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. 2013 Sep;87(17):9523-37.
doi: 10.1128/JVI.00861-13. Epub 2013 Jun 19.

Immune activation and regulation in simian immunodeficiency virus-Plasmodium fragile-coinfected rhesus macaques

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Immune activation and regulation in simian immunodeficiency virus-Plasmodium fragile-coinfected rhesus macaques

Kristin A Trott et al. J Virol. 2013 Sep.

Erratum in

  • J Virol. 2014 Nov;88(22):13516

Abstract

Human immunodeficiency virus (HIV) is characterized by immune activation, while chronic malaria is associated with elevated interleukin-10 (IL-10) levels. How these apparently antagonizing forces interact in the coinfected host is poorly understood. Using a rhesus macaque model of simian immunodeficiency virus (SIV)-Plasmodium fragile coinfection, we evaluated how innate immune effector cells affect the balance between immune activation and regulation. In vitro Toll-like receptor (TLR) responses of peripheral blood myeloid dendritic cells (mDC) and monocytes were temporarily associated with acute parasitemic episodes and elevated plasma IL-10 levels. Prolonged infection resulted in a decline of mDC function. Monocytes maintained TLR responsiveness but, in addition to IL-12 and tumor necrosis factor alpha, also produced IL-10. Consistent with the role of spleen in the clearance of parasite-infected red blood cells, coinfected animals also had increased splenic IL-10 mRNA levels. The main cellular source of IL-10 in the spleens of coinfected animals, however, was not splenic macrophages but T cells, suggesting an impairment of adaptive immunity. In contrast to those in spleen, IL-10-positive cells in axillary lymph nodes of coinfected animals were predominantly mDC, reminiscent of the immunosuppressive phenotype of peripheral blood mDC. Concurrent with IL-10 induction, however, SIV infection promoted elevated systemic IL-12 levels. The continuously increasing ratio of plasma IL-12 to IL-10 suggested that the overall host response in SIV-P. fragile-coinfected animals was shifted toward immune activation versus immune regulation. Therefore, SIV-P. fragile coinfection might be characterized by earlier manifestation of immune dysfunction and exhaustion than that of single-pathogen infections. This could translate into increased morbidity in HIV-malaria-coinfected individuals.

