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. 2020 Jun 11;16(6):e1008381.
doi: 10.1371/journal.ppat.1008381. eCollection 2020 Jun.

"V体育ios版" HIV infects astrocytes in vivo and egresses from the brain to the periphery

Victoria Lutgen  1 , Srinivas D Narasipura  1 , Hannah J Barbian  1 , Maureen Richards  1 , Jennillee Wallace  1 , Roshanak Razmpour  2 , Tetyana Buzhdygan  2 , Servio H Ramirez  2 , Lisa Prevedel  3 , Eliseo A Eugenin  3 , Lena Al-Harthi  1
Affiliations

VSports - HIV infects astrocytes in vivo and egresses from the brain to the periphery

"VSports app下载" Victoria Lutgen et al. PLoS Pathog. .

Abstract

HIV invades the brain during acute infection VSports手机版. Yet, it is unknown whether long-lived infected brain cells release productive virus that can egress from the brain to re-seed peripheral organs. This understanding has significant implication for the brain as a reservoir for HIV and most importantly HIV interplay between the brain and peripheral organs. Given the sheer number of astrocytes in the human brain and their controversial role in HIV infection, we evaluated their infection in vivo and whether HIV infected astrocytes can support HIV egress to peripheral organs. We developed two novel models of chimeric human astrocyte/human peripheral blood mononuclear cells: NOD/scid-IL-2Rgc null (NSG) mice (huAstro/HuPBMCs) whereby we transplanted HIV (non-pseudotyped or VSVg-pseudotyped) infected or uninfected primary human fetal astrocytes (NHAs) or an astrocytoma cell line (U138MG) into the brain of neonate or adult NSG mice and reconstituted the animals with human peripheral blood mononuclear cells (PBMCs). We also transplanted uninfected astrocytes into the brain of NSG mice and reconstituted with infected PBMCs to mimic a biological infection course. As expected, the xenotransplanted astrocytes did not escape/migrate out of the brain and the blood brain barrier (BBB) was intact in this model. We demonstrate that astrocytes support HIV infection in vivo and egress to peripheral organs, at least in part, through trafficking of infected CD4+ T cells out of the brain. Astrocyte-derived HIV egress persists, albeit at low levels, under combination antiretroviral therapy (cART). Egressed HIV evolved with a pattern and rate typical of acute peripheral infection. Lastly, analysis of human cortical or hippocampal brain regions of donors under cART revealed that astrocytes harbor between 0. 4-5. 2% integrated HIV gag DNA and 2-7% are HIV gag mRNA positive. These studies establish a paradigm shift in the dynamic interaction between the brain and peripheral organs which can inform eradication of HIV reservoirs. .

