Fig 1.
Increased number of macrophages and T cells observed in the liver during SIV infection.
CD68 macrophages and CD3 T cells were enumerated in the liver of uninfected, chronically SIV-infected, and chronically SIV-infected, cART infant or adult rhesus macaques by immunofluorescence microscopy. A) Fluorescent images at 600x (left image, scale bar = 10 um) and 100x (right image, scale bar = 100 um) depicting specific staining for macrophages (red) and T cells (green) in the liver (blue indicates nuclei). For quantification, eight random fields in the liver were imaged under 100x magnification and then analyzed by ImageJ software. B) Quantification of eight random fields of view for each animal to enumerate CD3 T cells by ImageJ Cell Counter analysis with adult and infant macaques graphed separately (top and bottom panels, respectively). C) Quantification CD68 macrophages in eight random fields of view for each animal using ImageJ Particle analysis. Data are graphed as the mean ± SEM. Statistical significance between groups was determined Mann Whitney T tests.
Fig 2.
The CCL2-CCR2 pathway is associated with liver monocyte/macrophage infiltration.
A) Levels of CCL2 (MCP-1) in the blood were determined by Luminex analysis. Data are graphed as the mean ± SEM. Adult (open symbols) and infant (closed symbols) macaques are denoted in each graph. B-C) Hepatic expression of CCL2 (B) and its receptor, CCR2, (C) were evaluated by quantitative PCR using cDNA prepared from RNA isolated from the liver of each animal. Data are graphed as log2 transformed relative expression levels determined by the comparative threshold method. Numbered macaques (1, 2, and 3) denote CCL2 and CCR2 expression levels for these three individual macaques between panels 2B and 2C. D) Correlation between the expression of CCR2 in the liver (y-axis) and the number of liver macrophages (x-axis) in uninfected (gray points) and SIV-infected (purple points) macaques was evaluated by Pearson Correlation analysis. E) Immunostaining for CCL2 in the livers of SIV-infected macaques depicting portal regions (100X magnification, scale bar = 100 um). A no primary CCL2 antibody negative control was included to show specific staining (100X magnification, scale bar = 100 um). F) Immunohistochemical staining of recently infiltrated Mac387-positive monocyte/macrophages (brown cells) localized in the portal regions of the liver for uninfected, SIV+, and SIV+ ART macaques (100X magnification, scale bar = 100 um). G) Human monocyte-derived macrophages were stimulated with poly I:C or ssRNA40 at 0.2 and 2 ug/mL for 12 hours in duplicate. Relative expression of CCL2 was assessed by qRT-PCR comparing poly I:C and ssRNA40 stimulated cells to control (unstimulated) monocyte-derived macrophages. Data are graphed as the mean fold change in CCL2 expression ± SEM. Statistical significance was determined using a Mann Whitney T test comparing technical replicates of each condition to unstimulated cells with p ≤ 0.05 denoted as *.
Fig 3.
CCR2-associated influx of T cells and monocytes/macrophages in the liver during SIV infection.
A) PBMC and liver cell suspensions were first gating on single, live, CD45+ cells followed by identification of T cells by CD3 expression and monocytes/macrophages by CD14 expression. T cells and monocytes/macrophages were each evaluated for CCR2 expression. Monocytes/macrophages were also assessed for CD16 expression, and the expression of Mac387 and CD16 on monocytes/macrophages expressing CCR2. B) Frequency of monocytes/macrophages and T cells expressing CCR2 in PBMC. C) Frequency of CD3+ T cells expressing CCR2 in the blood or in the liver. D) Levels of inflammatory CD16+CD14+ monocytes/macrophages in PBMC and in the liver. E) Phenotype of CCR2+ monocytes/macrophages in the blood and in the liver based on Mac387 and CD16 expression. F) Pearson Correlation evaluating the association between the levels of CCR2+CD16+ monocytes/macrophages in the liver (y-axis) and the number of CD68+ macrophages (x-axis) with uninfected macaques in gray and SIV-infected macaques in purple.
Fig 4.
Evaluation of CD68+ mature macrophages in the liver.
A) Freshly isolated macaque liver cells were incubated with fluorescently labeled E. coli bioparticles for 2 hours. After washing, cells were evaluated for phagocytosis of E.coli by flow cytometry. Gating first on single, live, CD45+, CD3- cells, CD68+ liver cells were found to be the primary cell subset to phagocytosis E. coli. B) Liver cells suspensions were first gated on single, live, CD45+, CD3- cells. Mature macrophages, identified as CD68+CD11b+, were evaluated for the expression of CD14 and CD16. C) Frequency of mature CD68+CD11b+ macrophages in the liver of uninfected (gray symbols) and SIV-infected (purple symbols) macaques. D) Phenotype of mature hepatic macrophages in uninfected (gray symbols) and SIV-infected (purple symbols) macaques based on CD14 and CD16 expression.
Fig 5.
Levels of microbial products in the liver and in the plasma during SIV infection.
