 5

Transcriptional changes in the brain tissues of CD34-NOG-hIL-34 with HIV-1 infection. a, b Alignment of reads to human genome (h19) comparing uninfected and HIV-1 infected CD34-NOG-IL-34 mice found 687 differentially expressed genes (DEG), and the pie chart (a) and the volcano plot (b) shows the proportion and fold change of upregulated and down-regulated genes, respectively. c, d Log fold change of top ranking differentially expressed human genes for uninfected vs HIV infected CD34-NOG-hIL-34 mice in human microglia and in brain. e, f Cellular pathways involving upregulated human genes (261) were related to interferon signaling, PRR and TLR signaling (e), and downregulated human genes (426) were highly associated with pathways of EIF2 signaling and oxidative phosphorylation (f). g Unmapped reads mapped to HIV-1_ADA genome indicated the expression levels of different HIV-1 genes in infected mouse brain