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Transplanted astrocytes survive and develop processes in neonate and adult NSG mice but spread out only in neonate NSG mice, without evidence for disruption of blood brain barrier or xenotransplanted astrocyte migration outside of the brain.
200,000 NHAs were injected into neonate brain at postnatal day 1 (PND1) or 50,000 NHAs into each striatum of adult (5–6 weeks) NSG mice. At 4 weeks (A), 8 weeks (B) and 12 weeks (C) post xenotransplantation in neonates, NHA expression is shown by human GFAP (huGFAP, red) with DAPI (blue) staining. Data is representative of 2 mice per time point. Representative images of neonatal prefrontal cortex (PFC, D), thalamus (TH, E), and hippocampus (HPC, F) immuno-stained for human GFAP (huGFAP, red) or human Nuclei (huNuclei, red) and DAPI (blue) at 12 weeks. Representative images of adult striatum immune-stained for human GFAP (G, red) or huNuclei (H, red) and DAPI (blue) at 4 weeks 5 days post xenotransplantation. Inset: higher magnification of square. (I) Representative image of negative staining control for rabbit or mouse IgG primary and corresponding secondary of adult non HIV-infected mouse striatum with DAPI (blue). Data is representative of 2 mice per time point. (J-K) Evaluation of cerebrovascular permeability in the striatum following intracranial implantation of astrocytes by microinjection in adult animals. Animals were transcardially perfused with a fluorescence tracer (FITC) conjugated to 10kDa dextrans in the following groups of animals: naïve, 4hrs after exposure to bacterial LPS (i.p 6mg/kg), 8hrs or 5 days following microinjections of transplanted exogenous astrocytes. (J) Representative confocal images of striatal areas at the injection coordinates (relative to Bregma +0.5 A/P, +1.8 M/L, −3.0 D/V). (K) Image analysis measurements of vascular permeability using pixel intensity (as a function of area) in the parenchymal areas of the injection site. Results are shown as the average ±SEM; multiple group comparisons were determined by one-way ANOVA with post-hoc Dunnett's test * = p<0.001, n = 3–4 per condition, NS, denotes no significance. (L) Adult mice were injected with NHAs without huPBMC reconstitution and real-time PCR hu cytoB mtDNA products extracted from the blood within 5 hours of injection were analyzed by electrophoresis. MS: blood from non-NHA injected animal, MS + NHA: blood from animal microinjected with NHAs, MS + huPBMCs: animals reconstituted with huPBMCs, NTC: no template control. (M) Adult mice were injected with NHAs without huPBMC reconstitution and real-time PCR DNA products (human GFAP, human GAPDH, mouse GFAP and mouse GAPDH) were analyzed by electrophoresis at day 5 post xenotransplantation. n = 3. MS: spleen from 1 non-NHA injected animal, HEK: human HEK293T cells. Scale bars: A-J, 50μm; G insert, 10μm.
Fig 2
Fig 2. HIV in the brain of neonate and adult NSG mice xenotransplanted with astrocytes.
NHAs were infected with GFP-expressing full length HIV virus (A, NLENG1-IRES 70, 1–3% GFP+ cells), or with HIVVSVg pseudotyped (B), VSVg-NLENG1-IRES 70, 20–30% GFP+ cells), both viruses express GFP. (C) Schematic of experimental design. HIV- or HIV+ NHAs were injected into mouse brain on PND1 (200,000 total NHAs) or adult striatum (5–6 weeks, 100,000 total astrocytes). At six weeks post PND1 xenotransplantation or 5 days post adult xenotransplantation, mice were reconstituted with huPBMCs. At four weeks post reconstitution animals were sacrificed for post-mortem analyses. Representative images of neonates (D-E) or adult (F-G) mice injected with NHAs or NHAs infected with HIV. (D) Representative images of neonate mice xenotransplanted with HIV- (top) or HIV+ (bottom) NHAs. Arrow indicates co-localization of huGFAP (red) and HIV (green) along with DAPI (blue) in mouse striatum. (E) Representative image of neonates xenotransplanted with NHAs (top) and NHA-HIV (bottom) for huNuclei (red), HIV (green) and DAPI (blue). Arrows indicate co-localization of huNuclei and HIV. (F) & (G) Representative images of adults injected with HIV- (top) or HIV+ (bottom) NHAs for either (F) huGFAP (red), HIV (green) and DAPI (blue) or (G) huNuclei (red), HIV (green) and DAPI (blue). Arrows indicate co-localization of human markers and HIV. Scale bars: B-C, E, G, 50μm; D, F, 10μm.