A) Plasma LBP levels were quantified in duplicate for each macaque by ELISA. Data are graphed as the mean LBP concentration (ng/mL) for each macaque ± SEM. Statistical significance was determined by Mann Whitney T tests. B-C) Levels of bacterial 16S DNA was quantified by qPCR for each liver sample assayed in duplicate. Nanograms (ng) of bacterial DNA present in 100 ng of total input DNA was determined using a standard curve and graphed as the mean of each macaque assayed in duplicate ± SEM. Infant (closed symbols) and adult macaques (open symbols) graphed together (B) or adult macaques graphed separately (C). Statistical significance was determined by Mann Whitney T tests. D) Correlation between the levels of 16S DNA in the liver (y-axis) and the number of liver macrophages (x-axis) in uninfected (gray points), SIV-infected (purple symbols), and SIV-infected cART macaques (green symbols) as evaluated by Spearman Correlation analysis.
Fig 6.
Liver macrophage number correlates with SIV levels.
A) SIV DNA copies in samples from the livers of both infant and adult macaques were determined by quantitative hybrid real-time/digital PCR and normalized per million cell equivalents, based on parallel analysis of a single copy rhesus macaque CCR5 target template. Macaques treated with cART are denoted with bolded font and a green line to the right of the table (BLD = below the limit of detection). B-C) Correlation analysis between copies of SIV DNA in the liver (left) or plasma SIV levels (right) and liver T cell number (B) or liver macrophage number (C) in SIV-infected (purple symbols) and SIV-infected cART (green symbols) macaques. For macaques having SIV plasma viral load and SIV DNA copies below the limit of detection, the log transformed limit of detection values (30 copies/mL for plasma or 10 copies/106 cells for liver) were used for correlation analysis. Adult (open symbols) and infant (closed symbols) macaques are denoted.
Fig 7.
SIV-infected cells localize around the portal triads.
A) RNAscope in situ hybridization was used to detect SIV RNA-positive cells in the liver. Images were captured from whole tissue scans at 5x magnification (images outlined in red, scale bar = 500 um) and depict SIV-infected cells around the portal triads. A representative 20x image of a portal triad was included of the SIV-RNA positive cells (brown cells).
Fig 8.
T cells are the primary cellular subset infected with SIV in the liver.
A-B) Liver tissue sections from SIV-infected untreated macaques were assessed for SIV RNA+ cells (red) by in situ RNAscope technology followed by antibody staining with CD3 to identify T cells (green) and CD68 to identify macrophages (pink). Nuclei were stained with Dapi (blue). SIV RNA+ signal was predominately found associated with CD3 T cells (A) and in rare cases CD68 macrophages (B).
Fig 9.
Liver macrophage number correlates with inflammatory and fibrosis mediators.
A-C) Quantitative PCR was used to evaluate the hepatic expression of macrophage-associated chemokines and cytokines using the comparative threshold method. Data are graphed as the Log2 transformed mean ± SEM with adult (open symbols) and infant (closed symbols) macaques are denoted in each graph. Statistical significance was determined by Mann Whitney T tests. Correlation analysis was used to evaluate the relationship of cytokine/chemokine expression with liver macrophage number. Data displaying normal distribution were analyzed by Pearson correlation with data not passing normality were assessed by Spearman correlation. A significant increase in TNFα (A), CCL3 (B), and TGFβ (C) was observed in livers of SIV-infected macaques that positively correlated with liver macrophage number in the uninfected (gray points) and SIV-infected (purple points) macaques. D) Human monocyte-derived macrophages were stimulated with poly I:C or ssRNA40 at 0.2 and 2 ug/mL for 12 hours in duplicate. Relative expression of TNFα, CCL3 and TGFβ was assessed by qRT-PCR comparing poly I:C and ssRNA40 stimulated cells to control (unstimulated) monocyte-derived macrophages. Data are graphed as the mean fold change in expression ± SEM. Statistical significance was determined using a Mann Whitney T tests comparing technical replicates of each condition to unstimulated cells with p ≤ 0.05 denoted as *.
Fig 10.
Heatmap of the global liver transcriptomic signature.
The liver transcriptome was evaluated by microarray analysis. Treatment groups (SIV+ or SIV+ cART) were compared against age-matched uninfected macaques to determine differentially expressed genes using the limma package in R/bioconductor. Co-expression analysis was performed using weighted gene coexpression network analysis (WGCNA) and heatmap packages in R/bioconductor using cutoffs of 1.5 fold change and 0.05 adjusted pvalues in at least one condition. Gene networks (modules) involved in immune response and inflammation are upregulated in SIV-infected macaques with antiviral RLR and interferon signaling highly upregulated in both adults and infants.
Table 1.
Top differentially expressed genes in SIV+ adult macaques.
Table 2.
Top differentially expressed genes in SIV+ infant macaques.
Table 3.
Top differentially expressed genes in SIV+ ART adult macaques.
Table 4.
Top differentially expressed genes in SIV+ ART infant macaques.
Table 5.
Characteristics of SIV-infected macaques.