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Figures

Fig 1
Fig 1
Correlation of peripheral blood parasitemia and myeloid dendritic cell (mDC) TLR4 responsiveness. TLR4 responsiveness (solid line) and parasitemia (dashed line) are shown for individual animals that were coinfected with SIV and P. fragile (A) or infected only with P. fragile (B). TLR4 responses were measured after in vitro stimulation of peripheral blood mononuclear cells with 1 μg/ml lipopolysaccharide (LPS) and subsequent multicolor flow-cytometric analysis. The percentage of TNF-α+ cells in the total mDC population (Lin CD11c+ HLA-DR+) is shown. Parasitemia is reported as the percentage of infected red blood cells.
Fig 2
Fig 2
TLR7/8 responses of peripheral blood mDC and monocytes. (A and B) The percentages of cytokine-positive mDC or monocytes, respectively, after in vitro stimulation with the TLR7/8 ligand R848. PBMC from animals in the various infections groups were analyzed for the production of TNF-α, IL-12, and IL-10. The experimental groups are ordered in the following order, from left to right on the x axis: SIV-P. fragile coinfection, SIV infection, and P. fragile infection. Individual animals in each group are represented by a specific symbol, as indicated in the graph. Results are shown for the time points corresponding to the time of SIV infection (DPI-SIV 0) or P. fragile (DPI-Pf 0/DPI-SIV 28) infection, the time of acute SIV infection (A-SIV and DPI-SIV 28; equivalent to the time of P. fragile infection in coinfected animals), acute P. fragile infection (A-Pf and DPI-SIV 35 to 49/DPI-Pf 7 to 21), and the chronic phase (Chr) of infection (DPI-SIV 84 to 112/DPI-Pf 42 to 84). See the text for further details.
Fig 3
Fig 3
Gene expression of specific TLR signaling molecules in tissues. Spleens and axillary lymph nodes collected at 3 months postinfection were analyzed for mRNA expression of TLR signaling molecules by a TLR PCR array. (A) Average fold regulation for TLR adapter molecules and transcription factors for SIV (empty bars)-infected, P. fragile (gray bars)-infected, and SIV-P. fragile-coinfected (black bars) animals. Data were analyzed using the manufacturer's online RT2 Profiler PCR array data analysis software. Changes in mRNA levels are expressed compared to the same mRNA levels in the same tissues collected from three uninfected healthy control animals. (B) Fold regulation for IL-10 and IL-12 mRNA levels in spleens and axillary lymph nodes.
Fig 4
Fig 4
IL-10 expression in spleen. Immunofluorescence was used to determine the phenotype of IL-10-producing cells in the red and white pulp of the spleen. Representative tissue sections of SIV-, P. fragile-, and SIV-P. fragile-infected animals at necropsy are shown. Images were stained with IL-10 (red) and the relevant lineage markers (green) for monocyte/macrophages (CD68), dendritic cells (CD205/DC-SIGN), T cells (CD3), or B cells (CD20). Panel A shows images for monocytes/macrophages and dendritic cells, and panel B includes the relevant images for T and B cells. Negative controls were performed via staining with appropriate isotype controls, and images were acquired and analyzed as positively stained sections (Fig. 6). White bars represent 50 μm. Despite the strong autofluorescence of red cells (closed white triangle) in the red pulp of the spleen, IL-10-positive cells could be clearly identified. Single cells positive for IL-10 are indicated by clear white block arrows, and lineage marker-positive cells are pointed out by dashed arrows. The boxed area and its enlargement in the SIV example shows a CD3-positive cell that is also positive for IL-10 (white arrow).
Fig 5
Fig 5
Cellular profile of IL-10-positive cells. (A) Total number of IL-10-positive cells in 100 fields of spleens (left) or axillary lymph nodes (right) of SIV-, P. fragile-, and SIV-P. fragile-infected animals that were determined by IHC (Fig. 4 and 6). Each bar represents an individual animal. The various patterns within the bars represent the number of Mo/Mph, mDC, T cells, and B cells. (B) Percentage of regulatory T cells within spleen (left) or axillary lymph node (right graph) cell suspensions. Each symbol represents an individual animal. The horizontal bar depicts the median percentage of Tregs within a specific infection group. (C) Longitudinal changes in peripheral blood regulatory T cells (black solid line) of SIV-P. fragile-coinfected animals in relation to plasma IL-10 levels (gray dotted line).
Fig 6
Fig 6
IL-10 expression in axillary lymph nodes. Immunofluorescence was used to determine the phenotype of IL-10-producing cells in the paracortex (T cell zone) and follicle (B cell area) of axillary lymph nodes. Representative tissue sections of SIV-, P. fragile-, and SIV-P. fragile-infected animals at necropsy are shown. Images show sections stained for IL-10 (red) and Mo/Mph or mDC markers (A) or sections stained for IL-10 and T or B cell markers (B). Single- and double-positive cells are identified as described in the legend to Fig. 4.
Fig 7
Fig 7
Apoptosis in lymphoid tissues. (A) Representative examples of tissue sections stained for caspase 3 (red) and CD68 (green) in axillary lymph nodes and spleens of SIV-, P. fragile-, and SIV-P. fragile-infected animals. Cells double positive for CD68 and caspase 3 in the axillary LN and the white pulp of SIV-P. fragile-infected animals are marked by a solid white arrow. Examples of Mo/Mph staining positive for CD68 are indicated by dashed white arrows, and representative caspase 3-positive cells are highlighted by the open arrows. Note that there is significant autofluorescence by red blood cells (color is more orange than that of red caspase 3-positive cells) within the red pulp; examples are shown by white filled triangles. (B) Summary of the data obtained by IHC analysis. Reported are the total frequencies of caspase 3-positive cells within a tissue and the frequencies of CD68-positive, caspase 3-positive cells (each per 100 fields analyzed). Each symbol represents an individual animal, and horizontal bars show the median values for all animals within one experimental group. (C) Flow-cytometric analysis of spleen and axillary lymph node cell suspensions for caspase 3-positive CD4 and CD8 T cells. Note that we only had spleen cells from one SIV-infected animal available to perform this analysis.
Fig 8
Fig 8
Apoptosis and T cell exhaustion in peripheral blood. Longitudinal blood samples of SIV-P. fragile-coinfected animals were analyzed for caspase 3 (A) and PD-1 (B) expression by flow-cytometric analysis. Shown are the percentages of caspase 3- or PD-1-positive CD3 T cells within an individual animal over time in relation to plasma IL-10 levels (gray dotted line). Data are reported for both CD4 (open circle) and CD8 (open triangle) T cells.
Fig 9
Fig 9
Plasma cytokine levels. Plasma IL-10 (left column) and IL-12 (middle column) levels were determined in longitudinally collected plasma samples by ELISA of SIV-P. fragile (A)-, SIV (B)-, or P. fragile (C)-infected animals. Individual animals are denoted by different symbols. The ratio of plasma IL-12 to IL-10 (with cytokine-negative samples being assigned a value of 5 pg/ml, the limit of detection) are shown in the right column. (D) The temporal association between plasma viremia or parasitemia with plasma IL-10 or IL-12 levels.

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