Fig 3
Fig 3. HIV egress from the brain into the periphery in the neonate astrocyte xenotransplantation model.
Neonate NSG mice were xenotransplanted with HIV- or HIV+ NHAs as described in Fig 2C and HIV detection outside of the brain was evaluated by Real-time PCR products (HIV DNA, A, or HIV RNA, B) from brain, cervical lymph node, spleen, splenocytes outgrowth assay and splenocytes outgrowth assay supernatant added to fresh PBMCs analyzed by electrophoresis and human GAPDH. Each column indicates individual animal. HIV- animals are shown to left of ladder, HIV+ animals shown to right for all gels shown. No template control (NTC) is the negative control. M/F indicates sex and number is animal number from that group. (C) GFP expression in splenocytes at day 14 day of culturing the cells and (D) flow cytometry of cultured splenocytes stained for CD3+/CD4+/GFP; dot blots (left) and cumulative data on right (p = 0.05, Mann-Whitney U-test). (E) Representative image of supernatant from splenocytes cultured for 14 days from neonates from HIV- animal (top) or HIV+ animal (bottom) on fresh PBMCs stimulated with soluble α-CD3 and α-CD28 IL-2. Egress for neonates was analyzed in n = 7 (HIV-) and 12 (HIV+) mice; n = 3 (HIV-) and 4 (HIV+) representative mice are shown here; n = 3 (HIV-) and 5 (HIV+) representative mice are shown here.
Fig 4
Fig 4. HIV egress from the brain into the periphery in the adult astrocyte xenotransplantation model.
Adult NSG mice were xenotransplanted with HIV- or HIV+ NHAs as described in Fig 2C and HIV detection outside of the brain was evaluated by Real-time PCR products (HIV DNA, A, or HIV RNA, B) from brain, cervical lymph node, spleen, peripheral lymph node, splenocytes outgrowth assay and splenocytes outgrowth assay supernatant added to fresh PBMCs analyzed by electrophoresis and human GAPDH. Each column indicates individual animal. HIV- animals are shown to left of ladder, HIV+ animals shown to right for all gels shown. No template control (NTC) is the negative control. (C) GFP expression in splenocytes at day 14 day of culturing the cells and (D) flow cytometry of cultured splenocytes stained for CD3+/CD4+/GFP; dot blots (left) and cumulative data on right (p = 0.04, Mann-Whitney U-test). (E) Representative image of supernatant from splenocytes cultured for 14 days from neonates from HIV- animal (top) or HIV+ animal (bottom) on fresh PBMCs stimulated with soluble α-CD3 and α-CD28 IL-2. Egress was analyzed in n = 7 (HIV-) and 9 (HIV+) mice; n = 3 (HIV-) and 5 (HIV+) representative mice are shown here.
Fig 5
Fig 5. HIV egress from the brain into the periphery in the adult astrocyte xenotransplantation model.
Adult NSG mice were xenotransplanted with HIV- or HIV+ astrocytes as described in Fig 2C and HIV detection outside of the brain was evaluated by electrophoresis. (A) HIV DNA and RNA from the brain and the spleen of n = 2 (HIV-) and 3 (HIV+) adult animals xenotransplanted with HIV- or HIV+ (non-pseudotyped) NHAs. (B) HIV DNA and RNA from brain and spleen from n = 2 (HIV-) and 3 (HIV+) adult animals xenotransplanted with HIV- or HIVVSVg+ U138MG astrocytoma cell line. (C) HIV DNA and RNA from brain and spleen from n = 2 (HIV-) and 5 (HIV+) adult animals xenotransplanted with HIV- or HIVIIIB+ NHAs. (D) HIV DNA and RNA from brain and spleen from n = 2 (HIV-) and 7 (HIV+) adult animals injected with HIV- or HIVVSVg+ free virus into the striatum in the absence of astrocytes. PC indicates a Positive Control for HIV DNA and HIV RNA.
Fig 6
Fig 6. HIV evolution in NSG mice.
(A) Highlighter plot denoting the locations of nucleotide substitutions in each gag sequence from 3 mice xenotransplanted with HIV-infected astrocytes (horizontal lines) in comparison to the virus inoculum (NL4-3, top sequence). Nucleotide substitutions are shown as color-coded tick-marks, and APOBEC-signature mutations are shown as pink circles. (B) Hamming distance plot demonstrating Poisson distribution and estimated days to most recent common ancestor (MRCA) with supporting statistics are shown. Actual days to MRCA (days post infection) were 33. Scale bars: 50μm.
