Immune Activations and Viral Tissue Compartmentalization During Progressive HIV-1 Infection of Humanized Mice

Abstract

Human immunodeficiency virus type one (HIV-1) tissue compartments are established soon after viral infection. However, the timing in which virus gains a permanent foothold in tissue and the cellular factors that control early viral-immune events are incompletely understood. These are critical events in studies of HIV-1 pathogenesis and in the development of viral reservoirs after antiretroviral therapy. Moreover, factors affecting the permanence of viral-tissue interactions underlie barriers designed to eliminate HIV-1 infection. To this end we investigated the temporal and spatial viral and host factors during HIV-1 seeding of tissue compartments. Two humanized NOD.Cg-Prkdc^scid IL2rg^tm1Wjl/SzJ mouse models were employed. In the first, immune deficient mice were reconstituted with human CD34+ cord blood hematopoietic stem cells (HSC) (hu-HSC) and in the second mice were transplanted with adult mature human peripheral lymphocytes (hu-PBL). Both, in measure, reflect relationships between immune activation and viral infection as seen in an infected human host. Following humanization both mice models were infected with HIV-1_ADA at 10⁴ 50% tissue culture infective doses. Viral nucleic acids and protein and immune cell profiles were assayed in brain, lung, spleen, liver, kidney, lymph nodes, bone marrow, and gut from 3 to 42 days. Peripheral CD4+ T cell loss began at 3 days together with detection of HIV-1 RNA in both mouse models after initiation of HIV-1 infection. HIV-1 was observed in all tested tissues at days 3 and 14 in hu- PBL and HSC mice, respectively. Immune impairment was most prominent in hu-PBL mice. T cell maturation and inflammation factors were linked directly to viral tissue seeding in both mouse models. We conclude that early viral tissue compartmentalization provides a roadmap for investigations into HIV-1 elimination.

Keywords: HIV-1 seeding; host immune responses; host inflammatory responses; humanized mice; immune activation; viral tissue compartments.

Figure 1

Human lymphocyte responses following HIV-1 infection of hu-HSC mice. (A) The illustrated experimental design for hu-HSC mice human cell reconstitution, HIV-1 infection, and serial animal sacrifice performed at days 0, 3, 5, 7, 14, 28, and 42. Animal numbers are N = 5, 5, 5, 5, 5, 3, and 3 at each of the time points. (B) Peripheral blood CD45+, CD4+, and CD8+ cell counts before/mock infection (blue) and after HIV-1 infection (red) for each of the time points by flow cytometry tests. Data are expressed as mean ± SEM and considered *, **, *** statistically different, at p < 0.05, p < 0.01, and p < 0.001.

Figure 2

Human lymphocyte responses following HIV-1 infection of hu-PBL mice. (A) The illustrated experimental design for hu-PBL mice human cell reconstitution, HIV-1 infection, and serial animal sacrifice performed at days 0, 3, 5, 7, and 14. Animal number are N = 4, 6, 5, 5, and 8 at each of the time points, respectively. (B) Peripheral blood CD45+, CD4+, and CD8+ cell counts before/mock infection (blue) and after HIV-1 infection (red) for each of the time points by flow cytometry tests. Data are expressed as mean ± SEM and considered **, *** statistically significant, at p < 0.01 and p < 0.001.

Figure 3

Peripheral viral loads during the course of HIV-1 infection. Plasma samples were collected following animal sacrifice from HIV-1 infected (A) hu-HSC and (B) hu-PBL mice. Fifty microliter mouse sera was collected then diluted to 1 ml with sterile filtered healthy human sera enabling a detection limit (DL) of 400 HIV-1 RNA copies/ml that is illustrated by the dashed line. Each dot represents an individual animal. The mean HIV-1 copy value from each group of animals was labeled.

Figure 4

Viral tissue compartments in HIV-1 infected hu-HSC mice. Gut, spleen, lung, liver, brain, and kidney tissues were collected at necropsy at times indicated from HIV-1 infected hu-HSC mice, followed by assay of viral DNA and RNA by qPCR. The kinetics of (A) HIV-1 DNA and (B) HIV-1 RNA in each tissue are shown by colored straight lines assigning viral copies/10⁶ hCD45+ cells (left Y axis) vs. time (X axis). The temporal relationship of viral load (acquired from Figure 3A) was plotted in red dashed line (right Y axis). Data are expressed as the means. (C) HIV-1 DNA and (D) HIV-1 RNA in tissues at single time point were listed with each dot representing an individual animal. Values below the horizontal line indicated that viral DNA and RNA were below the DL.

Figure 5

Viral tissue compartments in HIV-1 infected hu-PBL mice. Gut, spleen, lung, liver, brain, and kidney tissues were collected at necropsy at times indicated from HIV-1 infected hu-PBL mice, followed by assay of viral DNA and RNA by real-time qPCR. The kinetics of (A) HIV-1 DNA and (B) HIV-1 RNA in each tissue are shown by colored straight lines assigning viral copies/10⁶ hCD45+ cells (left Y-axis) vs. time (X axis). The temporal relationship of viral load (acquired from Figure 3B) was plotted in red dashed line (right Y axis). Data are expressed as the means. (C) HIV-1 DNA and (D) HIV-1 RNA in tissues at single time point were listed with each dot representing an individual animal. Values below the horizontal line indicated that viral DNA and RNA were below the limit of detection.

Figure 6

Viral RNA tissue expression in infected humanized mice. Spleen tissues of hu-HSC (A) and hu-PBL (B) mice were collected with formalin fixed and paraffin embedded at necropsy. Five micrometer thick slices were prepared for RNAscope assays. Representative images from each group were shown with HIV-1 RNA labeled as single or cluster of brown dots. Images were taken at 40 x objective magnifications.

Figure 7

HIV-1p24 expression in infected humanized mice. Spleen and lymph node samples were collected from (A) hu-HSC and (B) hu-PBL mice at necropsy then formalin fixed and paraffin embedded. Five micrometer thick sections were cut then stained with human HLA-DR and HIV-1p24 antibodies. Representative images from each group were selected and pictures were captured for both markers (shown as brown dots) from individual animals. Human HLA-DR images were taken at 20 x objective magnifications and HIV-1p24 images were captured at 40 x objective magnifications.

Figure 8

Expression of human immune activation markers in humanized mice. Total RNA was isolated from uninfected humanized mouse spleens and analyzed for the expression of markers for human immune activation. A total of 84 genes were evaluated and compared between the two animal models. (A) Heatmap depicted the differentially expressed genes (for 1–12) associated with immune activation in hu- PBL compared against HSC mice (for A–G). The log₂ fold change of the individual gene is listed in the bottom panel. (B) Differentially expressed genes with log₂ fold change of ≥3 are outlined that are expressed in hu-PBL spleens over what was found in HSC mice. A complete gene list can be found in Table S2.

Figure 9

Gene expression patterns analysis by IPA in humanized mice. (A) Canonical pathway gene analyses. The stacked bar chart demonstrates the percentage of upregulated (red) and downregulated (green), as well as non-overlapped (white) genes from the prestored genebank in IPA (numbers listed at the top of each bar). The right y-axis displays the –log of p-Value calculated by Fisher's Exact Test illustrates the significance of each canonical pathway. (B) Downstream biological effects prediction. The most relevant downstream effect predicted by IPA was lymphoid activation. A set of 65 genes that were differentially expressed between hu-PBL and hu-HSC mice co-regulate this pathway. The putative function was located in the center while the related regulator listed at the periphery. The type of interaction is indicated by red (prediction of activation), blue (prediction of inhibition), yellow (inconsistent), and gray (related, not predicted).