Fig 7
Fig 7. HIV egresses from the brain into the periphery likely through CD4+ T cells.
(A) Representative image of neonate hippocampus (HPC) immunostained for human T cells (CD3+, magenta), human astrocytes (huGFAP; red), HIV (green) and Nuclei (DAPI, blue) at week 10 post-xenotransplantation and week 4 reconstitution with huPBMCs. Arrows indicates detection of CD3+/HIV+ cell. Scale bar, 10μm. (B) Total PBMCs (HIV+) or monocyte/CD4+ T cell depleted PBMCs analyzed by flow cytometry for CD3+/CD4+ positive T cells (top) or CD14 positive or CD14/CD16 positive monocytes before injection into the adult mouse xenotransplanted with HIVVSVg + NHAs. HIV DNA (C) and RNA (D) from spleen from animals xenotransplanted with HIVVSVg+ NHAs and reconstituted with total PBMCs (HIV+) or PBMCs depleted of monocytes and CD4+ T cells (Mono/CD4). No analysis was performed on DNA as Mono/CD4 group had undetectable HIV DNA. RNA is depicted on the right (p = 0.08, Mann-Whitney U-test). n = 5–7 per group.
Fig 8
Fig 8. Astrocyte derived HIV egress under cART.
(A) Adult NSG mice were xenotransplanted with HIV+ astrocytes and treated with cART as depicted and cART treatment continued every other day until sacrifice at 3 or 4.5 weeks post reconstitution. cART interrupted animals were given 3 weeks of cART followed by 1.5 weeks of interruption before sacrifice. (B) HIV DNA was measured in the spleen by electrophoresis at time of sacrifice; with Kruskal-Wallis comparison (H = 9.185, p = 0.01) with Dunn post hoc test. * p < 0.05 from no cART, # p < 0.05 from cART interruption. HIV RNA was measured from spleen and shown in C; with Kruskal-Wallis test comparison (H = 5.904, p = 0.052). Data combined for three and four-and-a-half-week cART treatments as there were no statistical differences. n = 3–4 per group.
Fig 9
Fig 9. Evidence of HIV infection of astrocytes through systemic route of infection.
(A) Neonatal mice were xenotransplanted with uninfected NHAs and reconstituted with HIV+ huPBMCs and sacrificed 4 weeks later. (B) Representative image of neonate striatum of co-localization of human GFAP RNA (red) HIV RNA (green) and DAPI (blue) by RNAscope. (C) Representative image of neonate hippocampus with HIV DNA (green), huGFAP (red), HIV RNA (purple) and DAPI (blue). (D) Representative image from neonate hippocampus immunostained for human astrocytes (huGFAP; red), HIV p24 (green) and Nuclei (DAPI, blue). Arrows indicate co-localization of huGFAP and p24. n = 6 mice. 4 or 6 coronal sections were analyzed per mouse for RNAscope and immunofluorescence, respectively. Representative images chosen from 4 of 6 mice with observed HIV infection, with 1–4 infection events observed per animal. B, D scale bar, 20μm. B insert, 10μm. C scale bar, 5 μm.
Fig 10
Fig 10. HIV+ astrocytes detected in human brain of peripherally suppressed donors.
HIV+ astrocytes are detected in cortical and hippocampal brain tissue sections obtained from HIV infected individuals under effective ART viral suppression. Staining for these human tissue sections was performed for DNA (DAPI, blue), GFAP (Alexa 350, blue), Alu-repeats (probe for Alu, white), HIV-nef DNA (green), HIV-mRNA (red), and HIV-p24 protein (cyan). To separate all these fluorescent sequential scanning and spectral detection was used as well as each respective control. In addition, co-localization of DAPI, Alu-repeats, and HIV-DNA was considered a positive nuclear signal. In contrast, HIV-mRNA-gag, GFAP, and HIV proteins had minimal to no co-localization with nuclear markers. (A and B) Corresponds to a representative positive for HIV-DNA in GFAP positive astrocytes (see arrow). (C) No unspecific staining for HIV-nef DNA, HIV mRNA or viral proteins was detected in uninfected tissues. Data is quantified in Table 1. Scale bars: 80μm.
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